Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription

BACKGROUND: The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan (GAG) production and expression of type II collagen α1 (Col2a1) mRNA, and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1 enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element. CONCLUSIONS/SIGNIFICANCE: Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independent upregulation of Col2a1 transcription and upregulation of Sox9 mRNA expression under positive feedback system.