Hypoxia-inducible factor-1 is a positive regulator of Sox9 activity in femoral head osteonecrosis.

Legg-Calve-Perthes disease (LCPD) is a juvenile form of ischemic osteonecrosis of the femoral head leading to femoral head deformity and premature osteoarthritis. Femoral head osteonecrosis occurs due to blood supply disruption which results in hypoxic injury to the femoral head. Hypoxia-inducible factor-1 α (HIF-1α) is a master regulator of cellular response to hypoxia. A pig model of ischemic osteonecrosis of femoral head has been shown to have radiographic and histopathologic changes resembling LCPD. Our preliminary studies showed that the cartilage layer was thicker in the hypoxia group compared to the control group. The mechanism underlying this cartilage response is not known. To explore the hypoxia-induced downstream gene activity following the femoral head ischemia, porcine microarray analysis of gene profiles of chondrocytes from normal and ischemic femoral heads was performed. In the ischemic side, the expression of Sox9, a transcription factor required for chondrocyte differentiation, was upregulated along with HIF-1α. Expressions of Sox9 target genes, such as type II collagen and aggrecan, were also increased. Microarray results were confirmed by quantitative real-time RT-PCR. In addition, immunohistochemistry assay demonstrated that both HIF-1α and Sox9 were upregulated in chondrocytes in ischemic femoral heads compared with normal controls. To investigate the possible molecular mechanisms of hypoxia on Sox9 activity, we tested the effect of HIF-1α on Sox9 expression in vitro. We made a luciferase reporter construct driven by 2kb Sox9 promoter. Transient transfection assay showed that HIF-1α activated Sox9 promoter activity in a dose-dependent manner. Sox9 is known to activate type II collagen target gene expression. To test the effect of HIF-1α on Sox9-mediated transcription, HIF-1α was cotransfected with Sox9 in type II collagen reporter assay. Our results demonstrated that HIF-1α enhanced Sox9-mediated transcriptional activity. Moreover, coimmunoprecipitation assay showed that HIF-1α associated with Sox9 directly. Taken together, these findings indicate that HIF-1α activates Sox9 expression and enhances Sox9-mediated transcriptional activity and that HIF-1α physically interacts with Sox9. We speculate that HIF-1α upregulation of Sox9 activity may have a chondroprotective role following femoral head ischemia.

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