Hypoxia-inducible factor (HIF)-1α and CCN2 form a regulatory circuit in hypoxic nucleus pulposus cells: CCN2 suppresses HIF-1α level and transcriptional activity

The objective of the study was to investigate if hypoxia-inducible factor (HIF)-1α and connective tissue growth factor (CCN2) form a regulatory network in hypoxic nucleus pulposus (NP) cells. A decrease in CCN2 expression and proximal promoter activity was observed in NP cells after hypoxic culture. Analysis of both human and mouse CCN2 promoters using the JASPAR core database revealed the presence of putative hypoxia response elements. Transfection experiments showed that both promoter activities and CCN2 expression decreases in hypoxia in a HIF-1α-dependent fashion. Interestingly, deletion analysis and mutation of the hypoxia responsive elements individually or in combination resulted in no change in promoter activity in response to hypoxia or in response to HIF-1α, suggesting an indirect mode of regulation. Notably, silencing of endogenous CCN2 increased HIF-1α levels and its target gene expression, suggesting a role for CCN2 in controlling basal HIF-1α levels. On the other hand, treatment of cells with rCCN2 resulted in a decrease in the ability of HIF-1α transactivating domain to recruit co-activators and diminished target gene expression. Last, knockdown of CCN2 in NP cells results in a significant decrease in GAG synthesis and expression of AGGRECAN and COLLAGEN II. Immunohistochemical staining of intervertebral discs of Ccn2 null embryos shows a decrease in aggrecan. These findings reveal a negative feedback loop between CCN2 and HIF-1α in NP cells and demonstrate a role for CCN2 in maintaining matrix homeostasis in this tissue.