Differential and Reciprocal Regulation between Hypoxia-inducible Factor-α Subunits and Their Prolyl Hydroxylases in Pulmonary Arteries of Rat with Hypoxia-induced Hypertension

Hypoxia-inducible factor (HIF)-α subunits (HIF-1α,HIF-2α and HIF-3α),which play a pivotalrole during the development of hypoxia-induced pulmonary hypertension (HPH),are regulated through post-U'anslational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD 1,PHD2 and PHD3).PHDs could also be regulated by HIF.But differential and reciprocal regulation between HIF-α and PHDs duringthe development of HPH remains unclear.To investigate this problem,a rat HPH model was established.Meanpulmonary arterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling indexand right ventricular hypertrophy became evident after 14 d of hypoxia.HIF-1α and HIF-2α mRNA increasedslightly after 7 d of hypoxia,but HIF-3α increased significantly after 3 d of hypoxia.The protein expressionlevels of all three HIF-α were markedly upregulated after exposure to hypoxia.PHD2 mRNA and proteinexpression levels were upregulated after 3 d of hypoxia;PHD 1 protein declined after 14 d of hypoxia withoutsignificant mRNA changes.PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia,then themRNA remained at a high level,but the protein declined after 14 d of hypoxia.In hypoxic animals,HIF-lotproteins negatively correlated with PHD2 proteins,whereas HIF-2α and HIF-3α proteins showed negativecorrelations with PHD3 and PHD 1 proteins,respectively.All three HIF-α proteins were positively correlatedwith PHD2 and PHD3 mRNA.In the present study,HIF-α subunits and PHDs showed differential andreciprocal regulation,and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.     

Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro

Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.     

Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of hypoxia-inducible factor-alpha/Sima

Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-alpha/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima.     

Hypoxia-inducible factor 1α under rapid enzymatic hypoxia: Cells sense decrements of oxygen but not hypoxia per se

HIF1 (hypoxia-inducible factor 1α) is considered a central oxygen-threshold sensor in mammalian cells. In the presence of oxygen, HIF1 is marked by prolyl hydroxylases (PHDs) at the oxygen-dependent degradation (ODD) domain for ubiquitination followed by rapid proteasomal degradation. However, the actual mechanisms of oxygen sensing by HIF1 are still controversial. Thus, HIF1 expression correlates poorly with tissue oxygen levels, and PHDs are themselves target genes of HIF1 considered to readjust to new oxygen thresholds. In contrast to hypoxia chambers, we here establish an enzymatic model that allows both the rapid induction of stable hypoxia and independent control of H₂O₂. Rapid enzymatic hypoxia only transiently induced HIF1 in various cell types and the HIF1 was completely degraded within 8–12 h despite sustained hypoxia. HIF1 degradation under sustained hypoxia could be blocked by a competitive ODD–GFP construct and PHD siRNA, but also by cobalt chloride and micromolar H₂O₂ levels. Concomitant induction of PHDs further confirmed their role in degrading HIF1 under enzymatic hypoxia. The rapid and complete degradation of HIF1 under enzymatic hypoxia suggests that, in addition to hypoxia sensing, the HIF1/PHD loop may rather compensate for fluctuations of tissue oxygen staying tuned to other, e.g., metabolic, signals. In addition to hypoxia chambers, enzymatic hypoxia provides a valuable tool for independently studying the regulatory functions of hypoxia and oxidative stress in vitro.     

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.