The Scripps Research Institute, La Jolla, California, USA.
Objective: The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9.
Methods: Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element.
Results: SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser(181), and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9(Ser181) phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor beta treatment and mechanical compression.
Conclusion: These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation.
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