ECTS2013 Poster Presentations Chondrocytes and cartilage (20 abstracts)
Transcription factor Nkx3.2 plays crucial role in primary chondrogenesis by up-regulating type II collagen a1 transcription activity
Kosuke Ebina 1 , Yoshitaka Kawato 1 , Makoto Hirao 2 , Yui Honjo 1 , Tokimitsu Morimoto 1 , Jun Hashimoto 3 , Kenrin Shi 1 & Hideki Yoshikawa 1
1Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan; 2Department of Orthopaedic Surgery, Osaka Minami Medical Center, National Hospital Organization, Osaka, Japan; 3Department of Rheumatology, Osaka Minami Medical Center, National Hospital Organization, Osaka, Japan.
Objectives: Sox9 is a dominant but insufficient transcription factor to induce thorough primary chondrogenesis, so other factors which may induce primary chondrogenesis besides Sox9 have been assumed. The previously reported function of transcription factor Nkx3.2 is to maintain chondrogenic phenotype by suppressing Runx2, while recent studies demonstrated that mouse Nkx3.2 null mice shows severe metaphyseal dysplasia which is similar to that seen in type II collagen a1 (Col2a1) null mice. Therefore, we hypothesized that Nkx3.2 may play a crucial role in primary chondorogenesis besides Sox9.
Methods and results: Mouse multipotential mesenchymal C3H10T1/2 cells and mouse chondrogenic N1511 cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Over-expression of Nkx3.2 with wild-type Nkx3.2 (WT-Nkx3.2) plasmid up-regulated glycosaminoglycan (GAG) production and expression of Col2a1 mRNA, and these effects were evident before up-regulation of Sox9. RNAi-mediated inhibition of Nkx3.2 and Sox9 both abolished GAG production and Col2a1 mRNA expression. Interestingly, even when Sox9 is down-regulated, over-expression of WT-Nkx3.2 restored GAG production and Col2a1 mRNA/protein expression to a certain extent. Dual luciferase reporter assays revealed that WT-Nkx3.2 up-regulated Col2a1 enhancer activity in both C3H10T1/2 and N1511cells in a dose-dependent manner, although it fell short of WT-Sox9. Finally, ChIP assays revealed that Nkx3.2 bounds to the 48 bp Col2a1 enhancer element.
Conclusions: Our results demonstrated that Nkx3.2 is necessary not only in maintaining chondrogenic phenotype, but also in inducing primary chondrogenesis by up-regulating Col2a1 enhancer activity besides Sox9. Further investigation is expected to apply Nkx3.2-targeted treatment to cartilage regeneration.
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