Prolyl hydroxylase domain enzymes and their role in cell signaling and cancer metabolism

The prolyl hydroxylase domain (PHD) enzymes regulate the stability of the hypoxia-inducible factor (HIF) in response to oxygen availability. During oxygen limitation, the inhibition of PHD permits the stabilization of HIF, allowing the cellular adaptation to hypoxia. This adaptation is especially important for solid tumors, which are often exposed to a hypoxic environment. However, and despite their original role as the oxygen sensors of the cell, PHD are currently known to display HIF-independent and hydroxylase-independent functions in the control of different cellular pathways, including mTOR pathway, NF-kB pathway, apoptosis and cellular metabolism. In this review, we summarize the recent advances in the regulation and functions of PHD in cancer signaling and cell metabolism.     

Prolyl hydroxylase PHD3 activates oxygen-dependent protein aggregation

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.     

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.     

Overexpression of HIF Prolyl‐Hydoxylase‐2 transgene in the renal medulla induced a salt sensitive hypertension

Renal medullary hypoxia‐inducible factor (HIF)‐1α and its target genes, such as haem oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain‐containing proteins (PHDs) are major enzymes to promote the degradation of HIF‐1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF‐1α‐mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long‐term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF‐1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2‐treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2‐mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.     

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.     

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.     

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.