The central transcriptional response to hypoxia is mediated by the Prolyl Hydroxylase Domain protein (PHD):Hypoxia Inducible Factor (HIF) pathway. In this pathway, PHD prolyl hydroxylates and thereby negatively regulates the α-subunit of the transcription factor HIF (HIF-α). An important HIF target gene is that for Erythropoietin (EPO), which controls red cell mass. Recent studies have identified PHD2 as the critical PHD isoform regulating the EPO gene. Other studies have shown that the inducibility of the HIF pathway diminishes as a function of age. Thus, an important question is whether the PHD2:EPO pathway is altered in the aging. Here, we employed a mouse line with a globally-inducible Phd2 conditional knockout allele to examine the integrity of the Phd2:Epo axis in young (six to eight month old) and aging (sixteen to twenty month old) mice. We find that acute global deletion of Phd2 results in a robust erythrocytosis in both young and aging mice, with both age groups showing marked extramedullary hematopoiesis in the spleen. Epo mRNA is dramatically upregulated in the kidney, but not in the liver, in both age groups. Conversely, other Hif targets, including Vegf, Pgk1, and Phd3 are upregulated in the liver but not in the kidney in both age groups. These findings have implications for targeting this pathway in the aging.
Erythropoietin; Prolyl Hydroxylase Domain protein; Hypoxia Inducible Factor; Prolyl hydroxylation; Gene regulation
As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin—proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of transcription factors hypoxia-inducible factor (HIF) and NF-κB. We inhibited the UPP with proteasome-specific inhibitors and determined the activation of HIF and NF-κB as well as the expression and secretion of pro-angiogenic factors. HIF-1α was not detectable in ARPE-19 cells under normal culture conditions. However, when proteasome activity was inhibited, HIF-1α accumulated in RPE in a time-dependent manner. Consistent with accumulation of HIF-1α in the cells, levels of mRNA for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in RPE were up to 7-fold higher upon inhibition of the proteasome. Proteasome inhibition was also associated with a 2-fold increase in levels of mRNA for angiopoietin-1 (Ang-1). ARPE-19 cells secrete significant levels of VEGF under normal culture conditions. Inhibition of proteasome activity increased the secretion of VEGF by 2-fold. In contrast to the increase in HIF activity, NF-κB activation was reduced by proteasome inhibition. In addition, the expression and secretion of monocyte chemoattractant protein-1 (MCP-1) by RPE were substantially attenuated by the inhibition of proteasome activity. These data demonstrate that the UPP plays an important role in modulating the activities of HIF and NF-κB in the RPE. Consequences of an impairment of the UPP include accumulation of HIF-1α and diminished NF-κB activation, which lead to enhanced expression and secretion of pro-angiogenic factors and attenuated expression of MCP-1. Taken together, these data predict that the impairment of the UPP could lead to the development of AMD-related phenotypes.
age-related macular degeneration; angiogenesis; signal transduction; retinal pigment epithelium; ubiquitin; proteasome; hypoxia-inducible factor; vascular endothelial growth factor; monocyte chemoattractant protein-1
Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.
PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.
PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.
Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.
angiogenesis; HIF-1; hypoxia; pancreatic cancer; PHD3
Skeletal trauma and impaired skeletal healing is commonly associated with diminished vascularity. Hypoxia inducible factor alpha (HIF-1) is a key transcription factor responsible for activating angiogenic factors during development and tissue repair. Small molecule inhibitors of the prolyl hydroxylase enzyme (PHD), the key enzyme responsible for degrading HIF-1, have been shown to activate HIF-1, and are effective in inducing angiogenesis. Here we examined the effects of several commercially available PHD inhibitors on bone marrow mesenchymal stromal cells (MSCs) in vitro and in a stabilized fracture model in vivo. Three PHD inhibitors [Desferrioxamine (DFO), L-mimosine (L-mim), and Dimethyloxalylglycine (DMOG)] effectively activated a HIF-1 target reporter, induced expression of vascular endothelial growth factor (VEGF) mRNA in vitro, and increased capillary sprouting in a functional angiogenesis assay. DFO and DMOG were applied by direct injection at the fracture site in a stabilized murine femur fracture model. PHD inhibition increased the vascularity at 14 days and increased callus size as assessed by microCT at 28 days. These results suggest that HIF activation is a viable approach to increase vascularity and bone formation following skeletal trauma.
hypoxia inducible factor; fracture healing; vascularity; prolyl hydroxylase inhibitor
Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia inducible factor (HIF)-1α stability. In normoxia, PHD2 hydroxylates proline residues on HIF-1α, rendering it inactive. However in hypoxia, PHD2 is inactive, HIF-1α is stabilized and downstream effectors such as VEGF and FGF-2 are produced to promote angiogenesis. In the present study we utilize RNAi to PHD2 to promote therapeutic angiogenesis in a diabetic wound model, presumably by the stabilization of HIF-1α.
Stented wounds were created on the dorsum of diabetic Lpr db/db mice. Mice were treated with PHD2 siRNA or nonsense siRNA. Wounds were measured photometrically on days 0–28. Wounds were harvested for histology, protein, and RNA analysis.
Diabetic wounds treated with siRNA closed within 21 +/−1.2 days; sham treated closed in 28 +/−1.5 days. By day 7, Western blot revealed near complete suppression of PHD protein and corresponding increased HIF-1α. Angiogenic mediators VEGF and FGF-2 were elevated, corresponding to increased CD31 staining in the treated groups.
siRNA-mediated silencing of PHD2 increases HIF-1α and several mediators of angiogenesis. This corresponded to improved time to closure in diabetic wounds compared to sham treated wounds. These findings suggest that impaired wound healing in diabetes can be ameliorated with therapeutic angiogenesis.
diabetic wound; PHD2; HIF-1
The erythropoietin (EPO) belongs to the family of angiogenic factors, which is regulated by Hypoxia-inducible factor- 1α (HIF-1α). As known, EPO are expressed in human villi and decidua, but the function is not clear. In this study, we investigated the expression and roles of HIF-1α, EPO and its receptor (EPOR) in the biological functions of trophoblast and decidual stromal cell (DSC) in human early pregnancy. The expression of EPO, EPOR and HIF-1α was evaluated in the villi and deciduas by RT-PCR and immunohistochemistry. Thereafter, we silenced HIF-1α expression in HTR-8/SVneo cell line and decidual stromal cells (DSCs). The effects of EPO on the proliferation and apoptosis of trophoblasts and DSCs, and activation of signal molecules were investigated by BrdU proliferation assay, flow cytometry and western blot, respectively. We have observed that the HIF-1α silence results in the lower expression of EPO in trophoblasts and DSCs. The anti-EPO neutralizing antibody can inactivate the phosphorylation of STAT5 and activate p38 of these cells in a dosage-dependent manner. Furthermore, the expressions of EPO, EPOR and HIF-1α in the villi and decidua from the unexplained miscarriage were significantly lower than that of the normal early pregnancy. This study suggests that HIF-1α may regulate the expression of EPO, which plays a favorable regulatory role in the proliferation and survival of human first-trimester trophoblast cells and DSCs via inactivating p38 and activating STAT5 in an autocrine manner, while the inadequate EPO expression at maternal-fetal interface may lead to pregnancy wastage in humans.
EPO; HIF-1; trophoblast cell; decidual stromal cell; STAT5; p38
Studies of adaptive mechanisms to hypoxia led to the discovery of the transcription factor called hypoxia inducible factor (HIF). HIF is a ubiquitously expressed, heterodimeric transcription factor that regulates a cassette of genes that can provide compensation for hypoxia, metabolic compromise, and oxidative stress including erythropoietin, vascular endothelial growth factor, or glycolytic enzymes. Diseases associated with oxygen deprivation and consequent metabolic compromise such as stroke or Alzheimer's disease may result from inadequate engagement of adaptive signaling pathways that culminate in HIF activation. The discovery that HIF stability and activation are governed by a family of dioxygenases called HIF prolyl 4 hydroxylases (PHDs) identified a new target to augment the transcriptional activity of HIF and thus the adaptive machinery that governs neuroprotection. PHDs lose activity when cells are deprived of oxygen, iron or 2-oxoglutarate. Inhibition of PHD activity triggers the cellular homeostatic response to oxygen and glucose deprivation by stabilizing HIF and other proteins. Herein, we discuss the possible role of PHDs in regulation of both HIF-dependent and -independent cell survival pathways in the nervous system with particular attention to the co-substrate requirements for these enzymes. The emergence of neuroprotective therapies that modulate genes capable of combating metabolic compromise is an affirmation of elegant studies done by John Blass and colleagues over the past five decades implicating altered metabolism in neurodegeneration.
Hypoxia inducible factor; Prolyl 4-hydroxylase; Transcriptional regulation; Neuroprotection; Iron chelation
Venous thrombus resolution may be regulated by an angiogenic process that involves the surrounding vein wall. The aims of this study were to determine whether: (i) thrombosis stimulates activation of the angiogenic transcription factor, hypoxia-inducible factor (HIF) 1α, and downstream expression of growth factors in vein wall; and (ii) upregulation of HIF1α in vein wall leads to increased growth factor expression and enhanced thrombus resolution.
Materials and methods
HIF1α, vascular endothelial growth factor (VEGF), and placental growth factor (PLGF) were quantified in mouse inferior vena cava (IVC) at days 1, 3, 7, and 14 after thrombus formation (n = 10-13 per group). An additional group of thrombosed mice were treated with the prolyl-hydroxylase domain (PHD) inhibitor, L-mimosine (L-mim) or vehicle control. HIF1α, VEGF, and PLGF in IVC were measured at days 1 and 7; and vein recanalisation and thrombus resolution were measured at days 7 and 10 (n = 6-7 per group).
HIF1α was expressed in thrombosed IVC and its levels remained relatively constant throughout natural resolution. The levels of VEGF in thrombosed IVC were elevated at days 1 (P < 0.0001) and 3 (P < 0.05); and PLGF at days 1 (P < 0.0001), 3 (P < 0.0001), and 7 (P < 0.0001). Treatment with L-mim led to: increased HIF1α (P < 0.05), VEGF (P < 0.005), and PLGF (P < 0.001) levels in the IVC; decreased thrombus size (P < 0.01); and increased vein recanalisation (P < 0.001).
HIF1α levels in vein wall are not affected by thrombosis and it appears that the angiogenic drive in the vein surrounding resolving thrombus is regulated independently of HIF1α. Stimulating HIF1α levels in the vein wall leads to an increased angiogenic drive and promotes vein recanalisation and thrombus resolution.
HIF1α, hypoxia-inducible factor 1α; VEGF, vascular endothelial growth factor; PLGF, placental growth factor; IVC, inferior vena cava; L-mim, L-mimosine; Hypoxia; Hypoxia-inducible factor; Angiogenesis; Thrombus resolution
Idiopathic erythrocytosis (IE) is a rare condition in which there is an increase in red cell mass and hematocrit. As it is typically driven by elevated or inappropriately normal erythropoietin (Epo) levels, it has the potential to reveal the identities of proteins involved in the oxygen sensing pathway that regulates the transcription factor, Hypoxia Inducible Factor (HIF), and hence Epo production in humans. One example of this is provided by Chuvash polycythemia, a form of erythrocytosis due to a mutation in the von Hippel Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF for degradation. A recent report of familial erythrocytosis now implicates a different protein, Prolyl Hydroxylase Domain protein 2 (PHD2), which is an enzyme that hydroxylates HIF.
HIF; Hypoxia Inducible Factor; PHD2; EGLN1; HPH2; prolyl hydroxylation; idiopathic erythrocytosis; erythropoietin
Idiopathic erythrocytosis (IE) comprises a heterogeneous group of disorders characterized by hyperplasia of the erythroid lineage; however, in many cases, the molecular basis remains undetermined. Serum erythropoietin (EPO) levels can be raised, normal, or reduced, suggesting that there are at least two underlying etiologies involving either the control of EPO production or modulation of EPO-induced signaling. EPO production is regulated by the oxygen-sensing pathway via the hypoxia inducible transcription factor (HIF) complex. Proteasomal turnover of HIF is controlled by interactions with the von Hippel Lindau (VHL) and prolyl hydroxylase domain 2 (PHD2) proteins. Erythrocytosis-associated mutations have been detected in the oxygen sensing pathway indicating that EPO is regulated by the HIF-2alpha-PHD2-VHL axis (reviewed by McMullin ). Aberrant EPO-induced signaling in IE patients with subnormal serum EPO levels can arise from mutations in the EPO receptor (EpoR) gene which result in the receptor being hypersensitive to EPO with prolonged activation of the EPO-dependent signaling pathways (reviewed by Percy ).
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
Activation of hypoxia-inducible factors (HIFs), responsible for tumor angiogenesis and glycolytic switch, is regulated by reduced oxygen availability. Normally, HIF-α proteins are maintained at low levels, controlled by site-specific hydroxylation carried out by HIF prolyl hydroxylases (PHDs), and subsequent proteasomal degradation via the von Hippel-Lindau (VHL) ubiquitin ligase. Using a yeast-two hybrid screen, we identified an interaction between MAGE-11 cancer-testis antigen and the major HIF-α hydroxylating enzyme PHD2. The interaction was confirmed by pull-down assay, co-immunoprecipitation and co-localization in both normoxic and hypoxic conditions. Furthermore, MAGE-9, the closest homolog of MAGE-11, was also found to interact with PHD2. MAGE-11 inhibited PHD activity without affecting protein levels. This inhibition was accompanied by stabilization of ectopic or endogenous HIF-1α protein. Knock-down of MAGE-11 by siRNA results in decreased hypoxic induction of HIF-1α and its target genes. Inhibition of PHD by MAGE-11 and following activation of hypoxia-inducible factors is a novel tumor associated HIF regulatory mechanism. This finding provides new insights into the significance of MAGE expression in tumors and may provide valuable tools for therapeutic intervention because of the restricted expression of the MAGE gene family in cancers but not in normal tissues.
HIF; hypoxia; MAGE-11; PHD2
When humans are exposed to hypoxia, systemic and intracellular changes operate together to minimise hypoxic injury and restore adequate oxygenation. Emerging evidence indicates that the hypoxia-inducible factor (HIF) family of transcription factors plays a central regulatory role in these homeostatic changes at both the systemic and cellular levels. HIF was discovered through its action as the transcriptional activator of erythropoietin, and has subsequently been found to control intracellular hypoxic responses throughout the body. HIF is primarily regulated by specific prolyl hydroxylase-domain enzymes (PHDs) that initiate its degradation via the von Hippel-Lindau tumour suppressor protein (VHL). The oxygen and iron dependency of PHD activity accounts for regulation of the pathway by both cellular oxygen and iron status. Recent studies conducted in patients with rare genetic diseases have begun to uncover the wider importance of the PHD-VHL-HIF axis in systems-level human biology. These studies indicate that, in addition to regulating erythropoiesis, the system plays an important role in cardiopulmonary regulation. This article reviews our current understanding of the importance of HIF in human systems-level physiology, and is modelled around the classic physiological response to high-altitude hypoxia.
erythropoietin; erythropoiesis; polycythaemia; iron; oxygen sensing
The transcription factors hypoxia inducible factor 1 and 2 (HIF-1 and HIF-2) regulate multiple responses to physiological hypoxia such as transcription of the hormone erythropoietin (EPO) to enhance red blood cell proliferation, vascular endothelial growth factor (VEGF) to promote angiogenesis and glycolytic enzymes to increase glycolysis. Recent studies indicate that HIFs also regulate mitochondrial respiration and mitochondrial oxidative stress. Interestingly, mitochondrial metabolism, respiration and oxidative stress also regulate activation of HIFs. In this review we examine the evidence that mitochondria and HIFs are intimately connected to regulate each other resulting in appropriate responses to hypoxia.
mitochondria; HIF; ROS; respiration
Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1α O2-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-1α 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-1α 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 O2 depletion.
Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1α and/or Hif-2α (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel–Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1α. Here we show that conditional inactivation of Hif-2α in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2α but not Hif-1α and that the hypoxic induction of liver Epo in anemic adults was Hif-2α dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1α dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.
The purpose of the present study was to investigate the relationship of expression of hypoxia inducible factor (HIF)-1α-modifying enzymes prolyl hydroxylase (PHD)1, PHD2 and PHD3 to response of tumours and survival in breast cancer patients enrolled in a phase II trial of neoadjuvant anthracycline and tamoxifen therapy.
The expression of PHD1, PHD2 and PHD3 together with HIF-1α and the HIF-inducible genes vascular endothelial cell growth factor (VEGF) and carbonic anhydrase IX were assessed by immunohistochemistry using a tissue microarray approach in 211 patients with T2-4 N0-1 breast cancer enrolled in a randomised trial comparing single-agent epirubicin versus epirubicin and tamoxifen as the primary systemic treatment.
PHD1, PHD2 and PHD3 were detected in 47/179 (26.7%), 85/163 (52.2%) and 69/177 (39%) of tumours at baseline. PHD2 and PHD3 expression was moderate/strong whereas PHD1 expression was generally weak. There was a significant positive correlation between HIF-1α and PHD1 (P = 0.002) and PHD3 (P < 0.05) but not PHD2 (P = 0.41). There was a significant positive relationship between VEGF and PHD1 (P < 0.008) and PHD3 (P = 0.001) but not PHD2 (P = 0.09). PHD1, PHD2 and PHD3 expression was significantly increased after epirubicin therapy (all P < 0.000) with no significant difference in PHD changes between the treatment arms. There was no significant difference in response in tumours that expressed PHDs and PHD expression was not associated with survival.
Although expression of the PHDs was not related to response or survival in patients receiving neoadjuvant epirubicin, our data provide the first evidence that these enzymes are upregulated on therapy in breast cancer and that the biological effects independent of HIF make them therapeutic targets.
Oxygen-dependent regulation of the transcription factor HIF-1α relies on a family of prolyl hydroxylases (PHDs) that hydroxylate hypoxia-inducible factor 1α (HIF-1α) protein at two prolines during normal oxygen conditions, resulting in degradation by the proteasome. During low-oxygen conditions, these prolines are no longer hydroxylated and HIF-1α degradation is blocked. Hypoxia-induced miRNA-210 (miR-210) is a direct transcriptional target of HIF-1α, but its complete role and targets during hypoxia are not well understood. Here, we identify the enzyme glycerol-3-phosphate dehydrogenase 1-like (GPD1L) as a novel regulator of HIF-1α stability and a direct target of miR-210. Expression of miR-210 results in stabilization of HIF-1α due to decreased levels of GPD1L resulting in an increase in HIF-1α target genes. Altering GPD1L levels by overexpression or knockdown results in a decrease or increase in HIF-1α stability, respectively. GPD1L-mediated decreases in HIF-1α stability can be reversed by pharmacological inhibition of the proteasome or PHD activity. When rescued from degradation by proteasome inhibition, elevated amounts of GPD1L cause hyperhydroxylation of HIF-1α, suggesting increases in PHD activity. Importantly, expression of GPD1L attenuates the hypoxic response, preventing complete HIF-1α induction. We propose a model in which hypoxia-induced miR-210 represses GPD1L, contributing to suppression of PHD activity, and increases of HIF-1α protein levels.
Erythropoietin (Epo) and vascular growth factor (VEGF) are known to be involved in the regulation of cellular activity when oxygen transport is reduced as in anaemia or hypoxic conditions. Because it has been suggested that Epo could play a role in skeletal muscle development, regeneration, and angiogenesis, we aimed to assess Epo deficiency in both normoxia and hypoxia by using an Epo-deficient transgenic mouse model (Epo-TAgh). Histoimmunology, ELISA and real time RT-PCR did not show any muscle fiber atrophy or accumulation of active HIF-1α but an improvement of microvessel network and an upregulation of VEGFR2 mRNA in Epo-deficient gastrocnemius compared with Wild-Type one. In hypoxia, both models exhibit an upregulation of VEGF120 and VEGFR2 mRNA but no accumulation of Epo protein. EpoR mRNA is not up-regulated in both Epo-deficient and hypoxic gastrocnemius. These results suggest that muscle deconditioning observed in patients suffering from renal failure is not due to Epo deficiency.
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the α subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the α subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF α subunits. These enzymes hydroxylate specific prolines in HIF α subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1α. Furthermore, and unexpectedly, mutations can be tolerated at the −5, −2, and −1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1α is the hydroxylacceptor proline itself.
A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1α and HIF-2α and their negative regulator von Hippel–Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis.
The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions.
Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3.
We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.
The protein “amplified in osteosarcoma-9” (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.
One of the molecules regulated by the transcription factor, hypoxia inducible factor (HIF), is the hypoxia-responsive hematopoietic factor, erythropoietin (EPO). This may have relevance to the development of renal cell carcinoma (RCC), where mutations of the von Hippel-Lindau (VHL) gene are major risk factors for the development of familial and sporadic RCC. VHL mutations up-regulate and stabilize HIF, which in turn activates many downstream molecules, including EPO, that are known to promote angiogenesis, drug resistance, proliferation and progression of solid tumours. HIFs typically respond to hypoxic cellular environment. While the hypoxic microenvironment plays a critical role in the development and progression of tumours in general, it is of special significance in the case of RCC because of the link between VHL, HIF and EPO. EPO and its receptor, EPOR, are expressed in many cancers, including RCC. This limits the use of recombinant human EPO (rhEPO) to treat anaemia in cancer patients, because the rhEPO may be stimulatory to the cancer. EPO may also stimulate epithelial-mesenchymal transition (EMT) in RCC, and pathological EMT has a key role in cancer progression. In this mini review, we summarize the current knowledge of the role of EPO in RCC. The available data, either for or against the use of EPO in RCC patients, are equivocal and insufficient to draw a definitive conclusion.
Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB–mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF–related pathologies including ageing, ischemia, and cancer.
The mechanisms by which cells and organisms respond to oxygen are of extreme importance for development and also for certain pathologies such as cancer, ageing, and ischemia. These are mediated by a family of transcription factors called hypoxia inducible factor (HIF), a factor that coordinates expression of a great number of genes. Significantly, these processes are evolutionary conserved from worms to humans. It is known that regulation of HIF occurs to a great extent through protein degradation. However, other important mechanisms of HIF control are currently being investigated. In this study, we have uncovered a novel mechanism of HIF regulation that relies on the action of another transcription factor family called NF-κB. We have found that NF-κB controls the levels of HIF-1α and HIF-1β genes by direct regulation. Furthermore, through its control of HIF-1β, NF-κB indirectly controls HIF-2α. Importantly, we find that this mechanism is conserved in Drosophila and mice. These results suggest an alternative avenue for therapeutic intervention in the HIF pathway, which has important implications for many human diseases.