HIF-1a and HIF-2a 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)-1a and HIF-2a 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-a turnover in nucleus pulposus cells is not yet known. We show that in nucleus pulposus cells HIF-1a and HIF-2a, degradation was mediated through 26S proteasome irrespective of oxygen tension. It is noteworthy that HIF-2a degradation through 26S proteasome was more pronounced in hypoxia. Surprisingly, treatment with DMOG, a PHD inhibitor, shows the accumulation of only HIF-1a and induction in activity of its target genes, but not of HIF-2a. 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-1a degradation; again HIF-2a degradation was unaffected. We also show that the treatment with inhibitors of lysosomal proteolysis results in a strong accumulation of HIF-1a and to a much smaller extent of HIF-2a levels. It is thus evident that in addition to PHD2 catalyzed degradation, the HIF-1a turnover in nucleus pulposus cells is primarily regulated by oxygen-independent pathways. Importantly, our data clearly suggests that proteasomal degradation of HIF-2a 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-1a and HIF-2a in nucleus pulposus cells. (C) 2012 American Society for Bone and Mineral Research