Abstract
Background
Transcription factor nuclear factor E2p45-related factor 2 (Nrf2) is crucial for controlling the antioxidant response and maintaining cellular redox homeostasis. Binding of Nrf2 to antioxidant response elements (ARE) promotes the expression of anti-oxidative stress enzymes. In osteoblasts, Nrf2 directly interacts with Runx2, a strong transcriptional activator of osteoblast-specific genes. Sox9, a key regulator of chondrocyte differentiation is dominant over Runx2 in mesenchymal chondrogenic precursors. We therefore aimed to elucidate the role of Nrf2, and its regulation of Sox9, in chondrocytes.
Methods
ARE sites in SOX9 promoter fragments were inactivated and cloned into pGL3 prior to co-transfection with phRL-TK into C-28/I2 cells for dual luciferase assay (n=4). Analyses of Nrf2 and Sox9 expression (n=3), following Nrf2 RNA interference (RNAi) (Sigma-Mission shRNAs library), was performed by qPCR (Applied Biosystems) as well as by Nrf2 and Sox9 immunohistochemistry in femoral condyle cartilage of wild type (WT) and Nrf2-knockout (KO) mice with ethical approval.
Results
The Sox9 promoter region contains several putative antioxidant response elements. Mutagenesis of the ARE2 site reduced SOX9 promoter activity by 50%. Successful knock-down of Nrf2 using Nrf2-specific shRNAs in C-28/I2 chondrocytes also revealed parallel suppression of Sox9 mRNA. Furthermore, Nrf2-KO mice have fewer Sox9-positive-chondrocytes in their articular cartilage compared to WT littermates.
Conclusions
Successive deletion of two putative ARE sites in the SOX9 promoter region suggests that ARE2 positively regulates SOX9 transcription and is in line with Sox9 mRNA suppression upon Nrf-2-RNAi. Nrf2 binding may thus directly stimulate Sox9 expression. Nrf2-KO mice reveal reduced numbers of Sox9-positive hyaline chondrocytes, which may have important consequences for the extracellular matrix production in these animals. Our findings reveal a novel mechanism regulating extracellular matrix synthesis in chondrocytes and may improve cartilage regenerative medicine.
Level of evidence
Preclinical
