Regulation of Sox9 activity by crosstalk with nuclear factor-κB and retinoic acid receptors

INTRODUCTION: Sox9 and p300 cooperate to induce expression of cartilage-specific matrix proteins, including type II collagen, aggrecan and link protein. Tumour necrosis factor (TNF)-α, found in arthritic joints, activates nuclear factor-κB (NF-κB), whereas retinoic acid receptors (RARs) are activated by retinoid agonists, including all-trans retinoic acid (atRA). Like Sox9, the activity of NF-κB and RARs depends upon their association with p300. Separately, both TNF-α and atRA suppress cartilage matrix gene expression. We investigated how TNF-α and atRA alter the expression of cartilage matrix genes. METHODS: Primary cultures of rat chondrocytes were treated with TNF-α and/or atRA for 24 hours. Levels of transcripts encoding cartilage matrix proteins were determined by Northern blot analyses and quantitative real-time PCR. Nuclear protein levels, DNA binding and functional activity of transcription factors were assessed by immunoblotting, electrophoretic mobility shift assays and reporter assays, respectively. RESULTS: Together, TNF-α and atRA diminished transcript levels of cartilage matrix proteins and Sox9 activity more than each factor alone. However, neither agent altered nuclear levels of Sox9, and TNF-α did not affect protein binding to the Col2a1 48-base-pair minimal enhancer sequence. The effect of TNF-α, but not that of atRA, on Sox9 activity was dependent on NF-κB activation. Furthermore, atRA reduced NF-κB activity and DNA binding. To address the role of p300, we over-expressed constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 to increase p300 acetylase activity. caMEKK1 enhanced basal NF-κB activity and atRA-induced RAR activity. Over-expression of caMEKK1 also enhanced basal Sox9 activity and suppressed the inhibitory effects of TNF-α and atRA on Sox9 function. In addition, over-expression of p300 restored Sox9 activity suppressed by TNF-α and atRA to normal levels. CONCLUSION: NF-κB and RARs converge to reduce Sox9 activity and cartilage matrix gene expression, probably by limiting the availability of p300. This process may be critical for the loss of cartilage matrix synthesis in inflammatory joint diseases. Therefore, agents that increase p300 levels or activity in chondrocytes may be useful therapeutically.