Abstract
Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. TGF-β can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-β and that TGF-β stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-β. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-β. Here we show that p38 is required for TGF-β-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-β regulates Sox9 may lead to identification of therapeutic targets for OA.
Highlights
Members of the TGF-β superfamily are important regulators of chondrocyte function
RNA was isolated from cells that had been treated with TGF-β1 for 6 hours and was used for quantitative real time RT-PCR (QPCR) to determine expression levels of Sox[9] mRNA
The results suggest that TGF-βregulates Sox[9] protein stability in ATDC5 cells, similar to what we previously observed in bovine articular chondrocytes[32], supporting the use of ATDC5 cells as a model for cartilage homeostasis in this study
Summary
Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox[9], a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox[9]. Increased levels of TGF-βcan lead to osteophyte formation exacerbating the OA phenotype[21] For this reason, downstream targets of TGF-βthat regulate chondroprotective pathways must be identified to develop preventative and reparative therapies. Sex determining region Y (SRY) Box 9 (Sox9) is an important chondrogenic transcription factor It regulates formation of embryonic cartilage and is required for post-natal maintenance of the articular cartilage[22,23]. We previously showed that SOX9 is required for TGF-β1 mediated regulation of PAPSS2, an enzyme required for proper sulfation of proteoglycans, in bovine chondrocytes[31,32] For these reasons, we addressed the mechanism of how TGF-βregulates Sox[9] protein. The results provide evidence of a novel signaling pathway for TGF-βin chondrocytes
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