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PubMed2002

Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation.

Hypoxia-inducible factor 1 (HIF-1) plays a central role in cellular adaptation to changes in oxygen availability. Recently, prolyl hydroxylation was identified as a key regulatory event that targets the HIF-1alpha subunit for proteasomal degradation via the pVHL ubiquitination complex. In this report, we reveal an important function for ARD1 in mammalian cells as a protein acetyltransferase by direct binding to HIF-1alpha to regulate its stability. We present further evidence showing that ARD1-mediated acetylation enhances interaction of HIF-1alpha with pVHL and HIF-1alpha ubiquitination, suggesting that the acetylation of HIF-1alpha by ARD1 is critical to proteasomal degradation. Therefore, we have concluded that the role of ARD1 in the acetylation of HIF-1alpha provides a key regulatory mechanism underlying HIF-1alpha stability.

Acetylation↗
PubMed2002

HIF-1A, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors.

PURPOSE: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. METHODS: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, pimonidazole, IdUrd, and HIF-1alpha. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1alpha and pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. RESULTS: Measurement of all four parameters (diffusion-limited fraction, pimonidazole fraction, HIF-1alpha fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of pimonidazole and HIF-1alpha was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1alpha than pimonidazole. There was no significant correlation between the pimonidazole and HIF-1alpha fractions in the 10 tumors studied. CONCLUSIONS: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1alpha and pimonidazole indicate that HIF-1alpha might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.

Carcinoma, Squamous Cell↗
PubMed1999

mRNA cloning and expression studies of the quail homologue of HIF-2alpha.

Hypoxia inducible factors (HIF) are candidate transcriptional regulators of vascular development. Unlike HIF-1alpha - the founding member of the HIF family - which is expressed more or less ubiquitously, HIF-2alpha (also called HRF, HLF and EPAS1) is highly expressed by vascular endothelial cells and was shown to activate the transcription of endothelial cell-specific receptor tyrosine kinases (tie-2 and flk-1/VEGF receptor 2) and of vascular endothelial growth factor (VEGF). Therefore HIF-2alpha is a candidate dual regulator of vascular development. Here we describe the quail homologue of HIF-2alpha. Sequence analysis reveals that HIF-2alpha is highly conserved between birds and mammals. Like the murine HIF-2alpha, the quail molecule is highly expressed by endothelial cells but also detectable in certain epithelial cells such as in the endoderm.

Amino Acid Sequence↗
PubMed2004

HIF-1alpha induces cell cycle arrest by functionally counteracting Myc.

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1alpha, a basic helix-loop-helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1alpha for hypoxia-induced growth arrest and activation of p21(cip1), a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21(cip1) activation has been elusive. Here we demonstrate that HIF-1alpha, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21(cip1). The HIF-1alpha antagonism is mediated by displacing Myc binding from p21(cip1) promoter. Neither HIF-1alpha transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1alpha. In keeping with its antagonism of Myc, HIF-1alpha also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1alpha pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

Adenoviridae↗
PubMed2005

Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.

The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1alpha, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1alpha were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1alpha to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1alpha protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1alpha in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1alpha-linked upregulation of P450 2C11.

Animals↗
PubMed2004

Activation of the HIF pathway in childhood ALL, prognostic implications of VEGF.

Hypoxia-inducible factor 1 (HIF-1) controls angiogenesis and glycolysis, two leading characteristics of solid tumor invasion, metastasis, and lethality. Increased angiogenesis is also found in the bone marrow (BM) of leukemias. Less is known in leukemia about the role of HIF-1 and vascular endothelial growth factor (VEGF), the most important proangiogenic target gene of HIF-1. We show by immunohistochemistry that the oxygen-regulated component of HIF-1 (HIF-1alpha) is overexpressed in clusters of leukemic cells in BM specimens of childhood acute lymphoblastic leukemia (ALL) and absent in biopsies of normal BM. Half the HIF-1alpha-positive ALL biopsies exhibited VEGF coexpression. Among 96 children with relapsed ALL, diagnostic BM aspirates with high VEGF mRNA levels were associated with a significantly lower probability of event-free survival at 3 years (0.31+/-0.08 vs 0.65+/-0.07, P=0.003). Those with poor molecular response to therapy (evaluated by MRD assessment) had 2.2-fold higher VEGF levels than those responding well to chemotherapy (P=0.005). In conclusion, the data demonstrate activation of the HIF pathway in the BM of ALL patients and indicate that the expression of HIF target genes, such as VEGF, play an important role in leukemia progression, therapy response, and outcome.

Adolescent↗
PubMed2004

Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma.

Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1alpha proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1alpha expression and G250MN expression was observed. In contrast, no relationship between HIF-2alpha expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1alpha and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1alpha target gene in ccRCC.

Adenocarcinoma, Clear Cell↗
PubMed2001

HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1alpha is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1alpha. Under hypoxic conditions, HIF-1alpha is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1alpha, and that this regulation depends on a posttranslational modification of HIF-1alpha. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1alpha in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1alpha. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

Amino Acid Sequence↗
PubMed2001

Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor.

Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-alpha subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1alpha protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-alpha proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1alpha in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-alpha subunits.

Animals↗
PubMed2003

Role of iron (II)-2-oxoglutarate-dependent dioxygenases in the generation of hypoxia-induced phosphatidic acid through HIF-1/2 and von Hippel-Lindau-independent mechanisms.

Hypoxia-inducible factors (HIF-1/HIF-2) govern the expression of critical genes for cellular adaptation to low oxygen tensions. We have previously reported that the intracellular level of phosphatidic acid (PA) rises in response to hypoxia (1% O(2)). In this report, we have explored whether components of the canonical HIF/von Hippel-Lindau (VHL) pathway are involved in the induction of PA. We found that hypoxia induces PA in a cell line constitutively expressing a stable version of HIF-1alpha. PA induction was also found in HIF-1alpha- and 2alpha-negative CHO Ka13 cells, as well as in HIF-beta-negative HepaC4 cells. These data indicate that HIF activity is neither sufficient nor necessary for oxygen-dependent PA accumulation. PA generation was also detected in cells deficient for the tumor suppressor VHL, indicating that the presence of VHL was not required for the induction of PA. Here we show that PA accumulation also occurs at moderate hypoxia (5% O(2)), although to a lesser extent to that seen at 1% O(2), revealing that PA is induced at the same hypoxia range required to activate HIF-1. Prolyl hydroxylases (PHD) and asparaginyl hydroxylase (FIH) belong to the iron (II) and 2-oxoglutarate-dependent dioxygenase family and have been proposed as oxygen sensors involved in the regulation of HIFs. Chemical inhibition of these activities by treatment with iron chelators or 2-oxoglutarate analogs also results in a marked PA accumulation similar to that observed in hypoxia. Together these data show that PA accumulation in response to hypoxia is both HIF-1/2- and VHL-independent and indicate a role of iron (II)-2-oxoglutarate-dependent dioxygenases in the oxygen-sensing mechanisms involved in hypoxia-driven phospholipid regulation.

Cell Hypoxia↗
PubMed2005

The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia.

When oxygen supply is restricted, protein synthesis is rapidly abrogated owing to inhibition of global translation. However, HIF-1alpha protein expression can persist during hypoxia, owing to an internal ribosome entry site (IRES) in the 5'-untranslated region of its mRNA. Here, we report on the molecular mechanism of HIF-1alpha IRES-mediated translation during oxygen deprivation. Using RNA affinity chromatography and UV-crosslinking experiments, we show that the polypyrimidine tract binding protein (PTB) can specifically interact with the HIF-1alpha IRES, and that this interaction is enhanced in hypoxic conditions. Overexpression of PTB enhanced HIF-1alpha IRES activity, whereas RNA interference-mediated downregula-tion of PTB protein expression inhibited HIF-1alpha IRES activity. Furthermore, hypoxia-induced stimulation of the HIF-1alpha IRES was reduced in cells in which PTB function was downregulated. In agreement with these results, the IRES activity of HIF-1alpha IRES deletion mutants that are deficient in PTB-binding could not be stimulated by oxygen deprivation. All together, our data suggest that PTB plays a stimulatory role in the IRES-mediated translation of HIF-1alpha when oxygen supply is limited.

5' Untranslated Regions↗
PubMed2003

Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species.

Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance transporter P-glycoprotein (P-gp) has not been investigated. Herein, we demonstrate that with increasing size of DU-145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the alpha-subunit of HIF-1 (HIF-1alpha) and P-gp were up-regulated. Furthermore, P-gp was up-regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro-oxidants H2O2 and buthionine sulfoximine down-regulated HIF-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha and P-gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF-1beta. Chemical hypoxia robustly increased HIF-1alpha as well as P-gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P-gp in multicellular tumor spheroids is under the control of HIF-1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1alpha, and C/EBPalpha.

Hypoxia, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. Plasminogen activator inhibitor-1 (PAI-1) is an important factor suppressing fibrinolysis under conditions of low oxygen tension. We previously reported that hypoxia induced PAI-1 mRNA and antigen expression in murine macrophages secondary to increased de novo transcription as well as increased mRNA stability. We now show in RAW264.7 murine macrophages that the transcription factors early growth response gene-1 (Egr-1), hypoxia-inducible factor-1alpha (HIF-1alpha), and CCAAT/enhancer binding protein alpha (C/EBPalpha) are quickly activated in hypoxia and are responsible for transcription and expression of PAI-1. Murine PAI-1 promoter constructs, including Egr, HIF-1alpha, and/or C/EBPalpha binding sites, were transfected into RAW 264.7 murine macrophages. To identify the relative importance of each of these putative hypoxia-responsive elements, cells were exposed to normobaric hypoxia, and transcriptional activity was recorded. At 16 h of hypoxic exposure, murine PAI-1 promoter deletion constructs that included Egr, HIF-1alpha, and/or C/EBPalpha binding sites demonstrated increased transcriptional activity. Mutation of each of these three murine PAI-1 promoter sites (or a combination of them) resulted in a marked reduction in hypoxia sensitivity as detected by transcriptional analysis. Functional data obtained using 32P-labeled Egr, HIF-1alpha response element (HRE), and C/EBPalpha oligonucleotides revealed induction of DNA binding activity in nuclear extracts from hypoxic RAW cells, with supershift analysis confirming activation of Egr-1, HIF-1alpha, or C/EBPalpha. ChIP analysis confirmed the authenticity of these interactions as each of these transcription factors binds to chromatin under hypoxic conditions. Further, the induction of PAI-1 by Egr-1, HIF-1alpha, or C/EBPalpha was replicated in primary peritoneal macrophages. These data suggest that although HIF-1alpha appears to dominate the PAI-1 transcriptional response in hypoxia, Egr-1 and C/EBPalpha greatly augment this response and can do so independent of HIF-1alpha or each other. These studies are relevant to ischemic up-regulation of the PAI-1 gene and consequent accrual of microvascular thrombus under ischemic conditions.

Animals↗
PubMed2005

Mechanism of HIF-1alpha-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells.

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1alpha has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1alpha-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1alpha suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1alpha inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca(2+) concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X(L) levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1alpha-overexpressing OSCC line. Overexpression of HIF-1alpha also increased the levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings indicate that HIF-1alpha prevents apoptotic cell death through two mechanisms, including inhibition of cytochrome c release and activation of Akt and ERK.

Apoptosis↗
PubMed2004

HIF-1alpha reveals a binding activity to the promoter of iNOS gene after permanent middle cerebral artery occlusion.

Hypoxia inducible factor (HIF-1)-1alpha is a specific, oxygen-sensitive protein that regulates the activity of HIF-1, a transcriptional factor that increases after cerebral ischemia and may either promote or prevent neuronal survival. In this study to determine whether the inducible nitric oxide synthase (iNOS) gene containing the sequence of the hypoxia-responsive enhancer (HRE) was an HIF-1 target after cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO), electrophoretic mobility shift assay (EMSA) and iNOS western blot analysis were performed in the ischemic core, in the area surrounding the infarct and in the hippocampus ipsilateral and contralateral to the lesion. In addition, both HIF-1alpha mRNA and protein expression were examined in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the insult. Our results revealed that pMCAO up-regulates iNOS protein in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the lesion, and that the activation of iNOS expression is mediated by HIF-1. Moreover, HIF-1alpha mRNA and protein levels increased in the ischemic core and in the hippocampus ipsilateral to the lesion compared with the levels obtained in the corresponding areas of sham-operated controls or in the contralateral hemisphere. Particularly in the area surrounding the ischemic core, HIF-1alpha protein accumulated during pMCAO although mRNA did not increase. Our study suggests that the activation of HIF-1 might be involved in the mechanisms whereby iNOS promotes cell survival and/or death after cerebral ischemia.

Animals↗
PubMed2006

Heterozygous HIF-1alpha deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia.

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH-evoked cardio-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia-inducible factor-1 (HIF-1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF-1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF-1 and if so whether it contributes to cardio-respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild-type (WT) and heterozygous (HET) mice partially deficient in HIF-1alpha, the O2 regulated subunit of the HIF-1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h-1, 8 h day-1) or to 10 days of 21% O2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long-term facilitation (sLTF) of the chemoreceptor activity in CIH-exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH-exposed HET mice. Analysis of cardio-respiratory responses in CIH-exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH-exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH-induced increases in ROS but also CIH-evoked HIF-1alpha up-regulation in WT mice. These results indicate that: (a) HIF-1 activation is critical for eliciting CIH-induced carotid body-mediated cardio-respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF-1 and ROS.

Action Potentials↗
PubMed2002

NO and TNF-alpha released from activated macrophages stabilize HIF-1alpha in resting tubular LLC-PK1 cells.

Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1alpha (subjected to protein stability regulation) and constitutively expressed HIF-1beta. Besides hypoxia, diverse agonists are identified that stabilize HIF-1alpha during normoxia. Here we used a coculture system of RAW 264.7 macrophage cells and tubular LLC-PK(1) cells to establish that lipopolysaccharide- and interferon-gamma-stimulated but not resting macrophages elicited HIF-1alpha accumulation in LLC-PK(1) cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application of tumor necrosis factor-alpha (TNF-alpha)-neutralizing antibodies, we identified NO and TNF-alpha as signaling molecules. Working in concert, NO and TNF-alpha have a stronger response when allowed direct cell-to-cell contact instead of contact with only the cell supernatant of activated macrophages. We show that signal transmission by NO with TNF-alpha in LLC-PK(1) cells is mediated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is blocked by wortmannin or dominant-negative forms of PI3-K as well as protein kinase B. We conclude that NO and TNF-alpha, derived from activated macrophages, provoke HIF-1alpha stabilization in LLC-PK(1) cells under normoxic conditions, which underscores HIF-1alpha stabilization due to intercellular regulation.

Animals↗
PubMed1998

Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1.

Type II nitric oxide synthase (NOS) is upregulated in the pulmonary vasculature in a chronic hypoxia model of pulmonary hypertension. In situ hybridization analysis demonstrates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle in the lung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypoxia in pulmonary artery endothelial cells. Northern blot analyses demonstrate a two fold increase in HIF-1 alpha but not in HIF-1 beta RNA with hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial cells were subjected to hypoxia. This DNA binding complex was identified as HIF-1 using antibodies directed against HIF-1 alpha and HIF-1 beta. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compared with nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated the response to hypoxia. These results demonstrate that HIF-1 is essential for the hypoxic regulation of type II NOS gene transcription in pulmonary endothelium.

Animals↗