HIF-1α Hydroxylation Research Articles

Piceatannol, a hydroxystilbene natural product, stabilizes HIF-1α protein by inhibiting HIF prolyl hydroxylase

To investigate the mechanisms underlying the biological activity of piceatannol (PCT), a hydroxystilbene natural product that has anti-colitic properties, we examined whether PCT could modulate hypoxia-inducible factor (HIF)-1 activity in human colon carcinoma cells. PCT induced HIF-1α protein, leading to induction of its target gene products, vascular endothelial growth factor and heme oxygenase-1, which are involved in amelioration of colitis. PCT induction of HIF-1α resulted from HIF-1α protein stabilization, which occurred through inhibition of HIF-prolyl hydroxylase-2 (HPH-2). PCT inhibition of HPH-2 was reversed by addition of ascorbate, a cofactor of HPH-2, but not the cosubstrate, 2-ketoglutarate, to the reaction mixture of an in vitro von Hippel–Lindau (VHL) capture assay, and pretreatment with ascorbate abrogated PCT induction of cellular HIF-1α. Moreover, PCT prevented hydroxylation of cellular HIF-1α and attenuated coimmunoprecipitation of Flag-VHL protein and HA–HIF-1α over-expressed in human embryonic kidney 293 cells. Structural analysis using derivatives of PCT revealed that the catechol moiety in PCT was required for the stabilization of HIF-1α protein. Taken together, PCT activation of HIF-1 resulting from inhibition of HPH-2 may be a molecular mechanism for an anti-colitic effect of the natural product.

Nitric Oxide Produced Endogenously Is Responsible for Hypoxia-Induced HIF-1α Stabilization in Colon Carcinoma Cells

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.

AG490 Promotes HIF-1α Accumulation by Inhibiting Its Hydroxylation

AG490 is a tyrphostin originally described as a Janus Activated Kinase (JAK) 2 inhibitor. AG490 also inhibits epidermal growth factor receptor (EGFR) and guanylyl cyclases (GC). More recently, AG490 was associated with oxidative stress protection in experimental acute kidney injury models. We now show that AG490 is also a strong activator of the Hypoxia Inducible Factor (HIF)-1. Under normoxic conditions HIF-1α is degraded through hydroxylation, von Hippel Lindau protein (VHL)-mediated ubiquitin tagging and proteasomal degradation. AG490 increased HIF-1α protein, but not HIF-1α mRNA levels, dose- and time-dependently in cultured endothelial, vascular smooth muscle and kidney proximal tubular epithelial cells. AG490 increased HIF-1α protein half-life, suggesting that HIF-1α protein accumulation resulted from a decreased degradation. In this regard, AG490 prevented HIF-1α hydroxylation and increased HIF-1α protein levels in human renal carcinoma cells expressing VHL, but did not further increase HIF-1α in VHL negative cells. AG490 did not prevent the proteasomal degradation of other proteins. HIF-1α was not upregulated by dominant negative JAK2constructs, tyrphostin AG9, the EGFR inhibitors erbstatin and genistein, the GC inhibitor Ly83583 or cGMP analogues. Finally, AG490 also increased HIF-1α transcriptional activity evidenced by the increased HIF-1α-dependent VEGF expression. In conclusion, AG490 is a novel HIF-1α activator that increases HIF-1α half-life and protein levels through interference with HIF-1α hydroxylation and VHL-mediated degradation. This action may contribute to the cell and tissue protective effects of AG490.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Angiotensin II Differentially Regulates Morg1 Expression in Kidney Cells

Background: The mitogen-activated protein kinase organizer 1 (Morg1) belongs to the WD-40 repeat protein family and is a scaffold molecule for the extracellular regulated kinase signaling pathway. Morg1 also binds to prolyl-hydroxylase 3 (PHD3) and regulates the hypoxia-inducible factor-1α (HIF-1α) expression via PHD3 stabilization. Morg1 has been detected in the kidney as well as in other cell tissues but its expression in renal cells has not been well investigated. It has been widely shown that angiotensin II (ANG II) mediates renal damage. We have previously shown that ANG II downregulates the expression of PHD3 in PC12 cells. The aim of this study was to analyze whether ANG II regulates Morg1 expression in mouse mesangial cells (MMC), mouse proximal tubular cells (MTC) and in differentiated podocytes. The correlation between the expression of Morg1 and PHD3 activity was also addressed. Methods: Effect of ANG II on the Morg1 mRNA expression level was assessed by real-time PCR. Morg1 and HIF-1α cellular localization was analyzed by immunohistochemistry. HIF-1α promoter activity was investigated using a reporter gene system. PHD3 hydroxylase activity test was measured with a hydroxylation-coupled decarboxylation assay. Results: ANG II differentially regulates Morg1 expression in MMC, MTC and differentiated podocytes. We detected a biphasic effect of ANG II on Morg1 mRNA expression which was time dependent. While 9-hour ANG II treatment downregulated Morg1 expression in MMC, it induced Morg1 expression in MTC. Conversely, 24-hour ANG II stimulation upregulated the expression of Morg1 mRNA in MMC, but showed an opposite effect in MTC and differentiated podocytes. In addition, we found that ANG II signals mostly through the AT₁ receptor subtype in MMC and via the AT₂ subtype in MTC. PHD3 activity correlated to Morg1 expression patterns. Our data also demonstrate that HIF-1α transcriptional activity in MTC contrasted to PHD3 activity at 9 and 24 h, whereas in the MMC and in podocytes we did not find any correlation between PHD3 HIF-1α hydroxylation ability and HIF-1α transcriptional activation, suggesting a different mechanism of regulation in these cell types. Interestingly, the reduced expression of Morg1 in mesangial cells isolated from Morg1 (+/–) heterozygous mice correlated with a reduced PHD3 enzymatic activity and an increased HIF-1α transcriptional activity compared with mesangial cells originated from wild-type (Morg1 +/+) mice. Conclusions: We show for the first time in various renal cells that ANG II modulates Morg1 expression and HIF-1α transcriptional activity via cell type-specific mechanisms, demonstrating a novel mechanism by which ANG II may contribute to renal disease.

Factor Inhibiting HIF (FIH) Recognizes Distinct Molecular Features within Hypoxia-inducible Factor-α (HIF-α) versus Ankyrin Repeat Substrates

Factor Inhibiting HIF (FIH) catalyzes the β-hydroxylation of asparagine residues in HIF-α transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-α is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.

Evidence for inhibition of HIF-1α prolyl hydroxylase 3 activity by four biologically active tetraazamacrocycles

Hypoxia inducible factor 1 (HIF-1) is central to the hypoxic response in mammals. HIF-1α prolyl hydroxylase 3 (PHD3) degrades HIF through the hydroxylation of HIF-1α. Inhibition of PHD3 activity is crucial for up-regulating HIF-1α levels, thereby acting as HIF-dependent diseases therapy. Macrocyclic polyamines which display high stability on iron-chelating may well inhibit the enzyme activity. Thus inhibition and interaction on catalytic PHD3 by four biologically active tetraazamacrocycles (1-4), which have two types of parent rings to chelate iron(ii) dissimilarly, were studied. The apparent IC(50) values of 2.56, 1.91, 5.29 and 2.44 μM, respectively, showed good inhibition potency of the four compounds. K(I) values were 7.86, 3.69, 1.59 and 2.92 μM for 1-4, respectively. Different inhibition actions of the two groups of compounds were identified. Circular dichroism (CD) and fluorescence spectrometries proved that one type of compound has significant effects on protein conformation while another type does not. Computational methodology was constructed to employ the equilibrium geometry of enzyme active site with the presence of substrate competitive inhibitor. Iron(ii) coordination in the active site by inhibitors of this kind induces conformational change of the enzyme and blocks substrate binding.

Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-α subunits and a HIF-β subunit. Under normoxic conditions the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1α stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1α occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1α and shows less prolyl hydroxylase activity for its target HIF-1α than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1α predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.

Abstract P272: Physiological and Pathological Functions of Nox2 and Nox4 in Ischemia/Reperfusion Injury

NADPH oxidases (Noxes) represent an enzyme system whose primary function is to produce reactive oxygen species (ROS). Both Nox2 and Nox4 play an important role in regulating oxidative stress and growth/death of cardiomyocytes (CMs). However, the role of Nox2 and Nox4 during ischemia/reperfusion (I/R) is poorly understood. In order to elucidate the function of the Nox isoforms, wild type (WT), Nox2 KO (Nox2-/-), and cardiac specific Nox4 KO (cNox4-/-) mice were subjected to 30 minutes ischemia followed by 24 hours reperfusion. Myocardial infarction size/area at risk (MI/AAR) as evaluated by TTC staining was significantly smaller in Nox2-/- and cNox4 -/- than in WT mice (32 ± 3.1% and 28.0 ± 6.1% vs. 40.3 ± 8.3%, p < 0.05). O 2 - production in Nox2 -/- and cNox4 -/- hearts was significantly lower than in WT hearts, as evaluated with the lucigenin-chemiluminescence assay (433 ± 52, 580 ± 106 vs. 1250 ± 236 RLU, p < 0.05). The I/R experiment was also conducted with mice in which both Nox2 and Nox4 are deleted (double KO mice). O 2 - production in the double KO heart was significantly lower than in the single KO ones (257 ± 42 RLU, p < 0.05). The MI/AAR in double KO mice was, however, significantly greater than that in WT mice (58.0 ± 6.3% vs. 40.3 ± 8.3%, p < 0.05). These results raised the possibility that marked suppression of ROS by combined downregulation of Nox2 and Nox4 exacerbates I/R injury. To elucidate the underlying mechanism, we examined expression of hypoxia inducible factor-1alpha (HIF-1 α ), using DN-Nox transgenic (Tg) mice, which we have shown also exhibited marked suppression of O 2 - and significantly greater MI/AAR after I/R. HIF-1 α is lower in DN-Nox Tg mice than in WT mice in both the ischemic and non-ischemic areas. A genetic cross between DN-Nox Tg and mice lacking prolyl hydroxylase 2, an enzyme mediating hydroxylation and downregulation of HIF-1 α , partially rescued I/R injury in DN-Nox mice (35.8 ± 5.9% vs 65.2 ± 10.3%, p<0.05). Taken together, these data show that both Nox2 and Nox4 mediate increases in oxidative stress and myocardial injury in response to I/R. However, combined downregulation of Nox2 and Nox4 induces marked downregulation of ROS, which in turn exacerbates I/R injury possibly through downregulation of HIF-1α and consequent impairment of hypoxic adaptation.

Inactivation of prolyl hydroxylase domain (PHD) protein by epigallocatechin (EGCG) stabilizes hypoxia-inducible factor (HIF-1α) and induces hepcidin (Hamp) in rat kidney

HIF-1α plays a key role in iron uptake and transport in the liver, whose activity is tightly linked to the repression of hepcidin (Hamp). Hamp prevents intestinal iron uptake and cellular efflux by negatively modulating ferroportin. Hamp is also expressed in the kidneys, where transcriptional control by HIF-1α remains poorly understood. We show that the administration of epigallocatechin gallate (EGCG) results in a considerable Hamp expression in rat kidneys. We also provide evidence to show that EGCG inhibited prolyl hydroxylase (PHD) activity, essential for HIF-1α degradation in vivo and in vitro. Rats that were dosed with EGCG (60mg/kg, intraperitoneal) over a 7day time course stabilized HIF-1α protein in kidney tissues. Interestingly, Hamp gene expression was induced, even after subjecting rats to a 4h hypoxia treatment (8% oxygen). Using Hep3B cells, we determined that EGCG conferred its inhibitory action by complexing with PHD, altering its catalytic iron center and thus preventing HIF-1α hydroxylation. These data demonstrate EGCG’s therapeutic potential in modulating hepcidin expression in diseases associated with altered iron metabolism.

Activation of the HIF Prolyl Hydroxylase by the Iron Chaperones PCBP1 and PCBP2

Mammalian cells express dozens of iron-containing proteins, yet little is known about the mechanism of metal ligand incorporation. Human poly (rC) binding protein 1 (PCBP1) is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. We have identified the iron-dependent prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) that modify hypoxia-inducible factor α (HIFα) as targets of PCBP1. Depletion of PCBP1 or PCBP2 in cells led to loss of PHD activity, manifested by reduced prolyl hydroxylation of HIF1α, impaired degradation of HIF1α through the VHL/proteasome pathway, and accumulation of active HIF1 transcription factor. PHD activity was restored invitro by addition of excess Fe(II), or purified Fe-PCBP1, and PCBP1 bound to PHD2 and FIH1 invivo. These data indicated that PCBP1 was required for iron incorporation into PHD and suggest a broad role for PCBP1 and 2 in delivering iron to cytosolic nonheme iron enzymes.

HIF-1α in Epidermis: Oxygen Sensing, Cutaneous Angiogenesis, Cancer, and Non-Cancer Disorders

Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway.

Activation of hypoxia-inducible factor-1α (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model

AbstractThe oxygen-sensing transcription factor hypoxia-inducible factor-1α (HIF-1α) plays a critical role in the regulation of myeloid cell function. The mechanisms of regulation are not well understood, nor are the phenotypic consequences of HIF modulation in the context of neutrophilic inflammation. Species conservation across higher metazoans enables the use of the genetically tractable and transparent zebrafish (Danio rerio) embryo to study in vivo resolution of the inflammatory response. Using both a pharmacologic approach known to lead to stabilization of HIF-1α, and selective genetic manipulation of zebrafish HIF-1α homologs, we sought to determine the roles of HIF-1α in inflammation resolution. Both approaches reveal that activated Hif-1α delays resolution of inflammation after tail transection in zebrafish larvae. This delay can be replicated by neutrophil-specific Hif activation and is a consequence of both reduced neutrophil apoptosis and increased retention of neutrophils at the site of tissue injury. Hif-activated neutrophils continue to patrol the injury site during the resolution phase, when neutrophils would normally migrate away. Site-directed mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1α by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1α regulates neutrophil function in complex ways during inflammation resolution in vivo.

Abstract 3512: Regulation of hypoxia inducible factor alpha in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (CCRC) is the most common kidney cancer with high incidence of hypoxia-inducible factor 1 and 2 alpha (HIF-α) expression as a consequence of the nonfunctional Von Hippel-Lindau (VHL). The purpose of the study is: a) to investigate the expression levels and relationship between HIF-α and prolyl hydroxylases (PHDs), the negative regulators of HIF-α in tissue microarray (TMA) of human surgical specimens. b) to demonstrate whether methylselenenic acid (MSA) would degrade HIF-α, independent of VHL. c) to determine whether inhibition of HIF-α would enhance cytotoxicity of docetaxel.The results of immunohistochemical evaluation revealed that 92% (81 out of 88) combined incidence of both HIF-1α and HIF-2α with low levels of PHD2 (35%, 31 out of 88) and completely deficient with PHD3, the enzymes responsible for the hydroxylation of HIF-α and subsequent degradation in proteasome. In contrast, we found high incidence of PHD2 positive cases with low HIF-α in both head and neck (86% PHD2 with 38% HIF-α, n=210) and colorectal (90% PHD2 with 26% HIF-α, n=64) indicate high incidence of PHD2 is associated with low incidence of HIF-α. The expression of PHD3 was found in both head & neck (21%) and colorectal (50%) cancer specimens which have low HIF-α incidence compared to CCRC. These data indicate that there is an inverse relationship between HIF-α and PHD2 in CCRC clinical samples in addition to VHL deficiency. In agreement with the surgical specimens, CCRC cells RC2 and 786- O express PHD2 protein but not PHD3 with constitutive expression of HIF-1α in RC2 and HIF-2α in 786 O. We have demonstrated the degradation of both HIF-1α and HIF-2α by MSA that leads to the significant reduction of HIF-α transcriptionally regulated gene VEGF in RC2 and 786-O. MSA exhibits unique property of degrading both HIF-1α and HIF-2α that has not been reported earlier by any other agent. Proteasome inhibitor MG132 reversed the HIF-1α degradation by MSA indicates HIF-1α degradation is proteasome dependent and VHL independent since VHL is nonfunctional in CCRC. Based on these findings we hypothesize that the degradation of HIF-α by MSA is VHL independent. We found RC2 cells with constitutive HIF-1α are 5 fold resistant to docetaxel compared to RC2-VHL cells with no detectable HIF-1α due to overexpression of VHL. As we expected, the combination of MSA enhanced the docetaxel efficacy only in RC2 cells, but not in RC2-VHL shows that the effect of MSA is through HIF-1α. These findings demonstrated the therapeutic potential of selenium for the treatment of CCRC. Thus, the unique molecular properties of CCRC being VHL deficient, express high constitutive HIF-1α and HIF-2α protein with low PHD2 and deficient in PHD3 could provide the rational for the development and evaluation of selenium in combination with the clinically approved drugs for kidney cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2011-3512

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe + 4 ) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl 2). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.

Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1α)-dependent Mechanism

Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O(2)) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (∼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at -78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O₂ signaling in dictyostelium.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.

Abnormalities in Oxygen Sensing Define Early and Late Onset Preeclampsia as Distinct Pathologies

BackgroundThe pathogenesis of preeclampsia, a serious pregnancy disorder, is still elusive and its treatment empirical. Hypoxia Inducible Factor-1 (HIF-1) is crucial for placental development and early detection of aberrant regulatory mechanisms of HIF-1 could impact on the diagnosis and management of preeclampsia. HIF-1α stability is controlled by O2-sensing enzymes including prolyl hydroxylases (PHDs), Factor Inhibiting HIF (FIH), and E3 ligases Seven In Absentia Homologues (SIAHs). Here we investigated early- (E-PE) and late-onset (L-PE) human preeclamptic placentae and their ability to sense changes in oxygen tension occurring during normal placental development.Methods and FindingsExpression of PHD2, FIH and SIAHs were significantly down-regulated in E-PE compared to control and L-PE placentae, while HIF-1α levels were increased. PHD3 expression was increased due to decreased FIH levels as demonstrated by siRNA FIH knockdown experiments in trophoblastic JEG-3 cells. E-PE tissues had markedly diminished HIF-1α hydroxylation at proline residues 402 and 564 as assessed with monoclonal antibodies raised against hydroxylated HIF-1α P402 or P564, suggesting regulation by PHD2 and not PHD3. Culturing villous explants under varying oxygen tensions revealed that E-PE, but not L-PE, placentae were unable to regulate HIF-1α levels because PHD2, FIH and SIAHs did not sense a hypoxic environment.ConclusionDisruption of oxygen sensing in E-PE vs. L-PE and control placentae is the first molecular evidence of the existence of two distinct preeclamptic diseases and the unique molecular O2-sensing signature of E-PE placentae may be of diagnostic value when assessing high risk pregnancies and their severity.

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.