Abstract
Oxidative stress is implicated in osteoarthritis, and nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway maintains redox homeostasis. We investigated whether Nrf2/ARE signaling controls SOX9. SOX9 expression in human C-28/I2 chondrocytes was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap ‘‘n’’ collar homology-associated protein 1 (Keap1). To verify whether Nrf2 transcriptionally regulates SOX9, putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays. SOX9 promoter activities without and with Nrf2-inducer methysticin were compared. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Nrf2-specific RNAi significantly decreased SOX9 expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, but ARE1 excision did not. Functional ARE2 site was essential for methysticin-mediated induction of SOX9 promoter activity. Young Nrf2-knockout mice revealed significantly lower Sox9-positive chondrocytes, and old Nrf2-knockout animals showed thinner cartilage and more cartilage degeneration. Our results suggest Nrf2 directly regulates SOX9 in articular cartilage, and Nrf2-loss can develop mild osteoarthritis at old age. Pharmacological Nrf2 induction may hold the potential to diminish age-dependent cartilage degeneration through improving SOX9 expression.
Highlights
To the best of our knowledge, this is a first-time report indicating that the proximal upstream antioxidant response element (ARE) site (ARE2 ) plays a crucial direct role in the nuclear factor erythroid 2–related factor 2 (Nrf2)-mediated induction of the SOX9 promoter in human chondrocytes
We provided evidence that a systemic Nrf2 deficiency results in Sox9 suppression in murine articular chondrocytes in vivo
Nrf2-KO mice developed a mild osteoarthritic articular cartilage degeneration, which is in line with earlier reported proteoglycan loss upon postnatal
Summary
Osteoarthritis (OA), the most common joint disease, is characterized by cartilage damage and joint dysfunction [1]. Altered chondrocyte behavior such as chondrocyte hypertrophy, matrix ossification, and osteophyte formation in the joint is closely connected to its pathogenesis [2]. SOX9 is an essential transcription factor controlling cartilage extracellular matrix (ECM) homeostasis and a key regulator of chondrocyte differentiation through promoting collagen type II (Col II) expression [3]. SOX9 inactivation before mesenchymal condensation downregulated Col II expression, to affect cartilage formation in a mouse model [4]. A reduced SOX9 gene expression in articular chondrocytes
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