Hypoxia Up-regulates Prolyl Hydroxylase Activity

Abstract

The mechanism by which hypoxia induces gene transcription is now well established. Hypoxia reduces activity of prolyl hydroxylases (PHD) that hydroxylate specific proline residues in the oxygen-dependent degradation domain (ODD) of hypoxia-inducible factor-1␣ (HIF-1␣). As a consequence, HIF-1␣ accumulates and promotes hypoxic tolerance by activating gene transcription. This paper identifies the three forms of PHDs in rats and shows that a period of hypoxia selectively increases expression of PHD-2 mRNAs levels. We developed assays for PHD activity that used (i) the peptidespecific conversion of labeled 2-oxoglutarate into succinate and (ii) the binding of the von Hippel-Lindau protein to a glutathione S-transferase-ODD fusion protein. The two assays indicated a low enzymatic activity in normoxic and hypoxic cells and a rapid increase during reoxygenation. We also developed hydroxyprolinespecific antibodies that recognized hydroxylated forms of a fusion protein (ODD-green fluorescent protein) that combined the ODD domain of HIF-1␣ and the green fluorescent protein. Using this antibody, we demonstrated that reoxygenation induced a rapid hydroxylation of Pro-564, which was followed by a massive degradation of the proteins. The results suggest that a hypoxic upregulation of PHD (presumably PHD-2) acts as a feedback mechanism to stop hypoxic responses in reoxygenated cells. We propose that proline hydroxylation might play a role in hypoxic preconditioning.Cells respond to reduced oxygen tensions by up-regulating the expression of genes involved in angiogenesis (e.g. vascular endothelial growth factor), erythropoiesis (e.g. erythropoietin), and glycolysis. The transcriptional activation of target genes is induced by a common transcription factor, hypoxia inducible factor-1 (HIF-1).1 HIF-1 was first identified as a heterodimeric transactivator that recognizes a specific DNA sequence termed hypoxia-responsive element in the 3Ј-untranslated region of the erythropoietin gene (1). HIF-1 is composed of two subunits, HIF-1␣ and the aryl hydrocarbon nuclear translocator, both of which belong to the large family of basic helix-loop-helix-perarnt-sim transcription factors (2). The mechanism of the hypoxic induction of HIF-1␣ has recently been identified. Under normoxic conditions, specific HIF-1␣ prolyl hydroxylases (PHD) hydroxylate two proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation (ODD) domain of HIF-1␣ (3, 4). The von Hippel-Lindau protein (vHL) E3 ubiquitin ligase complex associates to hydroxylated proline residues and targets HIF-1␣ to proteasomal degradation. Under hypoxic conditions, oxygen becomes rate-limiting for proline hydroxylation. As a consequence, HIF-1␣ accumulates, migrates to the nucleus, and associates with the aryl hydrocarbon nuclear translocator and the complex interacts with hypoxia-responsive element of target genes (5, 6).Less attention has been given to the situation in which hypoxic cells are reoxygenated. Tissue reoxygenation is not an unusual condition. It is frequently...

Hsp70 and CHIP Selectively Mediate Ubiquitination and Degradation of Hypoxia-inducible Factor (HIF)-1α but Not HIF-2α

“…Therefore, Hsp70 plays a predominant role in HIF-1␣ degradation specifically under conditions of prolonged hypoxia. PHD2 expression is also induced by hypoxia, and an earlier study concluded it does not promote HIF-1␣ degradation until cells are reoxygenated (34), whereas a recent study implicated PHDs in HIF-1␣ degradation during prolonged hypoxia (35). However, this mechanism cannot explain the differential stability of HIF-1␣ and HIF-2␣, as both proteins are negatively regulated by PHD2.…”

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells

“…The teleologic reason for the differential expression of PHD2 between fetus and adult is unclear, but may relate to the critical role of the PHDs upon reoxygenation (8,15,16) to ensure rapid cessation of the hypoxic response. Similarly, PHD2 mRNA and protein levels might be relatively high in the term mammalian fetus to ready the fetus to rapidly silence the HIF-1-dependent 21 experiments) PA SMC, hypoxia increased HIF-1 mRNA expression ( * , P Ͻ 0.01, versus normoxia), whereas in adult (n ϭ 8 animals; 24 experiments) PA SMC hypoxia had no effect on HIF-1 mRNA expression.…”

“…PHD2 likely has the dominant role (15), because ''silencing'' of PHD2 with siRNAs stabilizes and activates HIF-1␣ in normoxia, whereas silencing of PHD1 and PHD3 has no effect on the stability of HIF-1␣. PHD2 and PHD3 expression are up-regulated by hypoxia by HIF-1, providing an autoregulatory mechanism of HIF-1 stability driven by oxygen tension (16,17). Paradoxically, the expression of these hydroxylases is induced in hypoxic cell culture despite the fact that the reduced availability of the O 2 cosubstrate renders them inactive, suggesting that the PHD function to rapidly terminate the HIF-1 response upon reoxygenation (8,16).…”

Differential Function of the Prolyl Hydroxylases PHD1, PHD2, and PHD3 in the Regulation of Hypoxia-inducible Factor

“…In the first instance, expression was defined at the mRNA level using RNase protection assays. Following reports of PHD mRNA regulation by hypoxia in certain cell lines (12,22,27,36), all cells were studied in normoxic culture and after exposure to hypoxia (0.5% oxygen) for 16 h. Fig. 1 shows results for cells derived from a variety of human tissues: BxPC-3 (pancreatic carcinoma); PC-3 (prostate carcinoma); MCF7, HS-587T, MDA-435, T47D, and ZR-75-1 (breast carcinoma); U-2 OS (osteosarcoma); OVCAR-3 (ovarian carcinoma); A549 (lung carcinoma); HT1080 (rhabdomyosarcoma); and JAR (choriocarcinoma).…”