Regulation Of Hypoxia-inducible Factor Research Articles

Human Placental Hypoxia-Inducible Factor-1α Expression Correlates with Clinical Outcomes in Chronic Hypoxia in Vivo

Placental hypoxia is causally implicated in fetal growth restriction and preeclampsia, with both occurring more frequently at high altitude (>2700 m; HA). The nuclear transcription factor hypoxia-inducible factor (HIF) may facilitate placental oxygen transport at HA by increasing erythropoiesis and placental angiogenesis. We therefore investigated HIF expression and its regulatory mechanisms in placentas from normal pregnancies at high (3100 m), moderate (1600 m), and sea level (75 m) altitudes. Moderate-altitude and sea level placentas did not differ, but HIF-1alpha and the von Hippel-Lindau tumor suppressor protein were overexpressed in HA placentas. The ability of von Hippel-Lindau tumor suppressor protein to form the E3 ubiquitin protein ligase complex, required for HIF-1alpha degradation, was unaltered in HA placentas. mRNA for factor-inhibiting HIF, a negative modulator of HIF-1alpha transactivation, was increased, but protein levels were diminished. Elevated HIF-1alpha likely contributed to the significant increase we report in HIF-1alpha downstream target proteins, transforming growth factor beta3 in the placenta, and vascular endothelial growth factor and erythropoietin in the maternal circulation. These circulating markers and lowered birth to placental weight ratios correlated with increased HIF-1alpha, thereby linking molecular and systemic physiological data. The HA response to chronic hypoxia resembles preeclampsia in several aspects, illustrating the utility of the HA model in understanding placental pathologies.

Postpneumonectomy lung expansion elicits hypoxia-inducible factor-1α signaling

We (42) previously reported differential regulation of hypoxia-inducible factors (HIF-1alpha, -2alpha, and -3alpha) mRNA in canine lungs during normal maturation and postpneumonectomy (PNX) compensatory growth in the absence of overt hypoxia. To test the hypothesis that lung expansion activates HIF signaling, we replaced the right lung of six adult foxhounds with inflated custom-shaped silicone prosthesis to keep the mediastinum in the midline and minimize lateral expansion of the remaining lung. After 3 wk of recovery and stabilization of perfusion, the prosthesis was acutely deflated in three animals, causing the remaining lung to expand by 114%. In three other animals, the prosthesis remained inflated. Three days following deflation, we observed significant elevation in the mRNA and nuclear protein levels of HIF-1alpha ( approximately 60%) as well as activation of its transcriptional regulator, the serine/threonine protein kinase B (phospho-Akt-to-total Akt ratio, 124%), and the mRNA and protein levels of its downstream targets, erythropoietin receptor (71-183%) as well as VEGF (33-58%) compared with the pre-PNX control lung from the same animal. The mRNA of HIF-2alpha, HIF-3alpha, and VEGF receptors did not change with acute deflation. We conclude that in vivo lung expansion by post-PNX deflation of space-occupying prosthesis elicits coordinated activation of HIF-1alpha signaling in adult lungs. This pathway could play an important role in mediating lung growth and remodeling during maturation and post-PNX compensation.

A novel approach of proteomics and transcriptomics to study the mechanism of action of the antioxidant–iron chelator green tea polyphenol (-)-epigallocatechin-3-gallate

Previous findings suggest that the antioxidant–iron chelator green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) may have a neurorescue impact in aging and neurodegenerative diseases to retard or even reverse the accelerated rate of neuronal degeneration. The present study sought a deeper elucidation of the molecular neurorescue activity of EGCG in a progressive neurotoxic model of long-term serum deprivation of human SH-SY5Y neuroblastoma cells. In this model, proteomic analysis revealed that EGCG (0.1–1 μM) affected the expression levels of diverse proteins, including proteins related to cytoskeletal components, metabolism, heat shock, and binding. EGCG induced the levels of cytoskeletal proteins, such as beta tubulin IV and tropomyosin 3, playing a role in facilitating cell assembly. In accordance, EGCG increased the levels of the binding protein 14-3-3 gamma, involved in cytoskeletal regulation and signal transduction pathways in neurons. Additionally, EGCG decreased protein levels and mRNA expression of the beta subunit of the enzyme prolyl 4-hydroxylase, which belongs to a family of iron–oxygen sensors of hypoxia-inducible factor (HIF) prolyl hydroxylases that negatively regulate the stability and degradation of several proteins involved in cell survival and differentiation. Accordingly, EGCG decreased protein levels of two molecular chaperones that were associated with HIF regulation, the immunoglobulin-heavy-chain binding protein and the heat shock protein 90 beta. Thus, the present study sheds some light on the antioxidative–iron chelating activities of EGCG underlying its neuroprotective/neurorescue mechanism of action, further suggesting a potential neurodegenerative-modifying effect for EGCG.

Interaction of Hydroxylated Collagen IV with the von Hippel-Lindau Tumor Suppressor

The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated alpha-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its beta-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with alpha-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions.

Cisplatin and doxorubicin repress Vascular Endothelial Growth Factor expression and differentially down-regulate Hypoxia-inducible Factor I activity in human ovarian cancer cells

Vascular Endothelial Growth Factor (VEGF ) and its transcriptional regulator Hypoxia-inducible Factor 1 (HIF-1) play an important role in the process of angiogenesis in many types of cancer, including ovarian cancer. We have examined whether the DNA-damaging drugs cisplatin and doxorubicin and the microtubule inhibitors docetaxel and paclitaxel can affect VEGF expression and HIF-1 activity in three human ovarian cancer cell lines. We demonstrate that cisplatin and doxorubicin abolish hypoxia-induced VEGF mRNA expression in all cell lines, while basal VEGF mRNA expression was also downregulated. Transient transfection with a HIF-1-responsive luciferase construct indicated that cisplatin and doxorubicin inhibited hypoxic activation of HIF-1. Cisplatin repressed HIF-1α protein expression in all cell lines. Stimulation of HIF-1α protein degradation by cisplatin was observed in the only cell line expressing wild-type p53. Cisplatin also inhibited the synthesis of HIF-1α protein for which p53 was dispensable. Interestingly, cisplatin strongly reduced the protein levels of the HIF-1 coactivators p300 and CREB-binding protein (CBP) under hypoxia in all cell lines. Although doxorubicin inhibited hypoxic activation of HIF-1, this drug had no significant effect on the expression levels of HIF-1α and hypoxic expression of p300 and CBP was only weakly reduced. Docetaxel and paclitaxel did neither influence VEGF expression nor hypoxia-induced HIF-1 activity. In total, our findings indicate that cisplatin and doxorubicin can repress hypoxic induction of VEGF expression by inhibiting HIF-1 through different mechanisms. This knowledge may be useful for future treatment schedules including agents that target the HIF-1 signalling pathway.

Regulation of hypoxia-inducible factors in human macrophages—involvement of the natural antisense of Hif-1α (AIHF)

An important aspect of the arteriosclerotic process is the migration of macrophages into hypoxic areas within the plaque. The involved hypoxia-triggered secretion of growth factors and cytokines causes inflammation and vascularization. Essential for this activation are the active α-subunits of the hypoxia-inducible factor (Hif-1α,-2α,-3α). But, little is known about the specific expression and regulation of the α-subunits in macrophages.

Selective inhibition of MEK1/2 reveals a differential requirement for ERK1/2 signalling in the regulation of HIF-1 in response to hypoxia and IGF-1

The transcription factor hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tumour growth and progression, and HIF-1 is regulated through a number of signalling pathways. Here, we investigated the involvement of the mitogen-activated protein kinase (MAPK) signalling pathway in HIF-1 regulation. We found that overexpression of wild-type (WT) extracellular signal regulated protein kinase 1 (ERK1) greatly potentiated HIF-1 activation in hypoxia and HIF-1alpha induced in response to insulin growth-like factor 1 (IGF-1). Conversely, treatment of tumour cells with the MEK1/2 inhibitors PD98059 or U0216, or expression of a dominant-negative form of ERK1 blocked HIF-1 activation in hypoxia without affecting HIF-1alpha induction, localization or binding of HIF-1beta. Interestingly however, the highly selective MEK1/2 inhibitor PD184352 did not inhibit HIF-1 activity or vascular endothelial growth factor (VEGF) induced in response to hypoxia but blocked HIF-1alpha protein and HIF-1 activity induced by IGF-1 stimulation without affecting HIF-1alpha mRNA levels. Finally, we found that ERK5 phosphorylation status was not significantly affected by hypoxia in the presence or absence of PD184352. Taken together, our data suggest that although ERK1/2 signalling is important for HIF-1alpha induction and HIF-1 activity in response to IGF-1, it is dispensable for the induction of HIF-1alpha and activation of HIF-1 in response to hypoxia.

Effect of Graft Preservation and Acute Rejection on Hypoxia-Inducible Factor-1 in Rat Cardiac Allografts

Hypoxia plays an integral part in cardiac transplantation as prolonged graft preservation is an individual risk factor for the development of cardiac allograft vasculopathy (CAV). In this study we characterized the role of hypoxia-inducible factor-1 (HIF-1) during prolonged graft preservation, ischemia-reperfusion (I/R), acute rejection, and chronic rejection. Heart transplantations were performed from Dark Agouti (DA) to Wister-Furth (allo) or DA to DA (syn) rats, without immunosuppression (acute rejection model, harvested at day 5) or with cyclosporine (chronic rejection model, harvested at day 60). To study the effect of preservation on HIF-1 regulation, normal DA hearts were subjected to different cold ischemia times with or without 45 minutes of additional warm ischemia. The role of I/R was studied by harvesting syngrafts at different time points after reperfusion. Real-time reverse-transcriptase polymerase chain reaction quantified total HIF-1 mRNA, while enzyme-linked immunosorbent assay and immunohistochemistry quantified and localized HIF-1 protein. Our results show that HIF-1 nuclear immunoreactivity is increased during graft preservation and I/R leads to loss of nuclear HIF-1 immunoreactivity. Acute rejection induced HIF-1 in mRNA level. Our findings thus indicated that HIF-1 is activated during transplantation and suggested that manipulation of the HIF-1 pathway might reveal new therapeutic options to manage CAV.

Cell-specific Regulation of Hypoxia-inducible Factor (HIF)-1α and HIF-2α Stabilization and Transactivation in a Graded Oxygen Environment

The hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are closely related, key transcriptional regulators of the hypoxic response, countering a low oxygen situation with the up-regulation of target genes associated with numerous processes, including vascularization and glycolysis. This involves a dual mechanism of control through both stabilization and transactivation, regulated via prolyl and asparaginyl hydroxylation. Despite high similarity with respect to protein sequence and activation pathway, a growing number of physiological and mechanistic differences between HIF-1alpha and HIF-2alpha are being reported. To further characterize this nonredundancy, the stabilization of endogenous proteins and regulation of the transactivation domains were compared in a graded oxygen environment across a series of cell lines. Although generally similar results were found, interesting and specific differences between the HIF-alpha proteins were observed within certain cell lines, such as rat adrenal PC12s, emphasizing the cell-specific nature of HIF-alpha regulation. We characterize a conserved amino acid substitution between HIF-1alpha and HIF-2alpha that contributes to the intrinsically higher FIH-1-mediated asparaginyl hydroxylation of HIF-1alpha and, hence, lower HIF-1alpha activity. In addition, our data demonstrate that the different cell lines can be classified into two distinct groups: those in which stabilization and transactivation proceed in conjunction (HeLa, 293T, and COS-1) and those cells in which HIF-alpha is stabilized prior to transactivation (PC12, HepG2, and CACO2). Interestingly, the initial stabilization of HIF-alpha prior to transactivation up-regulation predicted from in vitro derived hydroxylation data is only true for a subset of cells.

Cytokines and Hormones in the Regulation of Hypoxia Inducible Factor-1α (HIF-1α)

Hypoxia inducible factor-1 (HIF-1) is a central component of the oxygen sensing system that coordinates cellular responses to conditions of decreased oxygen availability. The hypoxia inducible transcription factor HIF-1 is a heterodimer composed of the helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1alpha and the aryl hydrocarbon nuclear translocator (ARNT) also known as HIF-1beta. Transactivation of HIF-1 transmits a hypoxic signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, an altered energy metabolism, as well as cell survival decisions by regulating a staidly growing number of target genes. Among recent advances are the discoveries that cytokines and growth factors make use of the 'hypoxic signaling system' under normoxia. Here we summarize current knowledge and existing concepts that help to understand how cytokines and hormones affect protein accumulation of HIF-1alpha and discuss potential implications of activating HIF-1 under normoxia. Considering the fundamental role of cytokines during inflammation may predict a role of HIF-1alpha in coordinating cellular responses to pathogens and point to the connection of cancer and inflammation. Moreover, we will address potential feed-back mechanisms showing an impact of HIF-1 on cytokine production. These considerations suggest an intimate signaling cross-talk between cytokines and the HIF-1 system.

HIF-1 regulates hypoxic induction of NHE1 expression and alkalinization of intracellular pH in pulmonary arterial myocytes

Vascular remodeling resulting from altered pulmonary arterial smooth muscle cell (PASMC) growth is a contributing factor to the pathogenesis of hypoxic pulmonary hypertension. PASMC growth requires an alkaline shift in intracellular pH (pH(i)) and we previously showed that PASMCs isolated from mice exposed to chronic hypoxia exhibited increased Na(+)/H(+) exchanger (NHE) expression and activity, which resulted in increased pH(i). However, the mechanism by which hypoxia caused these changes was unknown. In this study we tested the hypothesis that hypoxia-induced changes in PASMC pH homeostasis are mediated by the transcriptional regulator hypoxia-inducible factor 1 (HIF-1). Consistent with previous results, increased NHE isoform 1 (NHE1) mRNA and protein, enhanced NHE activity, and an alkaline shift in pH(i) were observed in PASMCs isolated from wild-type mice exposed to chronic hypoxia (3 wk at 10% O(2)). In contrast, these changes were absent in PASMCs isolated from chronically hypoxic mice with partial deficiency for HIF-1. Exposure of PASMCs to hypoxia ex vivo (48 h at 4% O(2)) or overexpression of HIF-1 in the absence of hypoxia also increased NHE1 mRNA and protein expression. Our results indicate that full expression of HIF-1 is essential for hypoxic induction of NHE1 expression and changes in PASMC pH homeostasis and suggest a novel mechanism by which HIF-1 mediates pulmonary vascular remodeling during the pathogenesis of hypoxic pulmonary hypertension.

The Novel WD-repeat Protein Morg1 Acts as a Molecular Scaffold for Hypoxia-inducible Factor Prolyl Hydroxylase 3 (PHD3)

Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive alpha- and a beta-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the alpha-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.

Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1)

The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, and effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors.

Involvement of HIF-1 in Invasion of Mum2B Uveal Melanoma Cells

The propensity of uveal melanoma cells for invasion and metastasis is critical factor for the clinical outcome of this form of cancer, and the essential biology of its aggressiveness is not completely understood. In the present study we investigated the involvement of hypoxia-inducible factor 1 (HIF-1) in uveal melanoma migration, invasion and adhesion, the hallmarks of aggressive behavior of cancer cells. We demonstrate that exposure to hypoxia increased migration, invasion and adhesion of uveal melanoma cells in in vitro assays. The "silencing" of HIF-1alpha, the oxygen-regulated subunit of HIF-1, using RNA interference technology resulted in a marked decrease of the uveal melanoma cell migration, invasion and adhesion. GeneChip microarray analysis revealed that a number of genes which regulate cancer invasion and metabolism such as CXCR4, angiopoietin-related protein, pyruvate dehydrogenase kinase 1 (PDK1) are also activated by hypoxia in a HIF-1-dependent manner in Mum2B uveal melanoma cells. We further demonstrate that serum deprivation resulted in HIF-1 and CXCR4 activation, suggesting specific metabolic regulation of HIF-1 in these cells. Microarray analysis of serum-deprived cells identified among the upregulated genes a number of cancer invasion-related genes, some of them being known HIF-1-regulated targets. Taken together, these results suggest that the involvement of HIF-1 in uveal melanoma tumorigenesis is significant and complex, and that metabolic regulation of HIF-1 activation in Mum2B uveal melanoma cells has its specificities.

Xanthine Oxidase–Dependent Regulation of Hypoxia-Inducible Factor in Cancer Cells

During chemical hypoxia induced by cobalt chloride (CoCl2), hypoxia-inducible factor 1alpha (HIF1-alpha) mediates the induction of a variety of genes including erythropoietin and vascular endothelial growth factor. We used glioma cells with oxidative phosphorylation-dependent (D54-MG) and glycolytic-dependent (U251-MG) phenotypes to monitor HIF1-alpha regulation in association with redox responsiveness to CoCl2 treatment. We showed that CoCl2 increased xanthine oxidase (XO)-derived reactive oxygen species (ROS), which causes accumulation of HIF1-alpha protein in U251-MG cells. Under these conditions, blockade of XO activity by pharmacologic (N-acetyl-L-cysteine or allopurinol) or molecular (by small interfering RNA) approaches significantly attenuated HIF1-alpha expression. Exogenous H2O2 stabilizes HIF1-alpha protein. XO was present in these cells and was the primary source of free radicals. We also showed higher XO activity in cells exposed to CoCl2 compared with cells grown in normoxia. From the experiments shown here, we concluded that ROS were indeed generated in D54-MG cells exposed to CoCl2 but it was unlikely that ROS participated in the hypoxic signal transduction pathways in this cell type. Possibly, cell type-dependent and stimulus-dependent factors may control ROS dependency or redox sensitivity of HIF1-alpha and thus HIF1-alpha activation either directly or by induction of specific signaling cascades. Our findings reveal that XO-derived ROS is a novel and critical component of HIF1-alpha regulation in U251-MG cells, pointing toward a more general role of this transcription factor in tumor progression.

Von Hippel-Lindau Tumor Suppressor Protein Regulates the Assembly of Intercellular Junctions in Renal Cancer Cells through Hypoxia-Inducible Factor–Independent Mechanisms

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.

Clusterin Is a Secreted Marker for a Hypoxia-Inducible Factor-Independent Function of the von Hippel-Lindau Tumor Suppressor Protein

Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose people to renal cancer, hemangioblastomas, and pheochromocytomas in an allele-specific manner. The best documented function of the VHL gene product (pVHL) relates to its ability to polyubiquitinate, and hence target for destruction, the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF). pVHL mutants linked to familial pheochromocyctoma (type 2C VHL disease), in contrast to classical VHL disease, appear to be normal with respect to HIF regulation. Using a simple method for identifying proteins that are differentially secreted by isogenic cell line pairs, we confirmed that the HIF targets IGBP3 and PAI-1 are overproduced by pVHL-defective renal carcinoma cells. In addition, cells lacking wild-type pVHL, including cells producing type 2C pVHL mutants, were defective with respect to expression and secretion of clusterin, which does not behave like a HIF target. Decreased clusterin secretion by pVHL-defective tumors was confirmed in vivo by immunohistochemistry. Therefore, clusterin is a secreted marker for a HIF-independent pVHL function that might be especially important in pheochromocytoma development.

The Tumor Suppressor von Hippel-Lindau Gene Product and Metastasis: New Thoughts on an Old Molecule

In this issue of The American Journal of Pathology, long-time von Hippel-Lindau (VHL) pioneer William G. Kaelin with Nakamura and colleagues1 demonstrates that cells lacking wild-type VHL are defective in the expression and secretion of the glycoprotein clusterin. This phenomenon was observed in cells capable of ubiquitylating hypoxia-inducible factor (HIF) and possessing Type 2C VHL mutants. The authors also found that pVHL-defective renal carcinoma cells overexpress insulin-like growth factor binding protein-3 (IGBP3) and plasminogen activator inhibitor type-1 in a HIF-dependent manner. Previous reports have described pVHL modulating other genes in a HIF-independent manner to promote its tumor suppressor function; however, the overexpression of clusterin by pVHL raises several questions and requires further discussion.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the alpha-subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel-Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Hypoxia-inducible factors: where, when and why?

Hypoxia-inducible factor (HIF) is a family of transcription factors that regulate the homeostatic response to oxygen deprivation during development, physiological adaptation, and pathological processes such as ischemia and neoplasia. Our understanding of the function of different HIF isoforms is being advanced by understanding the processes that regulate their activity, learning where and when they are expressed and what genes they regulate.