title: HIF-1α and HIF-2α degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc.
abstract: 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.
authors: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors: Nobuyuki
authors: N
authors: Fujita
authors: Kazuhiro
authors: K
authors: Chiba
authors: Irving M
authors: IM
authors: Shapiro
authors: Makarand V
authors: MV
authors: Risbud
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007403:Intervertebral Disc / Q000166:cytology* / Q000201:enzymology; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011393:Procollagen-Proline Dioxygenase; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C061553:ATP dependent 26S protease