Abstract
Several lines of evidence indicate that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation. Given the fact that SOX9 is essential for several steps of the chondrocyte differentiation pathway, we asked whether Ctgf (Ccn2) is the direct target gene of SOX9. We found that Ctgf mRNA was down-regulated in primary sternal chondrocytes from Sox9flox/flox mice infected with Ad-CMV-Cre. We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5′-end to 10 kb downstream of its 3′-end to determine SOX9 interaction site. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay. An important SOX9 regulatory element was found to be located in −70/−64 region of the Ctgf promoter. We found the same site for SOX9 binding to the Ctgf promoter in nucleus pulposus (NP) cells. The loss of Sox9 in growth plate chondrocytes in knee joint and in NP cells in intervertebral disc led to the decrease in CTGF expression. We suggest that Ctgf is the direct target gene of SOX9 in chondrocytes and NP cells. Our study establishes a strong link between two regulatory molecules that have a major role in cartilaginous tissues.
Highlights
SOX (SRY-related HMG box) proteins have critical functions in a number of developmental processes
Genetic studies in mice and in vitro studies of chromatin immunoprecipitation (ChIP)-on-chip analysis, reporter assay, and electrophoretic mobility shift assay (EMSA) have shown that SOX9 is required for the expression of typical cartilage matrix protein-coding genes (Col2a1, Col11a2, Aggrecan, CD-rap, etc.) which serve as direct targets of SOX922–27
We examined whether CTGF levels are restored when the SOX9 expression is rescued after its deletion by Ad-CMV-Cre
Summary
SOX (SRY-related HMG box) proteins have critical functions in a number of developmental processes. Genetic studies in mice and in vitro studies of chromatin immunoprecipitation (ChIP)-on-chip analysis, reporter assay, and electrophoretic mobility shift assay (EMSA) have shown that SOX9 is required for the expression of typical cartilage matrix protein-coding genes (Col2a1, Col11a2, Aggrecan, CD-rap, etc.) which serve as direct targets of SOX922–27. The Ctgf has been shown to stimulate extracellular matrix production by nucleus pulposus (NP) cells[28] and TGF-β, through Smad[3] and AP1, serves as a positive regulator of Ctgf expression in NP cells[29]. It is not known if Ctgf expression is regulated by SOX9 in disc tissues. We showed that SOX9 binds to the Ctgf promoter in NP cells and loss of CTGF expression was detected in Sox[9] deleted disc cells
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