title: Expression of prolyl hydroxylases (PHDs) is selectively controlled by HIF-1 and HIF-2 proteins in nucleus pulposus cells of the intervertebral disc: distinct roles of PHD2 and PHD3 proteins in controlling HIF-1α activity in hypoxia.
abstract: Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1β confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O(2), PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications.
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Fujita
authors.forename: Dessislava
authors.initials: D
authors.lastname: Markova
authors.forename: D Greg
authors.initials: DG
authors.lastname: Anderson
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Chiba
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Toyama
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D014161:Transduction, Genetic; 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; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C497444:Hif1a protein, rat