HIF‐1α and HIF‐2α degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc

Studies of many cell types show that levels of hypoxia inducible factor (HIF)‐1α and HIF‐2α are primarily controlled by oxygen‐dependent proteasomal degradation, catalyzed by HIF prolyl‐hydroxylases (PHDs). However, in the hypoxic niche of the intervertebral disc, the mechanism of HIF‐α turnover in nucleus pulposus cells is not yet known. We show that in nucleus pulposus cells HIF‐1α and HIF‐2α, degradation was mediated through 26S proteasome irrespective of oxygen tension. It is noteworthy that HIF‐2α degradation through 26S proteasome was more pronounced in hypoxia. Surprisingly, treatment with DMOG, a PHD inhibitor, shows the accumulation of only HIF‐1α and induction in activity of its target genes, but not of HIF‐2α. Loss and gain of function analyses using lentiviral knockdown of PHDs and overexpression of individual PHDs show that in nucleus pulposus cells only PHD2 played a limited role in HIF‐1α degradation; again HIF‐2α degradation was unaffected. We also show that the treatment with inhibitors of lysosomal proteolysis results in a strong accumulation of HIF‐1α and to a much smaller extent of HIF‐2α levels. It is thus evident that in addition to PHD2 catalyzed degradation, the HIF‐1α turnover in nucleus pulposus cells is primarily regulated by oxygen‐independent pathways. Importantly, our data clearly suggests that proteasomal degradation of HIF‐2α is not mediated by a classical oxygen‐dependent PHD pathway. These results for the first time provide a rationale for the normoxic stabilization as well as the maintenance of steady‐state levels of HIF‐1α and HIF‐2α in nucleus pulposus cells. © 2012 American Society for Bone and Mineral Research