HIF-1α AND HIF-2α DEGRADATION IS DIFFERENTIALLY REGULATED IN NUCLEUS PULPOSUS CELLS OF THE INTERVERTEBRAL DISC

摘要

Studies of many cell types show that level of HIF-1α and HIF-2α is primarily controlled by oxygen-dependent proteasomal degradation, catalyzed by HIF prolyl-hydroxylases (PHDs). However, in 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. Noteworthy, HIF-2α degradation through 26S proteasome was more pronounced in hypoxia. Surprisingly, treatment with DMOG, a PHD inhibitor, shows 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 demonstrate 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, 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 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.