Nkx3.2-induced suppression of Runx2 is a crucial mediator of hypoxia-dependent maintenance of chondrocyte phenotypes

Hypoxia is a key factor in the maintenance of chondrocyte identity. However, crucial chondrogenic transcription factors in the Sox families are not activated in this phenomenon, indicating that other pathways are involved.Nkx3.2 is a well-known chondrogenic transcription factor induced by Sonic hedgehog (Shh); it suppresses a key osteogenic transcriptional factor, Runt-related transcription factor 2 (Runx2), to maintain the chondrogenic phenotype in mesenchymal lineages. The purpose of this study was to examine the function of Nkx3.2 in hypoxia-dependent maintenance of chondrocyte identity.C3H10T1/2 pluripotent mesenchymal cells were cultured with rh-BMP2 (300 ng/ml) to induce chondrogenesis under normoxic (20% O2) or hypoxic (5% O2) conditions.Immunohistological detection of Nkx3.2 in a micromass cell culture system revealed that hypoxia promoted expression of the Nkx3.2 protein. Real-time RT-PCR analysis revealed that hypoxia promoted Nkx3.2 mRNA expression and suppressed Runx2 mRNA expression; however, Sox9 mRNA expression was not altered by oxygen conditions, as previously described. Over-expression of exogenous Nkx3.2 promoted glycosaminoglycan (GAG) production and inhibited Runx2 mRNA expression and, based on a dual luciferase assay, Runx2 promoter activity. Interestingly, downregulation of Nkx3.2 using RNAi abolished hypoxia-dependent GAG production and restored Runx2 mRNA expression and promoter activity.These results demonstrated that Nkx3.2-dependent suppression of Runx2 was a crucial factor in hypoxia-dependent maintenance of chondrocyte identity.

► We investigated the effect of Nkx3.2 on hypoxia-dependent chondrogenesis. ► Nkx3.2 protein was upregulated under hypoxia. ► Hypoxia upregulated Nkx3.2 mRNA and a Nkx3.2 reporter construct. ► Nkx3.2 was a positive regulator of hypoxia-dependent GAG production. ► Nkx3.2 downregulated Runx2 mRNA and a Runx2 reporter construct.