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Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca 2؉ /calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.Chondrogenesis is an important biological event for endochondral bone development, skeletogenesis, and tissue patterning (1, 2). The first step in chondrogenesis is the aggregation of mesenchymal cells into prechondrogenic condensations. These condensations start to express cartilage-specific genes and further differentiate into mature chondrocytes. In the growth plate, chondrocytes proliferate and further differentiate into hypertrophic chondrocytes. The control of chondrogenic differentiation and hypertrophy plays a pivotal role in the process. Dysregulation of either step leads to severe skeletal dysplasia in both mice and humans (3).The transcription factor Sox9 (SRY (sex-related Y)-type high mobility group box), which contains a SRY-related high mobility group box, has an essential role in the chondrocyte differentiation pathway (4, 5). Sox9 regulates the transcription of cartilage-specific extracellular matrix molecules, such as collagen type II (6), IX (7), and XI (8) and aggrecan (9). Heterozygous mutations in the SOX9 gene cause campomelic dysplasia characterized by severe chondrodysplasia (10). Sox9 heterozygous mutant mice and mice lacking SOX9 function show impaired endochondral bone formation (4, 5). Sox9 is also involved in the expression of Sox5 and So...

Bone morphogenetic protein(s) (BMP) are very powerful cytokines that induce bone and cartilage formation. BMP also stimulate osteoblast and chondrocyte differentiation. During bone and cartilage development, BMP regulates the expression and/or the function of several transcription factors through activation of Smad signalling. Genetic studies revealed that Runx2, Osterix and Sox9, all of which function downstream of BMP, play essential roles in bone and/or cartilage development. In addition, two other transcription factors, Msx2 and Dlx5, which interact with BMP signalling, are involved in bone and cartilage development. The importance of these transcription factors in bone and cartilage development has been supported by biochemical and cell biological studies. Interestingly, BMP is regulated by several negative feedback systems that appear necessary for fine-tuning of bone and cartilage development induced by BMP. Thus, BMP harmoniously regulates bone and cartilage development by forming network with several transcription factors.

Background: Molecular mechanisms controlling the late stages of endochondral ossification are unclear. Results: Osterix functions as a downstream and transcriptional partner of Runx2 and induces MMP13 during chondrocyte differentiation. Conclusion: Osterix is essential for late-stage endochondral ossification. Significance: Osterix affects the ossification of cartilage matrices and matrix vesicles and might be involved in the development of osteoarthritis and related disorders.

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Vinexin is a recently identified cytoskeletal protein and plays a key role in the regulation of cytoskeletal organization and signal transduction. Vinexin localizes at sites of cell-extracellular matrix adhesion in NIH3T3 fibroblasts and at sites of cell-cell contact in epithelial LLC-PK1 cells. Expression of vinexin promotes the formation of actin stress fiber, but the role of vinexin at sites of cell-cell contact is unclear. Here we identified lp-dlg/KIAA0583 as a novel binding partner for vinexin by using yeast two-hybrid screening. lp-dlg/KIAA0583 has a NH 2 -terminal coiled-coil-like domain, in addition to four PDZ domains, an Src homology (SH) 3 domain, and a guanylate kinase domain, which are conserved structures in membrane-associated guanylate kinase family proteins. The third SH3 domain of vinexin bound to the region between the second and third PDZ domain of lp-dlg, which contains a proline-rich sequence. lp-dlg colocalized with vinexin at sites of cell-cell contact in LLC-PK1 cells. Furthermore, lp-dlg colocalized with ␤-catenin, a major adherens junction protein, in LLC-PK1 cells. Co-immunoprecipitation experiments revealed that both endogenous and epitope-tagged deletion mutants of lp-dlg/KIAA0583 associated with ␤-catenin. We also showed that these three proteins could form a ternary complex. Together these findings suggest that lp-dlg/KIAA0583 is a novel scaffolding protein that can link the vinexin-vinculin complex and ␤-catenin at sites of cell-cell contact.Cell-cell adhesion is important for cell polarity, tissue morphogenesis development, and homeostasis (1-3). To this end, epithelial cells exhibit specialized structures involved in cell-cell contacts such as tight junctions and adherens junctions. Adherens junctions contain the transmembrane cell adhesion molecules, cadherins and nectins, which mediate the calcium-dependent and -independent cell-cell adhesion (1, 3, 4), respectively. The cytoplasmic domain of cadherin binds to ␤-catenin, which then binds to ␣-catenin. ␣-Catenin binds to actin and actin-binding proteins such as vinculin, ␣-actinin, and ZO-1, resulting in the link of cadherin to the actin cytoskeleton (3, 5, 6). The cytoplasmic domain of nectin binds to l-afadin, which then binds to actin and a vinculin-binding protein ponsin (4,7,8). Multiple protein complexes of these cytoplasmic proteins play important roles in communicating between cell adhesion systems, regulating cellcell adhesion, and transducing signals into cells.Vinexin is a protein localizing at cell-cell and cell-extracellular matrix junctions (9). There are at least two types of vinexin, vinexin ␣ and vinexin ␤, which share a common carboxyl-terminal sequence containing three SH (Src homology) 3 1 domains. The larger vinexin ␣ has an additional amino-terminal sequence containing a sorbin homology domain. Vinexin is a member of a novel adaptor protein family, including ArgBP2 and ponsin, all of which have a sorbin homology domain in the NH 2 -terminal half and three SH3 domains in the COOHterminal half (8 -12). Vin...

Osteoarthritis is a common disease in joint cartilages. Because the molecular pathogenesis of osteoarthritis remains elusive, early diagnostic markers and effective therapeutic agents have not been developed. To understand the molecular mechanisms, we attempted to identify transcription factors involved in the onset of osteoarthritis. Microarray analysis of mouse articular cartilage cells indicated that retinoic acid, a destructive stimulus in articular cartilage, up-regulated expression of sex-determining region Y-box (Sox)4, a SoxC family transcription factor, together with increases in Adamts4 and Adamts5, both of which are aggrecanases of articular cartilages. Overexpression of Sox4 induced a disintegrin-like and metallopeptidase with thrombospondin type 4 and 5 motif (ADAMTS4 and ADAMTS5, respectively) expression in chondrogenic cell lines C3H10T1/2 and SW1353. In addition, luciferase reporter and chromatin immunoprecipitation assays showed that Sox4 up-regulated ADAMTS4 and Adamts5 gene promoter activities by binding to their gene promoters. Another SoxC family member, Sox11, evoked similar effects. To evaluate the roles of Sox4 and Sox11 in articular cartilage destruction, we performed organ culture experiments using mouse femoral head cartilages. Sox4 and Sox11 adenovirus infections caused destruction of articular cartilage associated with increased Adamts5 expression. Finally, SOX4 and SOX11 mRNA expression was increased in cartilage of patients with osteoarthritis compared with nonosteoarthritic subjects. Thus, Sox4, and presumably Sox11, are involved in osteoarthritis onset by up-regulating ADAMTS4 and ADAMTS5.-Takahata, Y., Nakamura, E., Hata, K., Wakabayashi, M., Murakami, T., Wakamori, K., Yoshikawa, H., Matsuda, A., Fukui, N., Nishimura, R. Sox4 is involved in osteoarthritic cartilage deterioration through induction of ADAMTS4 and ADAMTS5.