Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment due to downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as Bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human IgG-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE prior to administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting NK-cell reactivity, resulting in enhanced anti-myeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes, or B-BiTE itself, appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using two different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with two mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematological malignancies.

DOI： 10.1182/blood.2022019082

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.

DOI： 10.3390/ijms241210186

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：English  

DOI： 10.1111/exd.14759

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.

DOI： 10.1021/acsami.2c03299

PubMed

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

DOI： 10.1038/s41598-022-12212-6

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Despite the high sensitivity of renal cell carcinoma (RCC) to immunotherapy, RCC has been recognized as an unusual disease in which CD8+ T-cell infiltration into the tumor beds is related to a poor prognosis. To approach the inner landscape of immunobiology of RCC, we performed multiplexed seven-color immunohistochemistry (CD8, CD39, PD-1, Foxp3, PD-L1, and pan-cytokeratin AE1/AE3 with DAPI), which revealed the automated single-cell counts and calculations of individual cell-to-cell distances. In total, 186 subjects were included, in which CD39 was used as a marker for distinguishing tumor-specific (CD39+) and bystander (CD39-) T-cells. Our clear cell RCC cohort also revealed a poor prognosis if the tumor showed increased CD8+ T-cell infiltration. Intratumoral CD8+CD39+ T-cells as well as their exhausted CD8+CD39+PD-1+ T-cells in the central tumor areas enabled the subgrouping of patients according to malignancy. Analysis using specimens post-antiangiogenic treatment revealed a dramatic increase in proliferative Treg fraction Foxp3+PD-1+ cells, suggesting a potential mechanism of hyperprogressive disease after uses of anti-PD-1 antibody. Our cell-by-cell study platform provided spatial information on tumors, where bystander CD8+CD39- T-cells were dominant in the invasive margin areas. We uncovered a potential interaction between CD8+CD39+PD-1+ T-cells and Foxp3+PD-1+ Treg cells due to cell-to-cell proximity, forming a spatial niche more specialized in immunosuppression under PD-1 blockade. A paradigm shift to the immunosuppressive environment was more obvious in metastatic lesions; rather the infiltration of Foxp3+ and Foxp3+PD-1+ Treg cells was more pronounced. With this multiplexed single-cell pathology technique, we revealed further insight into the immunobiological standing of RCC.

DOI： 10.1007/s00262-021-03006-2

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

A cutting edge therapy for future immuno-oncology is targeting a new series of inhibitory receptors (IRs): LAG-3, TIM-3, and TIGIT. Both immunogenomic analyses and diagnostic platforms to distinguish candidates and predict good responders to these IR-related agents are vital in clinical pathology. By applying an automated single-cell count for immunolabelled LAG-3, TIM-3, and TIGIT, we reveal that individual IR levels with exclusive domination in each tumour can serve as valid biomarkers for profiling human renal cell carcinoma (RCC). We uncover the immunogenomic landscape associated with individual IR levels in human RCC tumours with metastases in various organs and histological subtypes. We then externally validate our results and devise a workflow with optimal biomarker cut-offs for discriminating the LAG-3, TIM-3, and TIGIT tumour profiles. The discrimination of LAG-3, TIM-3, and TIGIT profiles in tumours may have a broad impact on investigations of immunotherapy responses after targeting a new series of IRs.

DOI： 10.1038/s41467-021-25865-0

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Two-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600-1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2-3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

DOI： 10.1038/s41467-021-23249-y

PubMed

researchmap

Language：English  

DOI： 10.1016/j.jdermsci.2021.03.004

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The use of donor-pi-acceptor (D-pi-A) skeletons is an effective strategy for the design of fluorophores with red-shifted emission. In particular, the use of amino and boryl moieties as the electron-donating and -accepting groups, respectively, can produce dyes that exhibit high fluorescence and solvatochromism. Herein, we introduce a dithienophosphole P-oxide scaffold as an acceptor-spacer to produce a boryl- and amino-substituted donor-acceptor-acceptor (D-A-A) pi-system. The thus obtained fluorophores exhibit emission in the near-infrared (NIR) region, while maintaining high fluorescence quantum yields even in polar solvents (e.g. lambda(em) = 704 nm and phi(F) = 0.69 in CH3CN). A comparison of these compounds with their formyl- or cyano-substituted counterparts demonstrated the importance of the boryl group for generating intense emission. The differences among these electron-accepting substituents were examined in detail using theoretical calculations, which revealed the crucial role of the boryl group in lowering the nonradiative decay rate constant by decreasing the non-adiabatic coupling in the internal conversion process. The D-A-A framework was further fine-tuned to improve the photostability. One of these D-A-A dyes was successfully used in bioimaging to visualize the blood vessels of Japanese medaka larvae and mouse brain.

DOI： 10.1039/d1sc00827g

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

High-speed two-photon microscopy can be used to analyze vascular dynamics in living animals and is essential for the understanding of brain diseases. Recent advances in fluorescent probes/optical systems have allowed successful imaging of the hippocampal vasculature in the deep brain of mice (1 mm from the brain surface) under low-speed conditions (1-2 fps); however, using high-speed techniques (>30 fps), observation of the deep-brain vasculature is still challenging. Here, a new nanoemulsion that encapsulates thousands of red-emissive pyrene dye molecules while maintaining their high two-photon brightness [1.5 x 10(2) GM (GM = 10(-50) cm(4)center dot s center dot photon(-1)center dot molecule(-1)) at 960 nm excitation] and delivers a large amount of such pyrene dyes (65 nmol) into the blood vessels of mice is developed. Remarkably, the nanoprobe is found to exploit the inherent performance of a commonly used Ti:sapphire excitation laser and a sensitive gallium arsenide phosphide nondescanned fluorescence detector to the limit, enabling visualization of the brain vasculature under the cortex region of mice (up to 1.5 mm) under very low-speed conditions. As a highlight, such a nanoprobe is successfully used to directly observe the blood flow in the hippocampal CA1 region (1.1 mm) through high-speed resonant scanning (120 fps).

DOI： 10.1002/adfm.202010698

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.

DOI： 10.1267/ahc.20-00020

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

© 2020, The Author(s). The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

DOI： 10.1038/s41598-020-61579-x

Scopus

PubMed

researchmap

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-β/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 μg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2-/- mice. The rhBMP2-induced ectopic bone in Smurf2-/- mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2-/- mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2-/- mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2-/- BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.

DOI： 10.1038/s41413-020-00115-z

PubMed

researchmap

Other Link： http://www.nature.com/articles/s41413-020-00115-z

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：Japanese   Publisher：(一社)日本脊椎脊髄病学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Transcriptional coactivator with a PDZ-binding motif (TAZ) is one of the mammalian orthologs of Drosophila Yorkie, a transcriptional coactivator of the Hippo pathway. TAZ has been suggested to function as a regulator that modulates the expression of cell proliferation and anti-apoptotic genes in order to stimulate cell proliferation. TAZ has also been associated with a poor prognosis in several cancers, including breast cancer. However, the physiological role of TAZ in tumorigenesis remains unclear. We herein demonstrated that TAZ negatively regulated the activity of the tumor suppressor p53. The overexpression of TAZ down-regulated p53 transcriptional activity and its downstream gene expression. In contrast, TAZ knockdown up-regulated p21 expression induced by p53 activation. Regarding the underlying mechanism, TAZ inhibited the interaction between p53 and p300 and suppressed the p300-mediated acetylation of p53. Furthermore, TAZ knockdown induced cellular senescence in a p53-dependent manner. These results suggest that TAZ negatively regulates the tumor suppressor functions of p53 and attenuates p53-mediated cellular senescence.

DOI： 10.3390/cells9010171

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Epithelial-mesenchymal transition (EMT) is a biological process by which epithelial cells acquire mesenchymal characteristics. In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties, leading to tumor progression. An inflammatory microenvironment is thought to be responsible for the development and progression of colorectal cancer (CRC); however, the precise role of inflammatory microenvironments in EMT-related CRC progression remains unclear. Here, we show the spatiotemporal visualization of CRC cells undergoing EMT using a fluorescence-guided EMT imaging system in which the mesenchymal vimentin promoter drives red fluorescent protein (RFP) expression. An inflammatory microenvironment including TNF-α, IL-1β, and cytokine-secreting inflammatory macrophages induced RFP expression in association with the EMT phenotype in CRC cells. In vivo experiments further demonstrated the distribution of RFP-positive CRC cells in rectal and metastatic tumors. Our data suggest that the EMT imaging system described here is a powerful tool for monitoring EMT in inflammatory microenvironment-CRC networks.

DOI： 10.1038/s41598-019-52816-z

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

The development of chimeric antigen receptor (CAR) and bispecific T-cell engager (BiTE) has led to the successful application of cancer immunotherapy. The potential reactivity mediated by CAR- and BiTE-redirected T cells needs to be assessed to facilitate the application of these treatment options to a broader range of patients. Here, we have generated CAR and BiTE possessing the same single chain fragment variable (scFv) specific for the HLA-A2/NY-ESO-1157-165 complex (A2/NY-ESO-1157). Using HLA-A2+NY-ESO-1+ myeloma cells and peptides presented by HLA-A2 molecules as a model, both sets of redirected T cells recognized and killed HLA-A2+NY-ESO-1+ myeloma cells in an A2/NY-ESO-1157-specific manner in vitro. Moreover, CAR- and BiTE-activated T cells showed similar functional avidity, as assessed by cytokine production and killing activity, both displaying antitumor reactivity against HLA-A2+NY-ESO-1+ myeloma cells in vivo. Interestingly, cross-reactivity for homologous peptides presented by HLA-A*02:01 and NY-ESO-1157 peptide presented by HLA-A2 alleles was not identical between CAR- and BiTE-redirected T cells, probably due to structural differences of modified antibodies. These results have demonstrated that both antitumor CAR- and BiTE-activated T cells have comparable potential to recognize tumors, while paying attention to unknown off-target reactivity that would differ for each antibody-based modality even if the same scFv was employed.

DOI： 10.1038/s41598-019-49834-2

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

DOI： 10.1016/j.cell.2019.04.007

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Bone is one of the preferred sites for the metastasis of malignant tumours, such as breast cancer, lung cancer and malignant melanoma. Tumour cells colonizing bone have the capacity to induce the expression of receptor activator of nuclear factor-κB ligand (RANKL), which promotes osteoclast differentiation and activation. Tumour-induced osteoclastic bone resorption leads to a vicious cycle between tumours and bone cells that fuels osteolytic tumour growth, causing bone pain and hypercalcaemia. Furthermore, RANKL contributes to bone metastasis by acting as a chemoattractant to bone for tumour cells that express its receptor, RANK. Thus inhibition of the RANKL-RANK pathway is a promising treatment for bone metastasis, and a human monoclonal anti-RANKL antibody, denosumab, has been used in the clinic. However, orally available drugs targeting RANKL must be developed to increase the therapeutic benefits to patients. Here we report the efficacy of the small-molecule RANKL inhibitor AS2676293 in treating bone metastasis using mouse models. Oral administration of AS2676293 markedly inhibited bone metastasis of human breast cancer cells MDA-MB-231-5a-D-Luc2 as well as tumour-induced osteolysis. AS2676293 suppressed RANKL-mediated tumour migration in the transwell assay and inhibited bone metastasis of the murine cell line B16F10, which is known not to trigger osteoclast activation. Based on the results from this study, RANKL inhibition with a small-molecule compound constitutes a promising therapeutic strategy for treating bone metastasis by inhibiting both osteoclastic bone resorption and tumour migration to bone.

DOI： 10.1038/s41413-018-0036-5

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Osteoarthritis (OA) is a chronic joint disorder involving degeneration of articular cartilage and subchondral bone in joints. We previously established a second harmonic generation (SHG) imaging technique for evaluating degenerative changes to articular cartilage in an OA mouse model. SHG imaging, an optical label-free technique, enabled observation of collagen fibrils, and characterized critical changes in the collagenous patterns of the joints. However, it still remains to be determined how morphological changes in the organization of tissue collagen fibrils should be quantified. In this study, we addressed this issue by employing an approach based on texture analysis. Image texture analysis using the gray level co-occurrence matrix was explored to extract image features. We investigated an image patch-based strategy, in which texture features were extracted on individual patches derived from original images to capture local structural patterns in them. We verified that this analysis enables discrimination of cartilaginous and osseous tissues in mouse joints. Moreover, we applied this method to OA cartilage pathology assessment, and observed improvements in the performance results compared with those obtained using an existing feature descriptor. The proposed approach can be applied to a wide range of conditions associated with collagen remodeling and diseases of cartilage and bone.

DOI： 10.1038/s41598-018-21005-9

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

DOI： 10.1038/s41598-018-35412-5

PubMed

researchmap

Language：English   Publisher：(一社)日本血液学会-東京事務局  

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Various fluorescence microscopy techniques require bright NIR-emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus-substituted rhodamine dyes (PORs) upon substitution at the 9-position with a 2,6-dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long-term and three-color imaging in the vis-NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye-labeled biomolecule by single-molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR-emitting PREX 710-dextran conjugate.

DOI： 10.1002/anie.201804731

PubMed

researchmap

Language：English   Publisher：日本がん免疫学会  

researchmap

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/cas.13544

Scopus

PubMed

researchmap

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/cas.13544

Language：English   Publishing type：Research paper (scientific journal)  

Double-stranded (ds) RNA-dependent protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. It was initially identified as an innate immune antiviral protein induced by interferon (IFN) and activated by dsRNA. PKR is recognized as a key executor of antiviral host defense. Moreover, it contributes to inflammation and immune regulation through several signaling pathways. In addition to IFN and dsRNA, PKR is activated by multiple stimuli and regulates various signaling pathways including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells pathways. PKR was initially thought to be a tumor suppressor as a result of its ability to suppress cell growth and interact with major tumor suppressor genes. However, in several types of malignant disease, such as colon and breast cancers, its role remains controversial. In hepatocellular carcinoma, hepatitis C virus (HCV) is the main cause of liver cancer, and PKR inhibits HCV replication, indicating its role as a tumor suppressor. However, PKR is overexpressed in cirrhotic patients, and acts as a tumor promoter through enhancement of cancer cell growth by mediating MAPK or signal transducer and activator of transcription pathways. Moreover, PKR is reportedly required for the activation of inflammasomes and influences metabolic disorders. In the present review, we introduce the multifaceted roles of PKR such as antiviral function, tumor cell growth, regulation of inflammatory immune responses, and maintaining metabolic homeostasis; and discuss future perspectives on PKR biology including its potential as a therapeutic target for liver cancer.

DOI： 10.1111/cas.13551

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Tissue intrinsic emission fluorescence provides useful diagnostic information for various diseases. Because of its unique feature of spectral profiles depending on tissue types, spectroscopic imaging is a promising tool for accurate evaluation of endogenous fluorophores. However, due to difficulties in discriminating those sources, quantitative analysis remains challenging. In this study, we quantitatively investigated spectral-spatial features of multi-photon excitation fluorescence in normal and diseased livers. For morphometrics of multi-photon excitation spectra, we examined a marker-controlled segmentation approach and its application to liver fibrosis assessment by employing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. We formulated a procedure of internal marker selection where markers were chosen to reflect typical biochemical species in the liver, followed by image segmentation and local morphological feature extraction. Image segmentation enabled us to apply mathematical morphology analysis, and the local feature was applied to the automated classification test based on a machine learning framework, both demonstrating highly accurate classifications. Through the analyses, we showed that spectral imaging of native fluorescence from liver tissues have the capability of differentiating not only between normal and diseased, but also between progressive disease states. The proposed approach provides the basics of spectroscopy-based digital histopathology of chronic liver diseases, and can be applied to a range of diseases associated with autofluorescence alterations.

DOI： 10.3389/fmed.2018.00350

PubMed

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively.

DOI： 10.1016/j.bbrep.2016.09.010

PubMed

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：Japanese   Publisher：(一社)日本消化器外科学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Spandidos Publications  

Breast cancer is the most common cancer in women. Although advances in diagnostic imaging for early detection, surgical techniques and chemotherapy have improved overall survival, the prognosis of patients with metastatic breast cancer remains poor. Understanding cancer cell dynamics in the metastatic process is important to develop new therapeutic strategies. Experimental animal models and imaging would be powerful tools for understanding of the molecular events of multistep process of metastasis. In the present study, to develop a new cancer cell line that is applicable to bioluminescence and fluorescence imaging, we transfected the expression vector of a green fluorescent protein ZsGreen1 into a metastatic cell line 5a-D-Luc, which is a subclone of the MDA-MB-231 breast cancer cell line expressing luciferase, and established a new tumor cell line 5a-D-Luc-ZsGreen expressing both luciferase and ZsGreen1. The 5a-D-Luc-ZsGreen cells proliferate more rapidly and have a more invasive phenotype compared with 5a-D-Luc cells following intracardiac injection. Metastasis sites were easily detected in the whole body by bioluminescence imaging and in excised tissues by ex vivo fluorescence imaging. The fluorescence of 5a-D-Luc-ZsGreen cells was not lost after formalin fixation and decalcification. It enabled us to easily evaluate tumor spread and localization at the cellular level in microscopic analysis. The strong fluorescence of 5a-DLuc- ZsGreen cells allowed for real-time imaging of circulating tumor cells in cerebral blood vessels of live animals immediately after intracardiac injection of cells using two-photon laser-scanning microscopy. These findings suggest that the 5a-D-Luc-ZsGreen cells would be a useful tool for research on mechanisms of metastatic process in animal models.

DOI： 10.3892/ijo.2015.3300

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor beta (TGF-beta) in certain kinds of cancer cells through the induction of Snail, a key regulator of EMT. We have previously found that TGF-beta remarkably induces Snail expression in cooperation with Ras signals; however, the underlying mechanism of this synergism has not yet been determined. Here, we demonstrate that signal transducer and activator of transcription 3 (STAT3) acts as a mediator that synergizes TGF-beta and Ras signals. The overexpression of STAT3 enhanced Snail induction, whereas siRNA-mediated knockdown of STAT3 inhibited it. The STAT3-gamma F mutant, which has Tyr 705 substituted with Phe, did not enhance Snail induction. Several STAT3 mutants lacking transcriptional activity also failed to enhance it; however, the putative STAT3-binding elements in the Snail promoter regions were not required for STAT3-mediated Snail induction. Protein inhibitor of activated STAT3 (PIAS3) inhibited the enhanced Snail promoter activity induced by TGF-beta and Ras. The interaction between PIAS3 and STAT3 was reduced by TGF-beta in cells harboring oncogenic Ras, whereas TGF-beta promoted the binding of PIAS3 to Smad3, a crucial mediator of TGF-beta signaling. Therefore, these findings suggest that STAT3 enhances Snail induction when it is dissociated from PIAS3 by TGF-beta in cooperation with Ras signals.

DOI： 10.1038/onc.2015.161

Web of Science

researchmap

Language：English   Publisher：WILEY-BLACKWELL  

Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation.

DOI： 10.1111/dgd.12252

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：The Japan Society for Industrial and Applied Mathematics  

<p><i>Abstract.</i> Texture image analysis plays a pivotal role in characterizing object properties. This technique numerically represents the spatial arrangement of the gray levels of digital images, and is used on problems of classifying image data sets. Second harmonic generation, an optical label-free technique based on nonlinear optical effects, is recognizing important for biomedical applications. Researches that integrate these two technologies are increasingly being used not only for advancing basic understanding of biological processes, but also for applications of clinical diagnosis. Here, we highlight recent advances in this combined research area, and describe how this approach is used in tackling medical problems.</p>

DOI： 10.11540/jsiamt.26.2_253

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公財)金原一郎記念医学医療振興財団  

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Arkadia, a positive regulator of Smad-dependent signalling via the transforming growth factor-beta (TGF-beta) family, is an E3 ubiquitin ligase that induces ubiquitylation and proteasome-dependent degradation of TGF-beta suppressors such as Smad7, c-Ski and SnoN. In this study, we examined the effects of Arkadia on bone morphogenetic protein (BMP)-induced osteoblast differentiation. Knockdown of Arkadia reduced mineralization and expression of osteoblast differentiation markers. Furthermore, we showed that Smad6, a BMP-specific inhibitory Smad, is a target of Arkadia: wild-type (WT) Arkadia, but not the C937A (CA) mutant lacking E3 ubiquitin-ligase activity, induced ubiquitylation and proteasome-dependent degradation of Smad6. Accordingly, protein levels of Smad6, Smad7 and c-Ski were elevated in MEFs from Arkadia KO mice. Finally, expression of Arkadia attenuated blockade of BMP signalling by Smad6 in a transcriptional reporter assay. These results demonstrate that Smad6 is a novel target of Arkadia, and that Arkadia positively regulates BMP signalling via degradation of Smad6, Smad7 and c-Ski/SnoN.

DOI： 10.1093/jb/mvv024

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.

DOI： 10.3390/ijms160715785

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Transforming growth factor-beta (TGF-beta) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (T(H)17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-beta receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, ROR gamma t encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in T(H)17 differentiation.

DOI： 10.1038/ncomms8600

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(株)医薬ジャーナル社  

生体蛍光イメージング技術は、生体で分子や細胞の挙動や機能を可視化するために有効な手法で、近年、急速に発展してきた。特に、新しい蛍光タンパク質や蛍光有機小分子の発見や改良、2光子励起顕微鏡など蛍光検出機器の性能の飛躍的向上や画像処理ソフトウェアの開発により、骨・軟骨組織においてもその応用が可能になりつつある。本稿では、最近の生物学における生体蛍光イメージング技術について、われわれの知見を中心に紹介する。さらに、生体蛍光イメージング技術の問題点と運動器疾患研究(骨・軟骨生物学)における応用の可能性について議論したい。(著者抄録)

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J02593&link_issn=&doc_id=20150810250010&doc_link_id=10.20837%2F4201508079&url=https%3A%2F%2Fdoi.org%2F10.20837%2F4201508079&type=%88%E3%8F%91.jp_%83I%81%5B%83%8B%83A%83N%83Z%83X&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00024_2.gif

Language：Japanese   Publisher：(一社)日本骨代謝学会  

researchmap

Language：Japanese   Publisher：(一社)日本消化器外科学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Bone modeling and remodeling are cellular events during which osteoblast lineage cells and osteoclasts interact. During these events, cells undergo drastic changes with time as they become differentiated. Their morphology, topology, and activity are affected by other cells and the extracellular matrices. Since the mechanisms underlying the cellular events of bone metabolism have not been elucidated, there is a need for systems to analyze these cellular networks and their microenvironments spatiotemporally at the cellular level. Here we report a novel in vitro system for reconstituting the bone cell network of osteoclasts, osteoblasts, and osteocytes in the mineralized nodule, allowing for observation of bone modeling and remodeling phenomena by 2-photon microscopy. Using this system, the change in morphology of osteoblasts from cuboidal to flat cells was observed and measured during the formation of mineralized nodules. Furthermore, the recruitment of osteoblasts to resorption pits and their replenishment by newly formed matrices were successfully observed, providing strong evidence for the coupling of bone resorption and bone formation at cellular level. During such remodeling cycle, flat osteoblasts that survived more than 7 weeks were recruited to resorption pits, where they became cuboidal osteoblasts that express osteocalcin. This novel system permitted the elucidation of cellular behavior during bone modeling and remodeling, and can be used to analyze cellular events involved in bone metabolism. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2015.02.030

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(一社)日本細胞生物学会  

researchmap

Language：Japanese   Publisher：(株)羊土社  

生きている動物の中で、細胞の機能をダイナミックに解析できる生体光イメージング技術に注目が集まっている。この手法を用いると、例えば、転移するがん細胞の細胞周期や細胞内シグナリングを画像化することが可能で、これまでは知り得なかった細胞内の現象や細胞間の相互作用が手に取るようにわかる。また、「見ること」で研究者の生命現象に対する理解が深まり、新しい診断や治療開発に関係するアイデアが浮かぶ。百聞は一見に如かずというが、まさしく生体光イメージング技術によって新たなシグナリング研究が拓かれようとしている。(著者抄録)

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We have previously reported that transforming growth factor (TGF-) plays an essential role in receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF- signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF- regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos. (c) 2014 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2418

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(一社)日本外科学会  

researchmap

Language：Japanese   Publisher：(公財)日本眼科学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OPTICAL SOC AMER  

Osteoarthritis (OA) restricts the daily activities of patients and significantly decreases their quality of life. The development of noninvasive quantitative methods for properly diagnosing and evaluating the process of degeneration of articular cartilage due to OA is essential. Second harmonic generation (SHG) imaging enables the observation of collagen fibrils in live tissues or organs without staining. In the present study, we employed SHG imaging of the articular cartilage in OA model mice ex vivo. Consequently, three-dimensional SHG imaging with successive image processing and statistical analyses allowed us to successfully characterize histopathological changes in the articular cartilage consistently confirmed on histological analyses. The quantitative SHG imaging technique presented in this study constitutes a diagnostic application of this technology in the setting of OA. (C) 2015 Optical Society of America

DOI： 10.1364/BOE.6.000405

Web of Science

PubMed

researchmap

Osteoporosis is a major bone disease that connotes the risk of fragility fractures resulting from alterations to bone quantity and/or quality to mechanical competence. Bone strength arises from both bone quantity and quality. Assessment of bone quality and bone quantity is important for prediction of fracture risk. In spite of the two factors contribute to maintain the bone strength, only one factor, bone mineral density is used to determine the bone strength in the current diagnosis of osteoporosis. On the other hand, there is no practical method to measure chemical composition of bone tissue including hydroxyapatite and collagen non-invasively. Raman spectroscopy is a powerful technique to analyze chemical composition and material properties of bone matrix non-invasively. Here we demonstrated Raman spectroscopic analysis of the bone matrix in osteoporosis model rat. Ovariectomized (OVX) rat was made and the decalcified sections of tibias were analyzed by a Raman microscope. In the results, Raman bands of typical collagen appeared in the obtained spectra. Although the typical mineral bands at 960 cm-1 (Phosphate) was absent due to decalcified processing, we found that Raman peak intensities of amide I and C-C stretching bands were significantly different between OVX and sham-operated specimens. These differences on the Raman spectra were statistically compared by multivariate analyses, principal component analysis (PCA) and liner discrimination analysis (LDA). Our analyses suggest that amide I and C-C stretching bands can be related to stability of bone matrix which reflects bone quality.

DOI： 10.1117/12.2078787

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Objective: Mechanical loading on the bone is sensed by osteocytes. Sclerostin is a molecule secreted by osteocytes that is downregulated by mechanical loading; therefore, its expression level is a potent sensor that indicates the spatial transduction of biomechanical properties in bone. This study applied macroconfocal microscopy to observe the spatial response of alveolar bone to orthodontic forces after immunofluorescence using anti-sclerostin antibodies.
Design: Orthodontic tooth movement with the Ni-Ti closed-coil spring was applied between the upper bilateral incisors and the left first molar of mice. Four days after this application, the animals were subjected to multimodal confocal fluorescence imaging analyses.
Results: Obvious downregulation of sclerotin in the osteocytic lacuna-canalicular system (LCS) was observed specifically in tensile sites of alveolar bone. Confocal-based three-dimensional fluorescence morphometry further quantitatively demonstrated that the distribution and expression of sclerostin in the tensile sites was significantly reduced compared to that observed in the corresponding control sites. Interestingly, the levels of sclerotin signals in the compression sites were significantly higher than those observed in the control sites, although the distribution of sclerotin was not significantly different.
Conclusions: Our observations suggest that spatial changes in the level and distribution of sclerostin in the alveolar LCS trigger successive bone remodelling due to orthodontic tooth movement. The multimodal confocal imaging analyses applied in this work will enhance comprehensive understanding regarding the spatial regulation of molecules of interest from the tissue to the cellular level. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2014.08.013

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE-INT SOC OPTICAL ENGINEERING  

Osteoporosis is a major bone disease that connotes the risk of fragility fractures resulting from alterations to bone quantity and/or quality to mechanical competence. Bone strength arises from both bone quantity and quality. Assessment of bone quality and bone quantity is important for prediction of fracture risk. In spite of the two factors contribute to maintain the bone strength, only one factor, bone mineral density is used to determine the bone strength in the current diagnosis of osteoporosis. On the other hand, there is no practical method to measure chemical composition of bone tissue including hydroxyapatite and collagen non-invasively. Raman spectroscopy is a powerful technique to analyze chemical composition and material properties of bone matrix non-invasively. Here we demonstrated Raman spectroscopic analysis of the bone matrix in osteoporosis model rat. Ovariectomized (OVX) rat was made and the decalcified sections of tibias were analyzed by a Raman microscope. In the results, Raman bands of typical collagen appeared in the obtained spectra. Although the typical mineral bands at 960 cm(-1) (Phosphate) was absent due to decalcified processing, we found that Raman peak intensities of amide I and C-C stretching bands were significantly different between OVX and sham-operated specimens. These differences on the Raman spectra were statistically compared by multivariate analyses, principal component analysis (PCA) and liner discrimination analysis (LDA). Our analyses suggest that amide I and C-C stretching bands can be related to stability of bone matrix which reflects bone quality.

DOI： 10.1117/12.2078787

Web of Science

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE  

We developed a near infrared fluorophore-conjugated anti-Carcinoembryonic antigen (CEA) antibody for mice bearing tumor of CEA expressing cells, and demonstrated in vivo optical imaging by macro zoom microscopy. In the result, tumors of CEA-expressing cancer cells were specifically detected in vivo. Furthermore, cancer-specific fluorescence images were acquired at subcellular level in vivo by two-photon microscopy. In preclinical applications, the lymph node micrometastasis was also successfully visualized by two-photon microscopy. These results suggest that two-photon excitation microscopy in combination with an immunoconjugated probe could be widely adapted to cancer detection in clinical settings.

DOI： 10.1117/12.2079092

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.

DOI： 10.1371/journal.pcbi.1003957

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Forster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from R-max and R-min values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.

DOI： 10.3390/ijms151119971

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio-conjugation of Alexa Fluor 594 to the anti-CEA antibody allowed visualization of tumor mass consisting of CEA-expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two-photon excitation microscope and the same fluorescent antibody resulted in subcellular-resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications.

DOI： 10.1111/cas.12500

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OPTICAL SOC AMER  

Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (&gt;700 mu m) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec. (C)2014 Optical Society of America

DOI： 10.1364/BOE.5.003311

Web of Science

PubMed

researchmap

Language：English   Publisher：日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background and Objective: Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury.
Materials and Methods: In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice.
Results: For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser.
Conclusion: The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future.(C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/lsm.22266

Web of Science

PubMed

researchmap

Language：English   Publisher：日本癌学会  

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：Japanese   Publisher：(一社)日本骨代謝学会  

researchmap

Language：Japanese   Publisher：日本骨形態計測学会  

researchmap

Language：Japanese   Publisher：日本骨形態計測学会  

researchmap

Language：Japanese   Publisher：(株)羊土社  

がん転移の標的臓器である骨は、がんの進展に能動的にかかわっている。がんの病態のさらなる理解のために、種々のイメージング技術を用いた骨髄がん微小環境の解析が試みられている。発光イメージングはマクロレベルでの骨転移病変の検出に優れ、2光子励起顕微鏡を用いた蛍光イメージングは、骨髄内における多チャンネルの解析を可能とする技術である。また、ラマン分光イメージングは無染色での病変の質的評価への応用可能性を秘めている。それぞれに克服すべき課題は多いが、今後の発展と普及が期待されている。(著者抄録)

researchmap

Language：Japanese   Publisher：(一社)日本外科学会  

researchmap

Language：Japanese   Publisher：(一社)日本病理学会  

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE  

Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage. © 2014 SPIE.

DOI： 10.1117/12.2041042

Scopus

researchmap

Language：Japanese   Publisher：(一社)日本血栓止血学会  

近年,生命科学分野における飛躍的な技術革新により,蛍光技術を用いて分子や細胞の動態や機能を解析するfluorescent imaging(蛍光イメージング)が急速に発展した.特に新しい蛍光タンパク質プローブの開発,2光子励起顕微鏡などの機器の開発とその性能の飛躍的向上や画像処理ソフトウエアの進歩により,蛍光イメージングの対象は培養細胞のみならず,生きているマウスまで拡大してきている.血管研究の分野でも蛍光イメージングの応用が進んでいが,一方で,動物が生きている状態(生体)での蛍光イメージングにおいては,生体組織による蛍光の吸収や散乱などのさまざまな光学的問題が生じ,特に深部観察が難しいという欠点がある.本稿では,血管研究における生体蛍光イメージング技術の現状と今後展望について,われわれのがん研究におけるデータを紹介しながら,生体蛍光イメージング技術の問題点を洗い出し,その解決策を探りたい.(著者抄録)

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2013&ichushi_jid=J02561&link_issn=&doc_id=20140108340005&doc_link_id=10.2491%2Fjjsth.24.582&url=https%3A%2F%2Fdoi.org%2F10.2491%2Fjjsth.24.582&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：English   Publishing type：Research paper (scientific journal)  

Members of the transforming growth factor-β (TGF-β) family, including TGF-βs, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. TGF-β family signalling is mainly mediated by membranous serine/threonine kinase receptors and intracellular Smad proteins. This signalling is tightly regulated by various post-translational modifications including ubiquitination. Several E3 ubiquitin ligases play a crucial role in the recognition and ubiquitindependent degradation of TGF-β family receptors, Smad proteins and their interacted proteins to regulate positively and negatively TGF-β family signalling. In contrast, non-degradative ubiquitin modifications also regulate TGF-β family signalling. Recently, in addition to protein ubiquitination, deubiquitination by deubiquitinating enzymes has been reported to control TGF-β family signalling pathways. Interestingly, more recent studies suggest that TGF-β signalling is not only regulated via ubiquitination and/or deubiquitination, but also it relies on ubiquitination for its effect on other pathways. Thus, ubiquitin modifications play key roles in TGF-β family signal transduction and cross-talk between TGF-β family signalling and other signalling pathways. Here, we review the current understandings of the positive and negative regulatory mechanisms by ubiquitin modifications that control TGF-β family signalling. © The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

DOI： 10.1093/jb/mvt097

Scopus

PubMed

researchmap

PubMed

researchmap

Language：Japanese   Publisher：(株)医薬ジャーナル社  

近年、動物が生きたままの状態で、多様な生命現象を解析できるintravital optical imaging(生体光イメージング)の技術が急速に発展しつつある。特に新しい蛍光タンパク質や蛍光有機小分子の発見や改良、2光子励起顕微鏡など蛍光検出機器の性能の飛躍的向上や画像処理ソフトウェアの開発により、これまで蛍光技術の応用が困難であった骨・軟骨組織においてもその応用が可能になりつつある。本稿では、生体光イメージング技術の最近の進歩とその骨・軟骨組織解析への応用について、われわれのデータを紹介しながら、現状とその問題点、さらにその将来や臨床応用の可能性について議論したい。(著者抄録)

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2013&ichushi_jid=J02593&link_issn=&doc_id=20131206240008&doc_link_id=10.20837%2F4201312075&url=https%3A%2F%2Fdoi.org%2F10.20837%2F4201312075&type=%88%E3%8F%91.jp_%83I%81%5B%83%8B%83A%83N%83Z%83X&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00024_2.gif

Language：English   Publisher：日本癌学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(株)医薬ジャーナル社  

血球のトラフィッキングの研究において、生体内で細胞の動態や機能を解析することが可能なin vivo imagingは必須の技術である。中でも蛍光タンパク質を駆使したin vivo fluorescent imagingは、経時的、リアルタイムに細胞動態を観察することが可能で、その細胞トラフィッキング解析への応用は急速に進んでいる。ただし、生体が高散乱体であるため、観察対象が生体深部に存在する時には、非線形光学を駆使した多光子励起顕微鏡など検出機器の技術革新が必要になり、それらの技術の今後の発展が期待される。(著者抄録)

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger Delta R/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.

DOI： 10.1038/srep02231

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：日本骨形態計測学会  

researchmap

Language：Japanese   Publisher：(一社)日本細胞生物学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC CELL BIOLOGY  

Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.

Web of Science

PubMed

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(NPO)日本レーザー医学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Smad7 is an inhibitory molecule induced by members of the transforming growth factor-beta (TGF-beta) family, including TGF-beta, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7-knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7-floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7-deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7-deficient MEF cells. Moreover, expression levels of TGF-beta type I receptor (ALK5) were higher in Smad7-deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7-deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7-deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo.

DOI： 10.1093/jb/mvs022

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(株)メディカルレビュー社  

血管新生は、発生期や創傷治癒から慢性炎症の増悪や癌の悪性化に至るまでさまざまな生命現象において重要なはたらきを担っている。より正確に血管新生を理解するためには、動物が生きたまま生体内の分子や細胞の動態や機能を解析するin vivo(生体)イメージングの血管新生研究への応用が有用である。血管標識用近赤外蛍光プローブを用いると、動物が生きている状態で癌の新生血管の変化を経時的に観察できる。本稿では、血管医学におけるin vivoイメージング、とくに蛍光技術を駆使したイメージングの現状と展望について、われわれの癌研究におけるデータを紹介しながら、その問題点と将来の可能性を探りたい。(著者抄録)

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Transforming growth factor-beta (TGF-beta) ligand is a multifunctional growth factor that regulates various cell behavior, such as cell proliferation, differentiation, migration, and apoptosis. Because TGF-beta is a potent growth inhibitor, abnormalities in TGF-beta signaling result in carcinogenesis. In addition to tumor suppressor function, TGF-beta acts as an oncogenic factor. In particular, TGF-beta signaling plays an important role during metastasis of breast cancer. Recently, epithelial-mesenchymal transition (EMT) has been shown to confer malignant properties such as cell motility and invasiveness to cancer cells and plays crucial roles during cancer metastasis. Moreover, breast stem-like cells exhibit EMT properties. Because TGF-beta is a potent regulator of EMT as well as cell stemness, TGF-beta signaling might play a crucial role in the regulation of breast cancer stem cells.

DOI： 10.1007/s12282-011-0321-2

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Phosphatidylinositol 3-kinase (PI3K) is regarded as a promising therapeutic target because it is often activated in cancer. We previously reported that ZSTK474, a specific PI3K inhibitor, inhibits tumour cell proliferation via G1 arrest of the cell cycle without inducing apoptosis in vitro. However, it remained unclear whether ZSTK474 induces G1 arrest to exert antitumour efficacy in vivo. We recently developed a live imaging system, named Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci), to visualise cell cycle distribution. Here, by using this system, we tested whether ZSTK474 induces G1 arrest in tumour cells in vivo, as well as in vitro. Fucci-introduced human breast cancer MCF-7 cells and cervical cancer HeLa cells were subcutaneously xenografted in nude mice. ZSTK474 was administered to the tumour-bearing mice for 5 days, and the cell cycle distribution in the xenografted tumours were analysed by monitoring fluorescence in live mice. We demonstrate that ZSTK474 induces G1arrest along with tumour suppression in vivo. Moreover, we show that ZSTK474 suppresses the tumour growth without inducing apoptosis. Interestingly, such increase in G1 cells and tumour suppression was maintained during long-term (3-month) administration of ZSTK474. These results suggest that ZSTK474 exerts its in vivo antitumour efficacy via G1 arrest but not via apoptosis as long as it is administered, and could be used for months as maintenance therapy for patients with advanced cancers. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ejca.2011.10.006

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.01.141

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(株)羊土社  

癌細胞は、原発巣で増殖・浸潤するのみならず、遠隔臓器に転移する。複雑な癌の転移メカニズムを解析するには、オーソドックスな生化学や病理学の手法に加え、生きたままの動物の中で細胞や分子の動態や機能を立体画像化し、経時的に解析することが可能な4Dイメージングが力を発揮する。本稿では、生物発光や蛍光を用いた時空間イメージングを駆使した癌研究について、最近のわれわれのデータを紹介し、癌研究の将来における4Dイメージングの可能性について考察したい。(著者抄録)

researchmap

Language：Japanese   Publisher：(株)メディカルレビュー社  

Fucciは、細胞の増殖サイクルのなかで細胞周期関連蛋白質の発現がユビキチン・プロテアソーム系によって厳密に制御されていることを利用して作製された、細胞周期をリアルタイムで可視化する画期的な蛍光イメージング技術である。Fucciを用いると、個体の発生、分化、再生、がん化など、細胞周期と関連する生命現象を細胞や動物が生きている状態で解析ができる。本稿では、Fucci技術を用いたがん研究について、その現状と将来性について議論する。(著者抄録)

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transforming growth factor-beta (TGF-beta) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-beta signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-beta signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-beta signaling, and knockdown of endogenous RB1CC1 attenuated TGF-beta beta-induced expression of target genes as well as TGF-beta-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-beta signaling by restricting substrate specificity of Arkadia.

DOI： 10.1074/jbc.M111.227561

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：愛媛医学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC HEMATOLOGY  

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-beta (TGF-beta)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-beta signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-beta response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-beta. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-beta signaling. Our results suggest that HBZ, by enhancing TGF-beta signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence. (Blood. 2011;118(7):1865-1876)

DOI： 10.1182/blood-2010-12-326199

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Specific regulation of target genes by transforming growth factor-beta (TGF-beta) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-beta. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4 alpha (HNF4 alpha) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4 alpha-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4 alpha binding regions under TGF-beta stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4 alpha bindings. MIXL1 was identified as a new combinatorial target of HNF4 alpha and Smad2/3, and both the HNF4 alpha protein and its binding motif were required for the induction of MIXL1 by TGF-beta in HepG2 cells. These findings generalize the importance of binding of HNF4 alpha on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-beta and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-beta-Smad signaling pathway.

DOI： 10.1074/jbc.M110.217745

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Previous studies have shown that transforming growth factor beta (TGF-beta) promotes receptor activator of nuclear factor-kappa beta ligand (RANKL) induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF-beta signals were blocked either by a specific inhibitor of TGF-beta type 1 receptor kinase activity, SB431542, or by introducing a dominant-negative mutant of TGF-beta type 2 receptor, RANKL-induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c-Ski markedly suppressed RANKL-induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6-TAB1-TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6-TAB1-TAK1 complex formation was not observed when TGF-beta signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6-TAB1-TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL-induced osteoclast differentiation. In summary, TGF-beta is indispensable in RANKL-induced osteoclastogenesis, and the binding of Smad3 to the TRAF6-TAB1-TAK1 complex is crucial for RANKL-induced osteoclastogenic signaling. (C) 2011 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.357

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：Japanese   Publisher：(株)医薬ジャーナル社  

近年、複雑かつ多様な生命現象を動物が生きたまま解析できるin vivo蛍光イメージング技術が急速に発展しつつある。特に新しい蛍光タンパク質や蛍光色素の発見やその改良、2光子励起顕微鏡など検出機器の性能の飛躍的向上やソフトウエアの開発により、これまで蛍光技術の応用が困難であった骨代謝分野においても、その応用が可能になりつつある。本稿では、in vivo蛍光イメージング技術について、我々のデータを紹介しながら、骨代謝研究における現状とその問題点、さらにその将来や臨床応用の可能性について議論したい。(著者抄録)

researchmap

Language：Japanese   Publisher：日本分子イメージング学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ski was originally identified as an oncogene based on the fact that Ski overexpression transformed chicken and quail embryo fibroblasts. Consistent with these proposed oncogenic roles, Ski is overexpressed in various human tumors. However, whether and how Ski functions in mammalian tumorigenesis has not been fully investigated. Here, we show that Ski interacts with p53 and attenuates the biological functions of p53. Ski overexpression attenuated p53-dependent transactivation, whereas Ski knockdown enhanced the transcriptional activity of p53. Interestingly, Ski bound to the histone deacetylase SIRT1 and stabilized p53-SIRT1 interaction to promote p53 deacetylation, which subsequently decreased the DNA binding activity of p53. Consistent with the ability of Ski to inactivate p53, overexpressing Ski desensitized cells to genotoxic drugs and Nutlin-3, a small-molecule antagonist of Mdm2 that stabilizes p53 and activates the p53 pathway, whereas knocking down Ski increased the cellular sensitivity to these agents. These results indicate that Ski negatively regulates p53 and suggest that the p53-Ski-SIRT1 axis is an attractive target for cancer therapy.

DOI： 10.1074/jbc.M110.177683

Web of Science

PubMed

researchmap

Language：Japanese   Publishing type：Research paper (scientific journal)  

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Arkadia is a positive regulator of transforming growth factor (TGF)-beta signalling that induces ubiquitin-dependent degradation of several inhibitory proteins of TGF-beta signalling through its C-terminal RING domain. We report here that, through yeast-two-hybrid screening for Arkadia-binding proteins, the mu 2 subunit of clathrin-adaptor 2 (AP2) complex was identified as an interacting partner of Arkadia. Arkadia was located in both the nucleus and the cytosol in mammalian cells. The C-terminal YXX Phi-binding domain of the mu 2 subunit associated with the N-terminal YALL motif of Arkadia. Arkadia ubiquitylated the mu 2 subunit at Lys130. In addition, Arkadia interacted with the AP2 complex, and modified endocytosis of epidermal growth factor receptor (EGFR) induced by EGF. Arkadia thus appears to regulate EGF signalling by modulating endocytosis of EGFR through interaction with AP2 complex.

DOI： 10.1093/jb/mvq127

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Mechanical stress rapidly induces Delta FosB expression in osteoblasts, which binds to interleukin (IL)-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs) also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription.
Methodology/Principal Findings: Mechanical loading by fluid shear stress (FSS) induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads), Smad1/5, in murine primary osteoblasts (mPOBs). FSS rapidly phosphorylated Y311 of protein kinase C (PKC)delta, and phosphorylated PKC delta interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKC delta siRNA or Y311F mutant PKC delta abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE) of IL-11 gene promoter and formed complex with Delta FosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS.
Conclusions/Significance: These results demonstrate that PKC delta-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKC delta-BR-Smads and Delta FosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.

DOI： 10.1371/journal.pone.0013090

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ER alpha and ER beta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ER alpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ER alpha inhibits TGF-beta signaling by decreasing Smad protein levels. ER alpha-mediated reductions in Smad levels did not require the DNA binding ability of ER alpha, implying that ER alpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ER alpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ER alpha

DOI： 10.1074/jbc.M109.093039

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1(f/f) mice and alpha 1(II) Runx1(f/f) mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and alpha 1(II) DKO mice, respectively]. Prx1 Runx1(f/f) mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, alpha 1(II) Runx1(f/f) mice and alpha 1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of alpha 1(II) collagen (Col2a1-Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of alpha 1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

DOI： 10.1242/dev.045005

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Molecular mechanism of mechanical stress-induced bone formation remains unclear. We demonstrate that mechanical unloading suppresses and reloading enhances Interleukin (IL)-11 gene expression in the hindlimb of mice in vivo. Mechanical stress to osteoblasts by fluid shear stress (FSS) in vitro rapidly and transiently enhances fosB gene transcription, stimulates binding of Delta FosB/JunD complex to activator protein (AP)-1 site of the IL-11 gene promoter, and enhances IL-11 gene transcription. Anti-IL-11 antibody blocks mechanical stress-induced enhancement of osteoblastogenesis and suppression of adipogenesis, suggesting the requirement of IL-11 for the stimulation of osteoblast differentiation by mechanical stress. Downregulation of Delta FosB/JtmD by small interfering RNA (siRNA) suppresses and overexpression of Delta FosB/JunD enhances IL-11 gene promoter activity. Consistent with our previous observations that up-regulation of Delta FosB depends upon activation of cyclic AMP response element-binding protein (CREB) via Ca(2+)-dependent activation of extracellular signal-regulated kinase (ERK) to phosphorylate CREB, mechanical stress-induced activation of IL-11 gene transcription is dependent upon Ca(2+)-ERK pathway. Present results also demonstrated that FSS to osteoblasts enhances canonical Wnt signaling in vitro, and that mechanical unloading induces and reloading suppresses the expression of a canonical Wnt signal inhibitor, dickkopf2 (Dkk2), in vivo. In addition, IL-11 siRNA enhances Dkk2 expression suppressed by FSS, and osteoblasts from IL-11 transgenic mice show reduced Dkk2 mRNA expression than those from wild-type mice. These observations are consistent with the notion that mechanical stress stimulates IL-11 gene transcription via an enhanced OFosB/JunD binding to the IL-11 gene promoter, and that increased IL-11 enhances canonical Wnt signal at least in part via a reduction in Dkk2 expression to stimulate osteoblast differentiation. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2009.07.087

Web of Science

PubMed

researchmap

Language：English  

Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre-existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors.

DOI： 10.1111/j.1349-7006.2009.01305.x

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Smad4, the common partner Smad, is a key molecule in transforming growth factor-beta (TGF-beta) family signaling. Loss of Smad4 expression is found in several types of cancer, including pancreatic cancer and colon cancer, and is related to carcinogenesis. Here we identified Smad4 binding sites in the promoter regions of over 25 500 known genes by chromatin immunoprecipitation on a microarray (ChIP-chip) in HaCaT human keratinocytes. We identified 925 significant Smad4 binding sites. Approximately half of the identified sites overlapped the binding regions of Smad2 and Smad3 (Smad2/3, receptor-regulated Smads in TGF-beta signaling), while the rest of the regions appeared dominantly occupied by Smad4 even when a different identification threshold for Smad2/3 binding regions was used. Distribution analysis showed that Smad4 was found in the regions relatively distant from the transcription start sites, while Smad2/3 binding regions were more often present near the transcription start sites. Motif analysis also revealed that activator protein 1 (AP-1) sites were especially enriched in the sites common to Smad2/3 and Smad4 binding regions. In contrast, GC-rich motifs were enriched in Smad4-dominant binding regions. We further determined putative target genes of Smad4 whose expression was regulated by TGF-beta. Our findings revealed some general characteristics of Smad4 binding regions, and provide resources for examining the role of Smad4 in epithelial cells and cancer pathogenesis. (Cancer Sci 2009).

DOI： 10.1111/j.1349-7006.2009.01299.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T) P motifs in the Smad linker region. (S/T) P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

DOI： 10.1074/jbc.M804659200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in some tumor cells. Here, we demonstrate the molecular mechanism whereby Snail, a key regulator of EMT, is induced by TGF-beta in tumor cells. Snail induction by TGF-beta was highly dependent on cooperation with active Ras signals, and silencing of Ras abolished Snail induction by TGF-beta in pancreatic cancer Panc-1 cells. Transfection of constitutively active Ras into HeLa cells led to induction of Snail by TGF-beta, while representative direct targets of TGF-beta, including Smad7 and PAI-1, were not affected by Ras signaling. Using mitogen-activated protein kinase inhibitors or Smad3 or Smad2 mutants, we found that phosphorylation at the linker region of Smad2/3 was not required for the induction of Snail by TGF-beta. Taken together, these findings indicate that Ras and TGF-beta-Smad signaling selectively cooperate in the induction of Snail, which occurs in a Smad-dependent manner, but independently of phosphorylation at the linker region of R-Smads by Ras signaling.

DOI： 10.1074/jbc.M804777200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Osteoblasts and chondrocytes arise from common bipotential mesenchymal progenitor cells. Although the differentiation of these two cell lineages can be induced by treatment with bone morphogenetic proteins (BMPs), the responses of mesenchymal progenitors to BMP differ from cell line to cell line. Here we demonstrate that C3H/10T1/2 cells preferred chondrogenic differentiation, primary bone marrow stroma cells (MSCs) tended to convert to osteoblasts, and ST-2 cells differentiated into both the osteoblastic and chondrocytic lineages simultaneously, suggesting that a molecular switch functions to select cell fate. Osterix, the secondary master regulator of osteoblastogenesis, was induced by BMP at high and low levels in MSCs and ST-2 cells, respectively; in contrast, C3H/10T1/2 cells demonstrated only faint expression. As osterix has been suggested as a negative regulator of chondrogenesis, we hypothesized that the intense chondrocyte differentiation of C3H/10T1/2 cells may have resulted from an absence of osterix. We therefore restored osterix gene expression in C3H/10T1/2 cells using an adenovirus vector. Following BMP treatment, infection with an osterix-encoding virus dramatically inhibited the chondrocytic differentiation of C3H/10T1/2 cells, resulting instead in prominent osteoblast differentiation. These results indicate the chondrogenic potential of C3H/10T1/2 cells was abrogated by osterix expression. Chondrocyte differentiation of MSCs, however, was not enhanced by silencing the osterix gene using lentivirus-mediated shRNA, despite successful suppression of osteoblast differentiation. These results suggest that the low levels of osterix expression remaining after knockdown are sufficient to block chondrogenesis, whereas higher expression may be required to promote osteoblastic differentiation.

DOI： 10.1007/s00774-008-0003-0

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Chromosomal amplification occurs frequently in solid tumors and is associated with poor prognosis. Several reports demonstrated the cooperative effects of oncogenic factors in the same amplicon during cancer development. However, the functional correlation between the factors remains unclear. Transforming growth factor (TGF)-beta signaling plays important roles in cytostasis and normal epithelium differentiation, and alterations in TGF-beta signaling have been identified in many malignancies. Here, we demonstrated that transcriptional co-repressors of TGF-beta signaling, SKI and MDS1/EVI1-like gene 1 (MEL1), were aberrantly expressed in MKN28 gastric cancer cells by chromosomal co-amplification of 1p36.32. SKI and MEL1 knockdown synergistically restored TGF-beta responsiveness in MKN28 cells and reduced tumor growth in vivo. MEL1 interacted with SKI and inhibited TGF-beta signaling by stabilizing the inactive Smad3-SKI complex on the promoter of TGF-beta target genes. These findings reveal a novel mechanism where distinct transcriptional co-repressors are co-amplified and functionally interact, and provide molecular targets for gastric cancer treatment.

DOI： 10.1074/jbc.M808989200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The differentiation of myoblasts is regulated by multiple extracellular and intracellular factors. Of the extracellular regulators, members of transforming growth factor-beta (TGF-beta) family play critical roles in the regulation of osteoblasts and myoblast differentiation. Little is known, however, about the regulation of Myostatin/TGF-beta signaling during myoblast differentiation. In this study, we examined the roles of Arkadia, an E3 ubiquitin ligase, in Myostatin/TGF-beta signaling and the regulation of myoblast differentiation. Knockdown of Arkadia reduced Myostatin/TGF-beta signaling and enhanced the differentiation of C2C12 myoblasts. In addition, exogenous overexpression of Arkadia enhanced Myostatin/TGF-beta signaling, preventing myoblast differentiation. In the absence of the activation of Myostatin/TGF-beta signaling, knockdown of Arkadia enhanced myoblast differentiation via upregulation of Ski protein, an intracellular enhancer of myoblast differentiation. Arkadia likely affected the differentiation of myoblasts in a Smad-independent fashion by inducing Ski degradation. Knockdown of Arkadia increased the Myostatin-induced phosphorylation of Smad2/3 in C2C12 cells. Arkadia bound Smad2/3 via Ski to induce the ubiquitination of Smad2/3. These results suggest that Arkadia targets Ski-bound, inactive phospho-Smad2/3 to regulate positively Myostatin/TGF-beta signaling. Taken together, this study indicates that Arkadia regulates myoblast differentiation through both Smad-dependent and Smad-independent pathways. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2008.09.013

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor beta (TGF-beta) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-beta-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-beta-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-beta-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-beta and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.

DOI： 10.1128/MCB.01038-08

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Although CCAAT/enhancer-binding protein beta (C/EBP beta) is involved in osteocalcin gene expression in osteoblast in vitro, the physiological importance of and molecular mechanisms governing C/EBP beta in bone formation remain to be elucidated. In particular, it remains unclear whether C/EBP beta acts as a homodimer or a heterodimer with other proteins during osteoblast differentiation. Here, deletion of the C/EBP beta gene from mice resulted in delayed bone formation with concurrent suppression of chondrocyte maturation and osteoblast differentiation. The expression of type X collagen as well as chondrocyte hypertrophy were suppressed in mutant bone, providing new insight into the possible roles of C/EBP beta in chondrocyte maturation. In osteoblasts, luciferase reporter, gel shift, DNAP, and ChIP assays demonstrated that C/EBP beta heterodimerized with activating transcription factor 4 (ATF4), another basic leucine zipper transcription factor crucial for osteoblast maturation. This complex interacted and transactivated osteocalcin-specific element 1 (OSE1) of the osteocalcin promoter. C/EBP beta also enhanced the synergistic effect of ATF4 and Runx2 on osteocalcin promoter transactivation by enhancing their interaction. Thus, our results provide evidence that C/EBP beta is a crucial cofactor in the promotion of osteoblast maturation by Runx2 and ATF4.

DOI： 10.1091/mbc.E08-03-0329

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ubiquitin-dependent protein degradation is involved in various biological processes, and accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are E3 ubiquitin ligases, which suppress transforming growth factor-beta (TGF-beta) family signaling through degradation of Smads and receptors for TGF-beta and bone morphogenetic proteins. In addition, Smurf1 has been reported to promote RhoA ubiquitination and degradation and regulate cell motility, suggesting the involvement of Smurf1 in cancer progression. However, the regulation and biological function of Smurf1 and Smurf2 in cancer development remain to be elucidated. In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. Of note, knockdown of Smurf1, but not of Smurf2, enhanced TGF-beta signaling in MDA-MB-231 cells, suggesting that increased an protein level of Smurf1 offsets the effect of Smurf2 knockdown on TGF-beta signaling. These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.

DOI： 10.1074/jbc.M710496200

Web of Science

PubMed

researchmap

Language：English   Publisher：WILEY-BLACKWELL  

Members of the transforming growth factor-beta (TGF-beta) family, including TGF-beta, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-beta family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin-proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-beta family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-beta family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCF(Fbw1a) have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-beta family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-beta family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-beta family signaling may lead to the development and progression of various cancers. (Cancer Sci 2008; 99: 2107-2112).

DOI： 10.1111/j.1349-7006.2008.00925.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Transforming growth factor (TGF)-beta is known to promote tumor invasion and metastasis. Although bone morphogenetic proteins ( BMPs), members of the TGF-beta family, are expressed in a variety of human carcinoma cell lines, their roles in tumor progression have not been fully clarified. In this study, we sought to determine the roles of BMPs in the progression of breast cancer bone metastasis using human breast cancer samples and a mouse xenograft model. Immunohistochemical analysis of samples from breast cancer patients as well as a mouse xenograft model of MDA-231-D, highly metastatic human breast cancer cells, revealed phospho-Smad2 and phospho-Smad1/5/8 staining in the nuclei of cancer cells in primary tumor and/or bone metastasis. Using a functional in vivo bioluminescence imaging system, we showed that TGF-beta- and BMP-induced transcriptional pathways are active in bone metastatic lesions in vivo. In addition, both TGF-beta 3 and BMP-2 promoted the motility and invasiveness of the MDA-231-D cells in vitro. Moreover, expression of dominant-negative receptors for TGF-beta and/or BMPs in the MDA-231-D cells inhibited invasiveness in vitro and bone metastasis in the xenograft model. These results suggest that BMPs as well as TGF-beta promote invasion and bone metastasis of breast cancer.

DOI： 10.1038/onc.2008.232

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The cell-cycle transition from G(1) to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G, phase nuclei red and those in S/G(2)/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

DOI： 10.1016/j.cell.2007.12.033

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Muscle cells are often exposed to bone morphogenetic proteins (BMPs) in pathological muscle and/or bone conditions. Because BMPs function as strong bone inducers as well as myogenesis inhibitors, certain molecules likely prevent muscle cells from converting into pathologic bone; without these molecules, de novo bone would form as observed in myositis ossificans traumatica. When C2C12 myoblasts are exposed to BMPs, they differentiate into osteoblastic cells but cannot mature into bone cells. As the Osterix gene, a transcription factor for osteoblast differentiation, is only transiently induced upon BMP stimulation in C2C12 cells, we hypothesized that unknown transcriptional repressor(s) inhibit Osterix expression and prevent complete osteoblastic differentiation. Gene microarray analyses were performed to identify putative inhibitors for osteoblastic differentiation, and the paired-like homeodomain transcription factor Pitx2 (also termed Rieg), which plays an important regulatory role in left-right asymmetry, was identified. Pitx2 was induced 2 days after BMP stimulation in C2C12 cells in concert with Osterix down-regulation. Overexpression of Pitx2 repressed Osterix expression and subsequent osteoblastic differentiation, whereas Runx2, the most upstream regulator of osteogenesis, was unaffected. Conversely, the induction of short hairpin RNA for Pitx2 in C2C12 cells enhanced Osterix expression and osteoblastic maturation upon BMP stimulation. Moreover, mouse embryonic fibroblasts containing myoblasts from Pitx2-null embryos showed enhanced Osterix expression upon BMP stimulation. These findings suggest that Pitx2 suppresses osteogenic signals induced by BMPs in myoblasts to prevent their osteoblastic conversion.

DOI： 10.1074/jbc.M708154200

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stral3) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.

DOI： 10.1158/0008-5472.CAN-07-1522

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transforming growth factor-beta (TGF-beta) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-beta signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-beta signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-beta signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-beta family signaling in target cells.

DOI： 10.1074/jbc.M701294200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.

DOI： 10.1074/jbc.M702100200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Transforming growth factor-beta (TGF-beta) binds to two different types of serine/threonine kinase receptors termed type II (T beta R-II) and type I (T beta R-I). TGF-beta is unable to bind to T beta R-I in the absence of T beta R-II, and initiates receptor assembly by binding with high affinity to T beta beta R-II. Previous structural analysis of the TGF-beta 3-T beta R-II complex has suggested that two charged amino acid residues, D55 and E142 of T beta R-II, are binding sites of TGF-beta. In the present study, we have shown that mutations of the amino-acid residues, D55 and E142 of T beta R-II, resulted in loss of TGF-beta binding and downstream signaling activity. Moreover, we found that 3,5,7,2',4'-pentahydroxyflavone (Morin) inhibits TGF-beta binding to T beta R-II, and suppresses phosphorylation of Smad2 and expression of a TGF-beta target gene Smad7 induced by TGF-beta. Our findings may thus provide useful information for designing therapeutic agents for various diseases induced by TGF-beta, including advanced cancers.

DOI： 10.1038/sj.onc.1210123

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Transforming growth factor (TGF)-beta signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF-beta type I receptor (T beta R-I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Ki26894 blocked TGF-beta signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-beta-responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-beta in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-beta. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/c nu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that T beta R-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.

DOI： 10.1111/j.1349-7006.2007.00357.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

c-Ski is a proto-oncogene product that induces morphologic transformation, anchorage independence, and myogenic differentiation when it is over-expressed in mesenchymal cells. c-Ski also inhibits signaling of transforming growth factor-beta (TGF-beta) superfamily members through interaction with Smad proteins. Although c-Ski is predominantly localized in the nucleus, aberrant cytoplasmic localization of it has also been reported in some tumor tissues and cell lines. In the present study, we identified the nuclear localization signal (NLS) in c-Ski. By introducing a mutation to abolish NLS activity, we examined the function of cytoplasmic c-Ski. Although cytoplasmic c-Ski suppressed TGF-beta superfamily-induced Smad signaling through sequestration of activated Smad complex to the cytoplasm, it failed to exhibit some of the activities that require nuclear localization of c-Ski, including suppression of basal transcription of the Smad7 gene. These findings indicate that subcellular localization of c-Ski affects its biologic activities. We also found that c-Ski accumulated in the cytoplasm when proteasome activity was inhibited. Mapping of the regions required for cytoplasmic accumulation by proteasome inhibitors suggests that subcellular localization of c-Ski may be regulated by proteasome-sensitive processes through amino acid residues 94-210 and 491-548.

DOI： 10.1111/j.1365-2443.2006.01018.x

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：日本癌学会  

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBP zeta) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBP beta and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBP beta, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBP beta and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.

DOI： 10.1128/MCB.02429-05

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Background. Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. We used adenovirus-mediated gene transfer of these natural inhibitors in a mouse model of breast cancer to examine the roles of TGF-beta superfamily signaling in tumor growth and metastasis. Methods: We systemically administered, by intravenous injection, adenoviruses (AdCMV) containing the mouse cDNAs for Smad7, Smad6, c-Ski, the c-Ski mutant c-Ski (ARPG), or LacZ (control) to nude mice (&gt;19 mice/group) bearing tumors derived from mouse mammary carcinoma JygMC(A) cells, which spontaneously metastasize to lung and liver, and examined their effects on survival and metastasis. High-throughput western blotting analysis was used to examine the expression levels for 47 signal transduction proteins in JygMC(A) cells and primary tumors. We also investigated the proliferation, migration, and invasion of JygMC(A) cells that stably overexpressed Smad6 or Smad7. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon's rank sum tests. Parametric comparisons were done by one-way analysis of variance or two-sided unpaired Student's t tests. All statistical tests were two-sided. Results: Control mice bearing tumors derived from JygMC(A) cells showed many metastases to the lung and liver; all animals died by 50 days after cell inoculation. By contrast, mice treated with AdCMV-Smad7 or AdCMV-c-Ski demonstrated a dramatic decrease in metastasis and statistically significantly longer survival than control mice (Smad7 versus LacZ: medium survival = 55 days versus 41 days, difference = 14 days [95% confidence interval {CI} = 6 days to 22 days], P&lt;.001), whereas mice treated with AdCMV-Smad6 or AdCMV-c-Ski (ARPG) did not. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and tight junctions, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells. Conclusion: Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers.

DOI： 10.1093/jnci/dji399

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Transforming growth factor (TGF)-beta signaling facilitates tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for the treatment of patients with such cancer. In this study, we synthesized and characterized a small molecule inhibitor, A-83-01, which is structurally similar to previously reported ALK-5 inhibitors developed by Sawyer et al. (2003) and blocks signaling of type I serine/threonine kinase receptors for cytokines of the TGF-beta superfamily (known as activin receptor-like kinases; ALKs). Using a TGF-beta-responsive reporter construct in mammalian cells, we found that A-83-01 inhibited the transcriptional activity induced by TGF-beta type I receptor ALK-5 and that by activin type IB receptor ALK-4 and nodal type I receptor ALK-7, the kinase domains of which are structurally highly related to those of ALK-5. A-83-01 was found to be more potent in the inhibition of ALK5 than a previously described ALK-5 inhibitor, SB-431542, and also to prevent phosphorylation of Smad2/3 and the growth inhibition induced by TGF-beta. In contrast, A-83-01 had little or no effect on bone morphogenetic protein type I receptors, p38 mitogen-activated protein kinase, or extracellular regulated kinase. Consistent with these findings, A-83-01 inhibited the epithelial-to-mesenchymal transition induced by TGF-beta, suggesting that A-83-01 and related molecules may be useful for preventing the progression of advanced cancers.

DOI： 10.1111/j.1349-7006.2005.00103.x

Web of Science

PubMed

researchmap

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smad4 mediates signaling by the transforming growth factor-&beta; (TGF-&beta;) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-&beta; receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated polyubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-&beta; signaling may be critical for proper physiological response to this pathway.

DOI： 10.1074/jbc.M414027200

Web of Science

PubMed

researchmap

Language：English   Publisher：ELSEVIER SCI LTD  

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, bind to two different serine/threonine kinase receptors, and mediate their signals through Smad-dependent and Smad-independent pathways. Receptor regulated-Smad (R-Smad) proteins specific for the BMP pathways interact with various proteins, including transcription factor Runx, and transmit specific signals in target cells. The recent development of DNA microarray techniques has allowed us to identify many BMP target genes. BMP signaling is modulated by various molecules, including inhibitory Smads (I-Smads). Moreover, recent findings have revealed that BMP pathways interact with other signaling pathways, and such signaling cross-talk plays pivotal roles in growth and differentiation of target cells. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cytogfr.2005.01.009

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Inhibitory Smad, Smad7, is a potent inhibitor of TGF-beta (transforming growth factor-beta) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-beta superfamily receptors. NEDD4-2 functions like Smurfs I and 2 in that it associates with TGF-beta type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-beta-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-beta and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-beta superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-beta superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.

DOI： 10.1042/BJ20040738

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Smad7 negatively regulates transforming growth factor (TGF)-beta superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism. To elucidate the regulatory mechanisms of TGF-beta signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system. One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-beta superfamily receptors. Using a TGF-beta-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-beta. Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-beta type I receptor to cause ubiquitination and degradation of the receptor. Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-beta. WWP1 thus negatively regulates TGF-beta signaling in cooperation with Smad7. However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN. Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs.

DOI： 10.1038/sj.onc.1207885

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Growth factors, cell-surface receptors, adhesion molecules, and extracellular matrix proteins play critical roles in vascular pathophysiology by affecting growth, migration, differentiation, and survival of vascular cells. In a search for secreted and cell-surface molecules expressed in the cardiovascular system, by using a retrovirus-mediated signal sequence trap method, we isolated a cell-surface protein named vasorin. Vasorin is a typical type I membrane protein, containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain. Expression analyses demonstrated that vasorin is predominantly expressed in vascular smooth muscle cells, and that its expression is developmentally regulated. To clarify biological functions of vasorin, we searched for its binding partners and found that vasorin directly binds to transforming growth factor (TGF)-beta and attenuates TGF-beta signaling in vitro. Vasorin expression was down-regulated during vessel repair after arterial injury, and reversal of vasorin down-regulation, by using adenovirus-mediated in vivo gene transfer, significantly diminished injury-induced vascular lesion formation, at least in part, by inhibiting TGF-beta signaling in vivo. These results suggest that down-regulation of vasorin expression contributes to neointimal formation after vascular injury and that vasorin modulates cellular responses to pathological stimuli in the vessel wall. Thus, vasorin is a potential therapeutic target for vascular fibroproliferative disorders.

DOI： 10.1073/pnas.0404117101

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Signals by cytokines of the transforming growth factor-beta( TGF-beta) superfamily are negatively regulated by inhibitory Smads ( I-Smads). Smad7 inhibits signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 inhibits TGF-beta signals less effectively. I-Smads have amino-terminal N domains and carboxyl-terminal Mad homology 2 (MH2) domains. The N domains are essential for specific inhibition of TGF-beta signaling by Smad7, whereas the MH2 domains of I-Smads are involved in the inhibition of TGF-beta superfamily signals through interaction with type I receptors. Here, we have identified four basic amino acid residues (Lys-312, Lys-316, Lys-401, and Arg-409) in the basic surface of the Smad7 MH2 domain that play important roles in interaction with type I receptors. Mutations of the four basic amino acid residues to acidic residues (K312E, K316E, K401E, and R409E) abolished the interaction of Smad7 with TGF-beta type I receptors, inhibition of Smad2 phosphorylation and transcriptional responses induced by TGF-beta, and induction of target genes of endogenous activin/Nodal signals in Xenopus early embryos. The K401E and R409E mutants of Smad7 were also unable to interact with BMP type I receptors ( BMPR-I), repress the Smad5 phosphorylation and transcription induced by BMP, and effectively inhibit endogenous BMP signals in Xenopus early embryos. However, the K312E and K316E mutants were able to interact with BMPR-I and retained the ability to inhibit BMP signaling. Thus, the MH2 domain of Smad7 plays important roles in specific inhibition of TGF-beta superfamily signals through differential interaction with type I receptors.

DOI： 10.1074/jbc.M313977200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Runx proteins regulate various biological processes, including growth and differentiation of hematopoietic cells, lymphocytes, osteoblasts, and gastric epithelial cells. Some of the biological activities of Runx proteins are reminiscent of those of transforming growth factor (TGF)-beta superfamily cytokines. Consistent with this notion, receptor-regulated Smads (R-Smads), signal mediators of the TGF-beta superfamily cytokines, and Runx proteins have been shown to physically interact with each other. R-Smads activated by TGF-beta and Runx proteins cooperatively induce synthesis of IgA in B lymphocytes, and those activated by bone morphogenetic proteins and Runx2 induce osteoblastic differentiation. Moreover, the R-Smad-Runx signaling pathways are regulated by an E3 ubiquitin ligase Smurf1, as well as a signal transducer of interferons, STAT1. Since Runxl and Runx3 are involved in the development of some cancers including acute leukemia and gastric cancer, it will be of interest to examine in detail whether TGF-beta-specific R-Smads and Runx proteins coordinately regulate growth and differentiation of hematopoietic cells and gastric epithelial cells.

DOI： 10.1038/sj.onc.1207131

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.

DOI： 10.1083/jcb.200311015

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-beta (TGF-beta) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-beta/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-beta-induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-beta/activin signaling.

DOI： 10.1091/mbc.E03-07-0478

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Smad proteins are intracellular signalling mediators of transforming growth factor-beta (TGF-beta) superfamily. In the nucleus, activated Smad complexes regulate transcriptional responses of the target genes in cooperation with transcriptional coactivators and corepressors. To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a coactivator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a coactivator for receptor-regulated Smads (R-Smads) for TGF-beta signalling pathways. GCN5 functions like PCAF, in that it binds to TGF-beta-specific R-Smads, and enhances transcriptional activity induced by TGF-beta. In addition, GCN5, but not PCAF, interacts with R-Smads for bone morphogenetic protein (BMP) signalling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo. Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-beta. In conclusion we identified GCN5 as a Smad-binding transcriptional coactivator which positively regulates both TGF-beta and BMP signalling pathways.

DOI： 10.1111/j.1365-2443.2004.00706.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Transforming growth factor-beta (TGF-beta), one of the most abundant cytokines in bone matrix, has positive and negative effects on bone formation, although the molecular mechanisms of these effects are not fully understood. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, induce bone formation in vitro and in vivo. Here, we show that osteoblastic differentiation of mouse C2C12 cells was greatly enhanced by the TGF-beta type I receptor kinase inhibitor SB431542. Endogenous TGF-beta was found to be highly active, and induced expression of inhibitory Smads during the maturation phase of osteoblastic differentiation induced by BMP-4. SB431542 suppressed endogenous TGF-beta signaling and repressed the expression of inhibitory Smads during this period, possibly leading to acceleration of BMP signaling. SB431542 also induced the production of alkaline phosphatase and bone sialoprotein, and matrix mineralization of human mesenchymal stem cells. Thus, signaling cross-talk between BMP and TGF-beta pathways plays a crucial role in the regulation of osteoblastic differentiation, and TGF-beta inhibitors may be invaluable for the treatment of various bone diseases by accelerating BMP-induced osteogenesis.

DOI： 10.1038/sj.emboj.7600067

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor-beta (TGF-beta) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF-beta and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly-ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF-beta receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF-beta receptors. In contrast to Smad7, expression of Arkadia is down-regulated by TGF-beta. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF-beta and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF-beta superfamily signalling under both physiological and pathological conditions.

DOI： 10.1093/emboj/cdg632

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 muM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 muM) as well as AG1296 (5 muM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smad4 is an essential signal transducer of all transforming growth factor-beta (TGF-beta) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono- ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R- Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R- Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono- ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.

DOI： 10.1074/jbc.M300159200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-beta type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.

DOI： 10.1091/mbc.E02-07-0441

Web of Science

PubMed

researchmap

Language：English   Publisher：JAPANESE CANCER ASSOC  

Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of most types of cells; therefore, perturbations of TGF-beta signaling are believed to result in progression of various tumors. On the other hand, TGF-beta has been shown to act as an oncogenic cytokine through induction of extracellular matrices, angiogenesis, and immune suppression. A wide variety of effects of TGF-P are mediated by physical interaction of signal transducer Smad proteins with various transcription factors. Among these, Runx3 plays a pivotal role in prevention of gastric cancer. TGF-beta signaling is regulated by various mechanisms in the cytoplasm and nucleus. Inhibitory Smads (I-Smads) repress TGF-beta signaling mainly by interacting with activated TGF-beta receptors. Smad ubiquitin regulatory factors (Smurfs) play important roles in facilitating the inhibitory signals induced by I-Smads. In addition, the transcriptional co-repressors c-Ski and SnoN interact with Smads, and repress transcription induced by TGF-beta. Abnormalities of these regulators of TGF-beta signaling may thus participate in the progression of various tumors.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-beta type I receptor, TbetaR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B. a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NFS mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-beta. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-beta signaling by Smad7.

DOI： 10.1074/jbc.M212663200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with growth factors, act as coreceptors and modulate growth factor activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as 22 tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell growth of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of growth factors by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/ijc.10856

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

c-Ski and SnoN are transcriptional co-repressors that inhibit transforming growth factor-beta signaling through interaction with Smad proteins. Among receptor-regulated Smads, c-Ski and SnoN bind more strongly to Smad2 and Smad3 than to Smad1. Here, we show that c-Ski and SnoN bind to the "SE" sequence in the C-terminal MH2 domain of Smad3, which is exposed on the N-terminal upper side of the toroidal structure of the MH2 oligomer. The "QPSMT" sequence, located in the vicinity of SE, supports the interaction with c-Ski and SnoN. Sequences similar to SE and QPSMT are found in Smad2, but not in Smad1. The N-terminal MH1 domain and linker region of Smad3 protrude from the N-terminal upper side of the MH2 oligomer toroid. Smurf2 induces ubiquitin-dependent degradation of SnoN, since it appears to be located close to SnoN through binding to the linker region of Smad2. In contrast, transcription factors Mixer and FoxH3 (FAST1) bind to the bottom side of the Smad3 MH2 toroid; therefore, c-Ski does not affect the interaction of Smads with these transcription factors. Our findings thus demonstrate the stoichiometry of how multiple molecules can associate with the Smad oligomers and how the Smad-interacting proteins functionally interact with each other.

DOI： 10.1074/jbc.C200596200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-1) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1-Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(DeltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(DeltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II-TbetaR-I complex. Moreover, although Smurf1(DeltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-1 and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.

DOI： 10.1074/jbc.M201901200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Germline mutations in,the BMPR2 gene encoding bone morphogenetic protein (BMP) type II receptor (BMPR-II) have been reported in patients with primary pulmonary hypertension (PPH), but the contribution of various types of mutations found in PPH to the pathogenesis of clinical phenotypes has not been elucidated. To determine the biological activities of these mutants, we performed functional assays testing their abilities to transduce BMP signals. We found that the reported missense mutations within the extracellular and kinase domains of BMPR-II abrogated their signal-transducing abilities. BMPR-II proteins containing mutations at the conserved cysteine residues in the extracellular and kinase domains were detected in the cytoplasm, suggesting that the loss of signaling ability of certain BMPR-II mutants is due at least in part to their altered subcellular localization. In contrast, BMPR-II mutants with truncation of the cytoplasmic tail retained the ability to transduce BMP signals. The differences in biological activities among the BMPR-II mutants observed thus suggest that additional genetic and/or environmental factors may play critical roles in the pathogenesis of PPH.

DOI： 10.1091/mbc.E02-02-0063

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (T betaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.

DOI： 10.1083/jcb.200106023

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in MS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors, Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells, These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

DOI： 10.1038/sj.onc.1204572

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Smads are signal mediators for the members of the transforming growth factor-beta (TGF-beta) superfamily. Upon phosphorylation by the TGF-beta receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-beta is degraded by the ubiquitin-proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. Am E3 ubiquitin ligase complex ROC1-SCFFbw1a consisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed beta TrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCFFbw1a is then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-beta /Smad3 signaling is thus irreversibly terminated by the ubiquitin-proteasome pathway.

DOI： 10.1091/mbc.12.5.1431

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which regulate the differentiation of osteoprogenitor cells, Here we show that among members of the BMP family, BMP-4 and growth/differentiation factor 5 (GDF-5) induce osteoblast differentiation through the activation of three receptor-regulated Smads (i.e. Smad1, Smad5 and Smad8). By contrast, BMP-6 and BMP-7 induce alkaline phosphatase activity through Smad1 and Smad5, but not through Smads, Consistent with these findings, BMP-4 induced phosphorylation and nuclear translocation of Smad1, Smad5 and Smads, but BMP-6 activated only Smad1 and Smad5. BMP-4 and GDF-5 are known to bind to activin receptor-like kinase 3 (ALK-3) and/or ALK-6 (also termed BMP type IA and type IB receptors, respectively), whereas BMP-6 and BMP-7 preferentially bind to ALK-2.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (T betaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with T betaR-I via Smad7, with subsequent enhancement of turnover of T betaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of T betaR-I through recruitment of an E3 ligase to the receptor.

DOI： 10.1074/jbc.C100008200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Cyclin-dependent kinase inhibitory proteins (CKIs) are negative regulators of the cell cycle. Of all CKls, only p57(Kip2) plays an essential role(s) that other CKIs cannot compensate for in embryonic development. Recently, we found that p57(Kip2) is degraded through the ubiquitin-proteasome pathway in osteoblastic cells stimulated to proliferation by transforming growth factor (TGF)-beta1 (Urano, T., Yashiroda, H., Muraoka, RI., Tanaka, K., Hosoi, T.:, Inoue, S., Ouchi, Y., and Toyoshima, H. (1999) J. Biol, Chem. 274, 12197-12200). We report here that TGF-beta1-induced p57(Kip2) proteolysis is mediated through transcription by the Smad pathway. When the constitutively active form of the TGF-beta type I receptor ALK-5(TD) was ectopically expressed in osteoblastic cells, p57(Kip2) that had been accumulated by serum starvation causing the cell-cycle arrest was rapidly degraded in a manner analogous to TGF-beta1 stimulation. Moreover, Smad2 or Smad3 with Smad4 enhanced the proteolytic pathway of p57(Kip2). Th, degradation of p57(Kip2) evoked by TGF-beta1 was blocked by forced expression of an inhibitory Smad called Smad7 or by the addition of actinomycin D or alpha -amanitin. These results indicate that accelerated degradation of p57(Kip2) by TGF-beta1/Smad signaling is mediated through a newly synthesized factor(s) that modifies p57(Kip2) or the ubiquitin-proteasome pathway.

DOI： 10.1074/jbc.M007499200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The activin receptor-like kinase 1 (ALK1) is a type I receptor for transforming growth factor-beta (TGF-beta) family proteins. Expression of ALK1 in blood vessels and mutations of the ALK1 gene in human type II hereditary hemorrhagic telangiectasia patients suggest that ALK1 may have an important role during vascular development, To define the function of ALK1 during development, we inactivated the ALK1 gene in mice by gene targeting. The ALK1 homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels. These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by deficient differentiation and recruitment of vascular smooth muscle cells. The blood vessel defects in ALK1-deficient mice are reminiscent of mice lacking TGF-beta 1, TGF-beta type II receptor (T beta R-II), or endoglin, suggesting that ALK1 may mediate TGF-beta 1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-beta 1 and T beta R-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-beta 1-dependent transcriptional activation mediated by the known TGF-beta 1 type I receptor, ALK5. Taken together, our results suggest that the balance between the ALK1 and ALK5 signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.

DOI： 10.1073/pnas.97.6.2626

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Background: Smad4 has a unique region of 35 amino acids between alpha-helices 3 and 4 (termed H3/4 loop) of the Mad homology (MH) 2 domain. In order to elucidate the functional importance of the H3/4 loop, we prepared chimeric constructs of Smad4 containing the region corresponding to the alpha-helix 3, H3/4 loop and alpha-helix 4 of different Smads, including a chimera containing that of Smad2 (Smad4-HL2).
Results: Smad4-HL2 constitutively induced the transcriptional activation of p3TP-Lux, a TGF-beta-responsive reporter construct. However, co-transfection of Smad2 with Smad4-HL2 did not induce a further increase in the activation of p3TP-Lux. Smad4-HL2 did not induce the activation of pAR3-Lux, which contains FAST1-binding sites and is activated by a complex composed of FAST1, Smad2 and Smad4. Smad4-HL2 formed a homo-oligomer more efficiently than wild-type Smad4 in mammalian cells. Moreover, Smad4-HL2 bound to DNA containing the Smad-binding sites with a gretaer affinity than the wild-type Smad4.
Conclusion: Smad4-HL2 spontaneously forms a homo-oligomer, which may bind to DNA with relatively high affinity and induce transcriptional activation of p3TP-Lux. The H3/4 loop of Smad4 may thus play a role in precluding the spontaneous oligomer formation of Smad4.

DOI： 10.1046/j.1365-2443.1999.00293.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

DOI： 10.1091/mbc.10.11.3801

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily. Smads bind to DNA and act as transcriptional regulators. Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction. Smads also associate with transcriptional coactivators such as p300 and CBP, E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition. Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction. One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region. p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner, E1A inhibits the interaction of Smad3 with a p300 mutant that contains SLD but lacks the E1A binding domain. We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby ELA antagonizes TGF-beta signaling.

DOI： 10.1074/jbc.274.40.28716

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEEP2/CBF) is a transcription factor complex composed of alpha and beta subunits. The alpha subunits of PEEP2/CBF, which contain the highly conserved Hunt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha subunits of PEBP2/CEF form complexes with R-Smads that act in TGF-beta/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2 alpha C/CBFA3/AML2 forms a complex with Smads and stimulates transcription of the germline Ig C alpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEEPS may thus be a nuclear target of TGF-beta/BMP signaling.

DOI： 10.1074/jbc.274.44.31577

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-beta superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8, In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation, BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells, Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells, Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smads was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smads by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Transforming growth factor beta (TGF-B) can inhibit epithelial cell growth and induce extracellular matrix formation through signal transduction via its two receptors and its downstream intracellular Smad proteins. We recently reported a germline mutation, i.e., substitution of methionine for threonine at codon 315 in the kinase subdomain IV, of the TGF-beta type II receptor gene in a kindred of hereditary nonpolyposis colorectal cancer without microsatellite instability and found that the mutant receptor abolished the signal transduction for growth inhibition by TGF-beta. In this study, we performed further functional amalysis of this mutant receptor. The results showed that, in contrast to its failure to mediate growth inhibition by TG;F-P, the mutant receptor still retained the ability to induce one of the extracellular matrix proteins, plasminogen activator inhibitor type 1, upon TGF-beta treatment. However, coincident with its failure to mediate growth inhibition by TGF-beta, the mutant receptor failed to transcriptionally upregulate one of the cyclin-dependent kinase inhibitors, p15(INK4B), in response to TG;F-P. These data suggest that threonine 315 of the TGF-beta type II receptor is dispensable. for extracellular matrix protein production, but is essential for the growth inhibition by TGF-beta, and that the lack of growth inhibition due to the mutant receptor is possibly mediated through its failure to upregulate p15(INK4B). (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.0788

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily that play a pivotal role in bone formation during embryogenesis and fracture repair. BMP signaling occurs via hetero-oligomeric serine/threonine kinase complexes of BMP type I (BMPR-IA or BMPR-IB) and type II receptors (BMPR-II). BMPR-IA and IB are closely related receptors, with sequence differences conserved between different species, suggesting that they serve distinct functions. Here we report the cDNA cloning of human BMPR1B and the chromosomal localization of all three BMPR genes. Using somatic cell hybrid and FISH analyses, the BMPR1A, BMPR1B, and BMPR2 genes were assigned to 10q23, 4q22-24, and 2q33-34, respectively. A processed BMPR1A pseudogene was mapped to 6q23.

DOI： 10.1007/s003359900990

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NEW YORK ACAD SCIENCES  

TGF-beta is a potent inhibitor of cell growth, and accumulating evidence suggests that perturbation of the TGF-beta signaling pathway leads to tumorigenesis. Smads are recently identified proteins that mediate intracellular signaling of the TGF-beta superfamily. Smads 2 and 3 are phosphorylated by the TGF-beta type I receptor. Smad4 was originally identified as a candidate tumor suppressor gene in pancreatic cancers. Smads 2 and 3 form complexes with Smad4 upon TGF-beta stimulation. The heteromeric Smad complexes translocate into the nucleus, where they activate expression of target genes. Our recent study demonstrated that Smads exist as monomers in the absence of TGF-beta. Smads 2 acid 3 form homo- as well as hetero-oligomers with Smad4 upon ligand stimulation. Both homo-oligomers and hetero-oligomers directly bind to DNA, suggesting that the signaling pathway of Smads may be muitiplex, Smads 2 and 3 associate with transcriptional coactivators such as p300 in a ligand-dependent manner. p300 enhances transactivation by TGF-beta, suggesting that coactivators link Smads to the basal transcriptional machinery. A missense mutation of Smad2 identified in colorectal and lung cancers was introduced to Smad3. The mutant, Smad3(DE), blocked the activation of wild-type Smad2 and Smad3, Thus, the missense mutation not only disrupts the function of the wild-type Smad but also creates a dominant-negative Smad, which could actively contribute to oncogenesis.

DOI： 10.1111/j.1749-6632.1999.tb09402.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

DOI： 10.1091/mbc.9.8.2145

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

The expression of bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) in the epiphyseal growth plate has not been clarified In this study,; we studied immunohistochemically the spatial and temporal localization of BMP-2/4, osteogenic protein-1 (OP-1, or BMP-7) and BMP receptors (BMPR-IA, BMPR-IB, and BMPR-II) in the epiphyseal plate of growing rats. The proximal parts of tibia in growing rats were observed. At 12 weeks after birth, BMP-2/4 and OF-I were expressed markedly in proliferating and maturing chondrocytes. BMPR-IA, LB and II were clearly co-expressed in proliferating and maturing chondrocytes, and the expression was decreased in hypertrophic chondrocytes. At 24 weeks, the expression of BMP-2/4 and OF-I war decreased, but BMPRs were still well-expressed in proliferating chondrocytes. The temporal and spatial expression of BMP and BMPR suggests that BMP and BMP receptors play roles in the multistep cascade of enchondral ossification in the epiphyseal growth plate.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II, On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone, On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels, BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our precious study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair, OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process, Thus, these findings suggested that BMPs acting through their BMP receptors mag play major roles in modulating the sequential events leading to bone formation, (C) 1998 by Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S8756-3282(98)00056-8

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOURNAL BONE JOINT SURGERY INC  

Activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and are thought to play an important role in modulating the formation of bone. Activins exert their cellular effects by way of activin type-I and type-II serine/threonine kinase receptors. Follistatin is an activin-binding protein that can suppress the biological effects of activins. In this study, the immunohistochemical expression of activin A, follistatin, and activin receptors was studied during fracture healing in the rat. Activin A was weakly detected in the periosteum near the fracture ends at an early stage but was absent in the chondrocytes around the fracture gap, where endochondral ossification took place. An antibody to follistatin stained osteogenic cells in the periosteum near the fracture ends; moderate and strong staining were observed in proliferating, mature, and hypertrophied chondrocytes at the sites of endochondral ossification. Levels of activin A and follistatin were high near the osteoblasts on the surface of the newly formed trabecular bone. In addition, an intense localization of activin A was noted where multinucleated osteoclast-like cells were present. This study suggests that the activin-follistatin system may contribute to cellular events related to the formation and remodeling of bone during fracture healing. Activin type-I and type-II receptors were co-expressed in intramembranous and endochondral ossification sites. The expression of activin type-I, type-II, and type-IIB receptors in the absence of activin A in the endochondral ossification suggests that other isoforms of activins may signal by way of these receptors.

DOI： 10.1002/jor.1100160307

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Members of the transforming growth factor-beta (TGF-beta) superfamily transduce signals via Smad proteins. Smad2 and Smad3 mediate TGF-beta signaling, whereas Smad1 and Smad5 transduce bone morphogenetic protein (BMP) signals. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 are recently identified members in the Smad family; they inhibit the signaling activity of the other Smad proteins. Here we show that expression of the Smad6 mRNA is dramatically induced by BMP-2 or osteogenic protein-1 (OP-1)/BMP-7 in various cells. BMP-2 induced expression of Smad7 in one cell type, although much less potently than that of Smad6. Smad6 message was induced by TGF-beta 1 in TGF-beta 1-responsive Mv1Lu cells, but the induction was transient in contrast to the induction by BMPs. These results indicate that Smad6 may form a feedback loop to regulate the signaling activity of BMPs. (C) 1998 Academic Press.

DOI： 10.1006/bbrc.1998.8200

Web of Science

PubMed

researchmap

Language：English   Publisher：ELSEVIER SCI LTD  

Bone morphogenetic proteins (BMPs) are multifunctional cytokines, which are members of the transforming growth factor-beta (TGF-beta) superfamily, Activities of BMPs are extracellularly regulated by BMP-binding proteins, Noggin and Chordin. BMPs bind to two different types of serine-threonine kinase receptors, type I and type II. Two BMP type I receptors and a BMP type LT receptor have been identified in mammals. Intracellular signals are transduced by Smad proteins. Smad1, Smad5 and probably MADH6, are activated by BMP receptors, form heteromeric complexes with Smad4, and translocate into the nucleus where they may activate transcription of various genes. Smad6 and Smad7 are inhibitory Smads, and may act as autocrine switch-off signals, In Drosophila melanogaster, Decapentaplegic (Dpp) is a homologue of mammalian BMPs, In this review, mechanism of action of Dpp will be discussed in comparison with that of BMPs, (C) 1998 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1359-6101(97)00036-1

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Background: Activin A is a multifunctional protein, which is a member of the transforming growth factor-beta (TGF-beta) superfamily. Smad proteins have recently been shown to transduce signals for the TGF-beta superfamily of proteins, and Smad2 was implicated in activin signalling in Xenopus embryos.
Results: We identified the receptors and Smad proteins activated by activin A in a human epidermal keratinocyte cell line, HaCaT. The major activin receptors expressed on HaCaT cells were activin type II receptor (ActR-II) and activin type IB receptor (ActR-IB). We have also shown that in HaCaT cells, activin A induced the phosphorylation of Smad3 and, to a lesser extent, of Smad2. On the other hand, TGF-beta induced an efficient phosphorylation of both Smad2 and Smad3, Activin A preferentially induced the nuclear translocation of Smad3 in HaCaT cells, whereas TGF-beta strongly induced the nuclear translocation of Smad2, as well as other Smads. Moreover, a constitutively active form of ActR-IB efficiently stimulated the formation of a heteromeric complex between Smad3 and Smad4 in COS cells transfected with Smad cDNAs.
Conclusions: These results suggest that activin A binds to a receptor complex of ActR-II and ActR-IB, and preferentially activates Smad3 in HaCaT human keratinocytes.

DOI： 10.1046/j.1365-2443.1998.00174.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOURNAL BONE JOINT SURGERY INC  

Ossification of the posterior longitudinal ligament is a human genetic disease in which pathological ectopic ossification of the spinal ligaments develops. This leads to myelopathy or radiculopathy due to compression of the spinal cord. In this study, we investigated the histological features of orthotopic ossification of the spinal ligaments of senile Zucker fatty rats. A remarkably high incidence of orthotopic ossification was observed mainly in the thoracic spinal ligaments as compared with controls The histopathological findings were similar to those for ossification of the human posterior longitudinal ligament. Bone morphogenetic proteins and activins, which exert their effects by way of specific type-I and type-II serine/threonine kinase receptors. play important roles in the formation of bone and cartilage. In the spinal ligaments of Zucker fatty rats, bone morphogenetic protein receptors and activin receptors were immunohistochemically detected around the ossified foci in a manner similar to that previously shown for the ossified tissue from patients who had ossification of the posterior longitudinal ligament. Thus, bone morphogenetic proteins and activin receptors might play important roles in orthotopic ossification of the spinal ligaments of Zucker fatty rats as well as in ossification of the posterior longitudinal ligament of humans. In addition, bone morphogenetic protein-receptor-IA was expressed in the nonossified ligament, suggesting that the spinal ligaments of the rats may have a predisposition to orthotopic ossification. In the controls, no expression of bone morphogenetic protein receptors or of activin receptors was observed. In conclusion, there is a great degree of similarity between orthotopic ossification of the spinal ligaments of Zucker fatty rats and ossification of the posterior longitudinal ligament of humans. Thus, the rats provide a useful animal model for the study of ossification of the human posterior longitudinal ligament.

DOI： 10.1002/jor.1100150606

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor-beta (TGF-beta) superfamily, Smad2 and Smad3 are structurally highly similar and mediate TGF-beta signals, Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation, Here we show that Smad2 and Smad3 interacted with the kinase-deficient TGF-beta type I receptor (TPR)-I after it was phosphorylated by TPR-II kinase, TGF-beta 1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells, Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-beta 1 stimulation, Similar results were obtained using HSC4 cells, which are also growth-inhibited by TGF-beta, Smads 2 and 3 interacted with Smad4 after T beta R activation in transfected COS cells, In addition, we observed T beta R-activation-dependent interaction between Smad2 and Smad3, Smads 2, 3 and 4 accumulated in the nucleus upon TGF-beta 1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter, Dominant-negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4, These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction.

DOI： 10.1093/emboj/16.17.5353

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients, In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the Ligamentum flavum to the bone, and limited expression was found in the smaller range, Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients. (C) 1997 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S8756-3282(97)00080-X

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

Ossification of the posterior longitudinal ligament (OPLL) is a pathological ossification tn the spinal ligament, with formation of ectopic bone mainly through endochondral ossification. Bone morphogenetic proteins (BMPs) and activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and that have been implicated in the formation of new bone and cartilage. BMPs and activins signal via type I and type II receptors for BMPs (BMPRs) and activins (ActRs), respectively. OP-1/BMP-7 binds to BMPR-II and ActR-II and forms complexes with BMPR-IA and -IB and ActR-I. We studied the expression of BMPR-IA, -IB, and -II, ActR-I, ActR-II, and OP-1/BMP-7 by immunohistochemistry in ossified ligament tissues of patients with OPLL and control ligament tissues cervical disc herniation. The expression of BMPRs and ActRs was elevated in OPLL compared with controls. Expressions of BMPR-IA, -IB, and -II were observed not only in chondrocytes at the fibrocartilage tissue around the calcified zone but also in fibroblast-like spindle cells at the nonossified ligament. ActR-I and -II were found co-localized in the hypertrophic chondrocytes near the calcified zone and in the ossified tissue. OP-1/BMP-7 was expressed in chondrocytes near the calcified zone. In the control cases, the BMPRs and ActRs were only weakly expressed in the fibrocartilage tissue at the site of ligament attachments to bone and OP1/BMP-7 was not detected. Enhanced expression of BMPRs at the nonossified ligament in OPLL patients suggests that these cells have a greater potential to differentiate into osteogenic cells than ligament cells from non-OPLL patients. The high expression of BMPRs and ActRs in the ectopic ossified ligament suggests that BMPs and activin may be tightly involved in the pathological ossification process of OPLL.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transforming growth factor-beta (TGF-beta) superfamily members are multifunctional cytokines that exert their effects via heteromeric complexes of two distinct serine and threonine kinase receptors. Drosophila mothers against decapentaplegic and related genes in Caenorhabditis elegans, Xenopus, and mammals were shown to function downstream in the intracellular signaling pathways of TGF-beta superfamily members. Here we report the cloning of a Mad-related protein, termed Sma- and Mad-related protein 2 (Smad2). TGF-beta stimulated the phosphorylation and nuclear translocation of Smad2 in nontransfected Mv1Lu cells. In addition, we demonstrated that TGF-beta and activin mediated phosphorylation of Smad2 after its overexpression with appropriate type I and II receptors in COS cells. Smad2 and Smad1 were found to be broadly expressed in human tissues. Smad2 is closely linked to DPC4 on chromosome 18q21.1, a region often deleted in human cancers. Cells that lack Smad2 may escape from TGF-beta-mediated growth inhibition and promote cancer progression.

DOI： 10.1074/jbc.272.5.2896

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Receptor serine-threonine kinases (RSTK) mediate inhibitory as well as stimulatory signals for growth and differentiation by binding to members of the transforming growth factor-beta (TGF-beta) superfamily. Over 12 different RSTKs have been isolated so far, displaying wide expression in peripheral tissues and in the nervous system. Here we report the isolation and characterization of a novel type I RSTK termed activin receptor-like kinase-7 (ALK-7) that, unlike other members of this receptor family, is predominantly expressed in the adult central nervous system. The ALK-7 gene encodes a 55-kDa cell-surface protein that exhibits up to 78% amino acid sequence identity in the kinase domain to previously isolated type I receptors for TGF-beta and activin. In the extracellular domain, however, ALK-7 is more divergent, displaying comparable similarities with all members of the ALK subfamily. RNase protection and in situ hybridization studies demonstrated a highly specific mRNA distribution restricted to neurons in several regions of the adult rat central nervous system, including cerebellum, hippocampus, and nuclei of the brainstem. Receptor reconstitution and cross-linking experiments indicated that ALK-7 can form complexes with type II RSTKs for TGF-beta and activin in a ligand-dependent manner, although direct binding of ALK-7 to ligand in these complexes could not be demonstrated. The specific expression pattern of ALK-7, restricted to the postnatal central nervous system, indicates that this receptor may play an important role in the maturation and maintenance of several neuronal subpopulations.

DOI： 10.1074/jbc.271.48.30603

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

We studied the immunohistochemical localization of TGF-beta in the ligament tissue of patients with ossification of the posterior longitudinal ligament(OPLL), and the effects of TGF-beta on cultured cells derive from the spinal ligament of patients with OPLL. ALP activity in the cultured cells was also examined. TGF-beta was present in the ossified matrix and in the chondrocytes in cartilaginous areas adjacent to OPLL. ALP activity was high in the cultured cells of OPLL patients. Exogenous TGF-beta inhibited proliferation in the OPLL cells but promoted proliferation in control cells. These results suggest that the spinal ligaments of OPLL patients have an osteogenetic predisposition and that TGF-beta may play a role in the ossification.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

We investigated the effects of various cytokines in the presence of human T-cell leukemia virus type I (HTLV-I) tax protein in murine clonal osteoblasts, MC3T3-E1 cells, Skeletal remodeling by osteoclasts and osteoblasts is coordinated by cytokines, which are activated by HTLV-I tax protein via nuclear factor-kappa B (NF-kappa B). MC3T3-E1 cells were cocultured with an irradiated HTLV-I-producing lymphocyte cell line, MT-2, After coculture, the tumor necrosis factor-alpha (TNF-alpha) level in the medium was markedly elevated during the 7 days of culture, and MC3T3-E1 cells underwent apoptotic cell death, Marked apoptosis was also observed in MC3T3-E1 cells treated with MT-2 culture medium and in HTLV-I tax-expressing MC3T3-E1 clones, which both expressed high levels of TNF-alpha. This apoptosis was prevented by treatment with neutralizing anti-TNF-alpha antibody (alpha TNF), HTLV-I tax protein and TNF-alpha induced activation of NF-kappa B in apoptotic MC3T3-E1 cells, Decreased NF-kappa B activation was observed in HTLV-I tax-expressing MC3T3-E1 cells treated with alpha TNF, Our results suggest that HTLV-I tax activated NF-kappa B and subsequently TNF-alpha, leading to apoptosis of osteoblasts.

DOI： 10.1002/jbmr.5650110209

Web of Science

PubMed

researchmap

Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transforming growth factor-beta 1 (TGF-beta 1) is the prototype of a large family of molecules that regulate a variety of biological processes. The type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta 1 are transmembrane serine/threonine kinases, forming a heteromeric signaling complex. Recent studies have shown that T beta R-II is a constitutively active kinase and phosphorylates T beta R-I upon Ligand binding, suggesting that T beta R-I is the effector subunit of the receptor complex, which transduces signals to intracellular targets. This model has been further confirmed by the identification of constitutively active T beta R-I that mediates TGF-beta 1-specific cellular responses in the absence of Ligand and TPR-II. To investigate signaling by TGF-beta 1, we have sought to isolate proteins that interact with the cytoplasmic region of T beta R-I. One of the proteins identified was the alpha subunit of farnesyl-protein transferase (FT alpha) that modifies a series of peptides including Ras. T beta R-I specifically interacts with FT alpha in the yeast two-hybrid system. Glutathione S-transferase-T beta R-I fusion proteins bind FT alpha translated in vitro. T beta R-I also phosphorylates FT alpha. We further show that the constitutively active T beta R-I interacted with FT alpha very strongly whereas an inactive form of T beta R-I did not. These results suggest that FT alpha may be one of the substrates of the activated T beta R-I kinase.

DOI： 10.1074/jbc.270.50.29628

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE PUBL INC CAMBRIDGE  

Type I receptors for bone morphogenetic proteins (BMPs), i.e., BMPR-IA and BMPR-IB, are transmembrane serine/threonine kinases, that bind osteogenic protein-1 (OP-1, also termed BMP-7) and BMP-4. Using antibodies specific to BMPR-IA and -IB, we have studied the expression of BMP type I receptors in the bone formation process during embryonic development and fracture healing, In the mouse embryo, both BMPR-IA and -IB were expressed in condensing mesenchymal cells at 13.5 days post coitum (p.c.). At 15.5 days p.c., expression of BMPR-IB, but not of BMPR-IA, was observed in the cells in perichondrium of developing cartilage. At 17.5 and 19.5 days p.c., expression of both receptors was observed in chondrocytes and in osteoblasts. In normal rat adult bone, expression of BMPR-IA, but not of BMPR-IB, was observed in osteoblasts in the periosteum. Three days after the femoral fracture, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum. On day 7, both receptors were found in fibroblast-like spindle cells and chondrocytes in the endochondral ossification sites, and osteoblasts in the newly formed trabecular bone. Expression of BMPR-IA was higher than that of BMPR-IB in osteogenic layer on day 3 and in osteoblasts in the trabecular bone on day 7. On day 14, expression of BMP type I receptors was observed at similar sites, albeit with lower expression levels than were observed on day 7, The present data suggest that expression of BMP type I receptors is up-regulated during bone formation, and that they may play important roles in bone morphogenesis.

DOI： 10.1002/jbmr.5650101107

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Synovial cell proliferation is one of the pathological bases of rheumatoid arthritis (RA). Several cytokines including IL-1 and IL-6 and growth factors have been shown to be involved in the synovial cell proliferation in RA, Thrombin is a multifunctional protease and acts as a mitogen for several cell types through its specific receptor. To assess whether thrombin is involved in overproliferation of rheumatoid synovial cells, we measured the concentration of thrombin-antithrombin III (ATIII) complex (TAT) in synovial fluid obtained from patients with RA or osteoarthritis (OA). We also examined the effect of thrombin or thrombin receptor agonist peptide (TRAP) on cell growth of synovial cell clones (SCCs) established from an RA patient. The concentrations of TAT in the synovial fluid from patients with RA were significantly higher than in those with OA. Moreover, both thrombin and TRAP enhanced proliferation of synovial cells in vitro. We also characterized the expression of thrombin receptor mRNA by reverse transcription-PCR. The expression of mRNA for thrombin receptor was up-regulated by thrombin or TRAP stimulation. Thrombin receptor antigen was also detected on both SCCs and synovial tissue from RA patients by immunostaining using a monoclonal antibody against thrombin receptor. These findings indicate that thrombin may act as a mitogen for synovial cells through thrombin receptor and may play some role in synovial overproliferation and remodeling in RA. (C) 1995 Academic Press, Inc.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily. Several members of this family have been shown to transduce their signals through binding to type I and type II serine(threonine) kinase receptors. Here we report the cDNA cloning and characterization of a human type II receptor for BMPs (BMPR-II), which is distantly related to DAF-4 a BMP type II receptor from Caenorhabditis elegans. In transfected COS-1 cells, osteogenic protein (OP)-1/BMP-7, and. less efficiently BMP-4, bound to BMPR-II. BMPR-II bound ligands only weakly alone, but the binding was facilitated by the presence of previously identified type I receptors for BMPs. Binding of OP-1/BMP-7 to BMPR-II was also observed in nontransfected cell lines. Moreover, a transcriptional activation signal was transduced by BMPR-II in the presence of type I receptors after stimulation by OP-1/BMP-7.

DOI： 10.1073/pnas.92.17.7632

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：West-Japanese Society of Orthopedics & Traumatology  

To study the interactions between TGF-β and decorin, we experimentally induced femoral fractures in rats and immunohistologically examined changes in the distribution of TGF-β and Decorin in the femur 3, 7, 14 days after fracture. In areas where endochondral ossification occurred, Decorin was detected during differentiation of mesenchymal cells into proliferating chondrocytes, but no TGF-β was observed during the same period. After the chondrocytes had hypertrophied, Decorin was no longer observed, while the matrix around the hypertrophic chondrocytes was positively stained for TGF-β. The disappearance of Decorin, almost simultaneously accompained by the appearance of the active type TGF-β, suggests that Decorin regulates the action of TGF-β.

DOI： 10.5035/nishiseisai.44.729

researchmap

Other Link： http://search.jamas.or.jp/link/ui/1995210628

Language：English   Publishing type：Research paper (scientific journal)  

We present an immunohistochemical study on extra-cellular matrix (ECM), decorin, in the epidermis and dermis of patients with ossification of the posterior longitudinal ligament (OPLL) to clarify the abnormality in the extracellular matrix of the skin of OPLL. Localization of decorin and TGF-β in the skin was studied. All patients with OPLL showed a diffuse strong staining of decorin in the entire epidermal layer. The epidermis was also positive for TGF-β antibody staining. Patients with cervical spondylotic myelopathy showed weak or no staining of decorin. Abnormality in the regulating system of TGF-β may be involved in the precipitation of extracellular matrix.

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

To study interactions between TGF-β and decorin, we experimentally induced femoral fracture in rats and immunohistologically examined changes in the distribution of TGF-β and decorin in the femur 3, 7 and 14 days after fracture. In the areas where endochondral ossification occurred, decorin was detected during differentiation of mesenchymal cells into proliferating chondrocytes, while no TGB-β was observed during same period. Once chondrocytes had hypertrophied, decorin was no longer observed, while the matrix around the hypertrophic chondrocytes was positively stained for TGB-β. The disapperance of decorin, almost simultaneously accompanied by the appearance of the active type TGB-β, suggests that decorin regulates the actions of TGB-β.

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT-RAVEN PUBL  

Dissociated motor loss occurring in the upper extremities with and without lower extremity myelopathy was evaluated in patients with cervical spondylosis. The presence of dissociated motor loss without attendant myelopathy was correlated with selected compression of the anterior nerve root in the lateral spinal canal, close to the intervertebral foramen. The clinical and radiologic feature differentiating these two dissociative syndromes were reviewed.

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：West-Japanese Society of Orthopedics & Traumatology  

We evaluated the effects of etretinate therapy on the spinal ligaments of patients. Nine patients who were treated with etretinate for disorders of keratinization received radiographic evaluations. Five patients showed ossification of the cervical spinal ligament. We observed the patients who developed ossification within therapy. We suspect that ossification of the spinal ligament was etretinate induced.

DOI： 10.5035/nishiseisai.42.1540

researchmap

Language：Japanese   Publisher：West-Japanese Society of Orthopedics & Traumatology  

X-ray examinations were performed on 239 hemodialyzed patients to detect abnormal findings of the Spine. Destructive Spondylarthropathy (DSA) was observed in 21 cases (8.8%); 9 males (6.7%) and 12 females (11.5%), and largely found in the cervical vertebrae. Rugger jersey spine was observed in 26 cases (10.9%); 16 males (11.9%) and 10 females (9.6%). The rate of DSA and Rugger jersey spine was markedly high in cases with long periods of hemodialysis. A high incidence of ossification of the posterior longiludinal ligament in the cervical spine was observed in a total of 18 cases (7.5%); 13 males (9.6%) and 5 females (4.8%). Our study showed radiographic findings of the spine in hemodialyzed patients to be extremely varied.

DOI： 10.5035/nishiseisai.40.1441

researchmap

Other Link： http://search.jamas.or.jp/link/ui/1993136142

Language：Japanese   Publisher：West-Japanese Society of Orthopedics & Traumatology  

It is still controversial whether stimulation by electromagnetic fields is effective in the treatment of bony nonunion. We conducted experiments to study the optimal stimulation of electromagnetic fields for nonunion and used an electromagnetic fields pulse generator manufactured by the Institute of Physical and Chemical Reserch. Ten patients with nonunion (9 traumatic nonunion, one congenital pseudoarthrosis) were entered in the study. The site of nonunion was the femur in 4 patients, tibia in 3, humerus in 2, and radius in one. Results were as follows: The rate of bone union was 80%. For traumatic nonunion cases the period to union ranged from 3 to 5 months. However, it took 8 months to obtain bone union in the case of congenital pseudoarthrosis. Stimulation by electromagnetic fields is an effective method of treatment for nonunion.

DOI： 10.5035/nishiseisai.41.275

researchmap

Language：Japanese   Publisher：West-Japanese Society of Orthopedics & Traumatology  

Limb-salvage operations in the treatment of malignant bone tumors were analyzed with particular attention given to functional results.<br>This atudy evaluates twelve patients (8 males, 4 females). The location of the primary tumor was the humerus in 3 patients, femur in seven and tibia in three. Histological diagnosis included conventional osteosarcoma in 7, chondrosarcoma in 3 and malignant fibrous histiocytom (MFH) in 2. Using Enneking's criteria surgical stage was evaluated as I A in one, I B 4 and II B in 7. Curative wide excision or wide excision was applied in all cases.<br>At an average thirty six-month follow-up, no patients showed any evidence of local recurrence and complication. However, four patients had lung metastases, three of them dying from this diseaes. Postoperative function was analyzed according to Enneking's functional criteria. The over-all rating was good in one and fair in 2 for the upper extremity; excellent in 2, good in 6 and fair in one for the lower extremity. Good results in ADL were obtained in both the upper and lower extremity except for arthrodesis of the knee joint.<br>Limb-salvage operation in malignant bone tumor is justified from both an oncological and a functional standpoint.

DOI： 10.5035/nishiseisai.41.493

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(株)医薬ジャーナル社  

＜文献概要＞BMPとTGF-βはファミリーを形成し,骨・軟骨形成や骨格筋形成からその恒常性の維持まで重要な働きを担っている。BMPとTGF-βは,細胞表面のI型とII型の2種類のセリン/スレオニンキナーゼ型レセプターおよび細胞内のSmadタンパク質を介して,シグナルを伝達する。細胞外では活性化やアンタゴニスト,細胞膜ではコレセプター,細胞内では,ホスファターゼによる脱リン酸化やユビキチン・プロテアソームシステムによる分解や修飾がBMPとTGF-βシグナルを制御している。本稿では,BMPとTGF-βおよびファミリー分子とシグナル伝達の運動器系組織における機能と役割,疾患について概説する。

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

DOI： 10.11477/mf.2425200665

J-GLOBAL

researchmap

Language：Japanese   Publisher：愛媛医学会  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：The Japanese Society of Medical Imaging Technology  

Main advantages of the optical imaging technique achieving high spatio-temporal resolution, non-radiation, less cytotoxicity and low cost with small devices as compared to conventional clinical imaging techniques such as CT, MRI and PET. In particular, near infrared (NIR) fluorescence imaging enables us to visualize deep portion of tissues due to the excellent penetration &lt;i&gt;in vivo&lt;/i&gt;. We developed a NIR fluorophore-conjugated anti-Carcinoembryonic antigen (CEA) antibody for mice bearing tumor of CEA expressing cells, and demonstrated &lt;i&gt;in vivo&lt;/i&gt; optical imaging by two-photon excitation microscopy. In the result, CEA-expressing cancer cells were specifically detected &lt;i&gt;in vivo&lt;/i&gt;. In preclinical applications, the lymph node micrometastasis was also successfully visualized by two-photon excitation microscopy. These results suggest that two-photon excitation microscopy in combination with the immunoconjugated NIR fluorescence probe could be widely adapted to cancer detection in clinical settings or various cancer metastasis model to reveal the mechanism of cancer metastasis and to visualize microenvironment which may consist of cancer cells and stroma cells.

DOI： 10.11409/mit.34.89

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：(公社)日本整形外科学会  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本工業出版  

CiNii Books

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本骨形態計測学会  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

DOI： 10.1117/12.2079092

Web of Science

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本骨形態計測学会  

researchmap

Language：Japanese   Publisher：一般社団法人日本外科学会  

CiNii Books

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

Web of Science

researchmap

Language：Japanese  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

Web of Science

researchmap

Web of Science

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publisher：日本癌学会  

researchmap

Language：Japanese  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2012183211

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2011-2655

Web of Science

researchmap

Language：Japanese   Publisher：寺田国際事務所先端医療技術研究所  

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2012010657

Language：Japanese  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2010340505

Language：English   Publisher：日本癌学会  

researchmap

Language：Japanese  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2010249446

Language：Japanese   Publisher：オーム社  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2009118728

Language：Japanese  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2009043831

Language：Japanese  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2009023289

Language：English   Publisher：日本癌学会  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本医学会  

researchmap

Language：Japanese   Publisher：オーム社  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2008055738

Language：Japanese  

CiNii Books

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

Web of Science

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

Web of Science

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

Web of Science

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

Web of Science

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2007136612

Language：Japanese   Publisher：医薬ジャーナル社  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2007341602

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本癌学会  

researchmap

Language：Japanese   Publisher：日本癌学会  

researchmap

Language：Japanese   Publisher：日本癌学会  

researchmap

Language：Japanese   Publisher：共立出版