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PubMed2004

HIF at the crossroads between ischemia and carcinogenesis.

Tissue hypoxia occurs where there is an imbalance between oxygen supply and consumption in both, solid tumors as a result of exponential cellular proliferation and in atherosclerotic diseases as a result of inefficient blood supply. Hypoxia-inducible factor 1 (HIF-1) is central in normal angiogenesis and cancer angiogenesis. HIF-1 is a transcriptional activator composed of an O(2)- and growth factor-regulated HIF-1alpha subunit and a constitutively expressed HIF-1beta subunit. Upon activation, HIF-1 drives the expression of genes controlling cell survival and governing the formation of new blood vessels. A better understanding of the regulation of HIF-1alpha levels by the receptor tyrosine kinases/phosphatidylinositol 3-kinase signaling pathway and by the HIF prolyl hydoxylases has provided new insights into the development of anticancer and revascularization therapeutics. We will focus on the potential of a new pharmacology for regulating HIF pathways in both, cancer and ischemic cardiac diseases. The consequences of the switch of HIF activation in these two disease states and the signaling pathway overlap that atherosclerosis and cancer angiogenesis share are discussed.

Animals↗
PubMed2001

Role of protein kinase Cdelta in transmitting hypoxia signal to HSF and HIF-1.

An hypoxic microenvironment is an important modulator of gene expression in many pathophysiological conditions. In this study, we show a coordinate activation of heat shock transcription factor (HSF) and hypoxia-inducible factor-1 (HIF-1) in RIF tumor cells by hypoxia. Since heat shock protein (hsp) and angiogenic factor genes that are regulated by HSF and HIF-1 are thought to contribute to the malignant progression of hypoxic tumor cells, it was of our major interest to identify the components that are responsible for the activation of both HSF and HIF-1. Our finding that a bioflavonoid quercetin (QCT), a well known inhibitor of hsp gene expression, significantly inhibited the transcriptional activation of HSF and HIF-1 strongly suggests that QCT-sensitive molecule(s) is involved in the transcriptional activation of HSF and HIF-1 by hypoxia. Our results revealed that PCKalpha, delta and epsilon isoforms are expressed in RIF cells, but only PKCdelta was specifically translocated to the membrane by hypoxia. Our results also revealed that the translocation of PKCdelta was completely abrogated by QCT. Moreover, inhibiting the PKCdelta activation, either pharmacologically with phorbol 12-myristate 13-acetate or with bisindolymaleimide II or genetically by transient transfection of a dominant negative PKCdelta, significantly inhibited the transcriptional activation of HSF and HIF-1 by hypoxia. These results strongly substantiate a view that the PKCdelta isozyme is the QCT-sensitive molecule that plays an important role in transmitting hypoxia signals to both HSF and HIF-1. Here we show that the membrane translocation of PKCdelta is dependent on the activation of phosphoinositol 3-kinase (PI3K). Treatment with PI3K inhibitor, wortmannin or LY294002, abrogated not only PKCdelta translocation but the subsequent transcriptional activation of HSF and HIF-1 by hypoxia. Together, our study shows that the PKCdelta isozyme acts as a shared component in transmitting hypoxia-induced signals to both HSF and HIF-1, and that the upstream regulator of PKCdelta is PI3K.

Cell Hypoxia↗
PubMed1999

Characterization of an oxygen/redox-dependent degradation domain of hypoxia-inducible factor alpha (HIF-alpha) proteins.

Hypoxia-inducible factors are heterodimeric DNA-binding complexes that control the hypoxia responses of several genes and regulate the adaptive responses to the lack of oxygen. The complex is composed of two b-HLH protein subunits, HIF-1beta (ARNT), that is constitutively expressed, and a HIF-alpha subunit, that is present only in hypoxic cells. HIF-alpha proteins are continuously synthesized, but are rapidly degraded by the ubiquitin-proteasome system under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-alpha proteins, allowing the formation of the transcriptionally active HIF complex. However, the sequences and mechanisms involved in the regulated degradation of the alpha protein subunits are poorly understood. Analysis of the available cloned sequences of human and mouse members of the HIF-alpha family of proteins revealed an area of about 15 amino acids with strong sequence conservation between all the members. This area corresponds to the region encompassing amino acids 557-571 of the hHIF-1alpha subunit. Fragments of HIF-1alpha and HIF-3alpha proteins containing this conserved sequence were able to confer hypoxia regulation when expressed as fusion proteins in Hep-3B cells. Regulation was observed with all the known hypoxia "mimics," including the reducing thiol donor N-mercaptopropionylglycine (NMPG). Selective alanine substitutions of amino acids 561-568 stabilized the protein in normoxic conditions. Furthermore, transfection with an expression vector containing a fragment of hHIF-1alpha comprising amino acids 540-580 enhanced transactivation activity of the full-length hHIF-1alpha protein. These results suggest that the above-mentioned conserved sequences are likely involved in the hypoxic stabilization of HIF-alpha proteins. The mechanisms and the interacting ubiquitin-ligases involved in the selective degradation process remain unknown.

Amino Acid Sequence↗
PubMed2002

Developmental stage-specific expression of the alpha and beta subunits of the HIF-1 protein in the mouse and human fetus.

Erythropoietin (Epo) is a glycoprotein hormone that is the primary regulator of erythropoiesis. Transcription of the Epo gene increases in response to hypoxia or anemia. Epo is synthesized in the liver in fetal life and in the kidney later in gestation. In the mammalian fetus the switch in Epo production from the liver to the kidney occurs in the third trimester. Hypoxia-inducible factor (HIF-1) is a heterodimeric transcription factor consisting of an alpha and beta subunit that binds under hypoxic conditions to an enhancer element in the 3' region of the Epo gene. In order to determine if there is a relationship between expression of HIF-1 alpha and beta subunits with the shift in expression of the Epo gene from the liver to the kidney or with the transitional events occurring at birth we analyzed the expression of these mRNAs in mouse and human fetuses at different stages of gestation. Total RNA was extracted from the brain, heart, kidney, liver, and lungs of mice at P15, P17, and P19 of gestation, from newborn mice at Days 1 and 3, from an adult and an anemic adult mouse as well as from human fetuses at 14-22 weeks of gestation. RNA was analyzed by Northern blot and slot-blot hybridization using appropriate cDNA probes. HIF-1alpha and -beta mRNA were expressed in all tissues tested and at all stages of gestation in the mouse and human fetus. Expression of HIF-1alpha and -beta in the mouse fetus was highest in the brain followed by heart, kidney, lung, and liver. Expression in the fetal and newborn mice was higher versus the adult and expression was higher in the anemic versus the normal adult mouse. In the human fetus a higher expression of HIF-1alpha was noted in the brain followed by heart, kidney, lung, and liver. There was a small trend toward a decrease in expression with advancing gestational age. HIF-1beta was expressed to a similar extent in all human tissues examined. Our studies indicate that expression of HIF-1alpha and -beta subunits was not related to the switch in Epo gene expression from the liver to the kidney. Although expression of HIF-1alpha and -beta did not decrease immediately after birth, it is possible that the HIF-1 protein is involved in the various events that occur during transition after birth.

Animals↗
PubMed2000

Regulation of the hypoxia-inducible factor-1 alpha. ARNT is not necessary for hypoxic induction of HIF-1 alpha in the nucleus.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1 alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents ubiquitination and proteasomal degradation of HIF-1 alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. To examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1 alpha in hypoxia, we used ARNT-mutant mouse hepatoma and ARNT-deficient embryonic stem cells. As shown by immunofluorescence, HIF-1 alpha accumulation in the nucleus of hypoxic cells did not require ARNT, demonstrating that nuclear translocation is intrinsic to HIF-1 alpha. During biochemical separation, both HIF-1 alpha and ARNT tend to leak from the nuclei in the absence of the corresponding subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

Animals↗
PubMed2005

The effect of age and calorie restriction on HIF-1-responsive genes in aged liver.

Hypoxia inducible factor-1 (HIF-1) regulates transactivation of several genes in response to hypoxia condition. We explore hepatic HIF-1 responsive gene regulation during aging and the age-related changes of the HIF-1 related gene activation in young and old rats. Results indicate that the aging process induces the activation of HIF-1alpha, which is accompanied by increased HIF-1 DNA binding. This increased binding activity is accompanied by the increase of HIF-1-dependent genes, heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS), which all showed remarkable up-regulation during aging process. In contrast, the increased HIF-1 related gene expression was effectively blunted by the anti-oxidative action of calorie restriction in aged rat liver. We propose that age-related HIF-1 binding activity may well be influenced by the increased pro-oxidative conditions of aged animals, which up-regulate HIF-1-dependent gene expression.

Aging↗
PubMed2006

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.

Cell Hypoxia↗
PubMed2006

Desferrioxamine, an iron chelator, enhances HIF-1alpha accumulation via cyclooxygenase-2 signaling pathway.

Cyclooxygenase-2 (COX-2) is an important inducible enzyme in inflammation and is overexpressed in a variety of cancers. Evidence is rapidly accumulating that chronic inflammation may contribute to carcinogenesis through increase of cell proliferation, angiogenesis, and metastasis in a number of neoplasms, including colorectal carcinoma. In the present study, we investigated some mechanistic aspects of DFX-induced hypoxia-driven COX-2 expression. Desferrioxamine (DFX), an iron chelator, is known to upregulate inflammatory mediators. DFX induced the expression of COX-2 and accumulation of HIF-1alpha protein in dose-dependent manners, but hypoxia mimetic agent cobalt chloride (CoCl2) induced accumulation of HIF-1alpha protein but not increase of COX-2 expression. DFX-induced increase of COX-2 expression and HIF-1alpha protein level was attenuated by addition of ferric citrate. This result suggested that the iron chelating function of DFX was important to induce the increase of COX-2 and HIF-1alpha protein. PD98059 significantly inhibited the induction of COX-2 protein and accumulation of HIF-1alpha, suggesting that DFX-induced increase of HIF-1alpha and COX-2 protein was mediated, at least in part, through the ERK signaling pathway. In addition, pretreatment with NS-398 to inhibit COX-2 activity also effectively suppressed DFX-induced HIF-1alpha accumulation in human colon cancer cells, providing the evidence that COX-2 plays as a regulator of HIF-1alpha accumulation in DFX-treated colon cancer cells. Together, our findings suggest that iron metabolism may regulate stabilization of HIF-1alpha protein by modulating cyclooxygenase-2 signaling pathway.

Colonic Neoplasms↗
PubMed2004

New anticancer strategies targeting HIF-1.

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by up-regulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

Adaptation, Physiological↗
PubMed2005

Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han.

The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.

Actins↗
PubMed2006

IL-4 enhances hypoxia induced HIF-1alpha protein levels in human transformed intestinal cells.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that mediates the adaptive response to hypoxia. Increasing evidence suggests a crucial role for HIF-1 in immune reactions. Here we investigated the effect of the Th2 type cytokines IL-4 and IL-10 on HIF-1alpha mediated response in normoxia (21% O2) and hypoxia (1% O2). Incubation of human transformed intestinal cells (HT-29) with IL-4 significantly increased HIF-1alpha protein levels during hypoxia but not during normoxia. Mechanisms involved are IL-4 induced up-regulation of HIF-1alpha gene transcription and the PI3K signaling pathway. The increase in hypoxia-induced accumulation of HIF-1alpha protein after IL-4 treatment did not result in up-regulation of HIF-1 DNA-binding activity or HIF-1 dependent gene expression. IL-10 did not affect HIF-1alpha protein levels.

Cell Hypoxia↗
PubMed2005

NO restores HIF-1alpha hydroxylation during hypoxia: role of reactive oxygen species.

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2005

Cytokines and the regulation of hypoxia-inducible factor (HIF)-1alpha.

Hypoxia-inducible factor (HIF)--an oxygen sensor? The HIF-oxygen sensing association type of dogma is, unequivocally, well anchored. But this is only one face of, at least, a double-sided coin. Current concepts charge HIF of taking sides with a yet not well-founded identity--an immunologic sensor and/or regulator. Or, is it really a sensor, put it more correctly, a key player in sensing mechanisms? The evolving association between HIF and immunity emanates from an established linkage that bonds oxidative stress and inflammation--notably the 'biologic response modifiers', or cytokines. HIF is a redox(y)-sensitive transcription factor, and so are cytokines. Recently, cytokines emerged as major regulators of HIF, under physiologic conditions extending the realm of hypoxia. Alternatively, can HIF, like the so infamous inflammatory transcription factor NF-(kappa)B, prove itself as a key player in the regulation of cytokines and, subsequently, the inflammatory process. The targeting of HIF would be, at least theoretically, of therapeutic value, but does it make sense given its intricate role in hypoxia signaling? It is the theme of HIF being an immunologic sensor that will be explored therein--with special emphasis on the regulatory role of cytokines.

Animals↗
PubMed2003

Differential regulation of HIF-1 alpha prolyl-4-hydroxylase genes by hypoxia in human cardiovascular cells.

Three HIF-alpha prolyl-4-hydroxylases (PHDs) (named PHD1, PHD2, and PHD3) effect the proteasome-mediated degradation of HIF by catalyzing the hydroxylation of key proline residues in the HIF-1 alpha subunit under normoxic conditions. When oxygen tension is reduced, PHD-mediated hydroxylation cannot occur, HIF-1 alpha accumulates in the nucleus, resulting in HIF-mediated gene transcription. In the present study, the expression and regulation of PHD mRNA and HIF protein expression was examined in human tissues and primary cells of cardiovascular origin. Treatment of human cardiac myocytes, smooth muscle cells, and endothelial cells with hypoxia or CoCl(2), a hypoxia mimic, resulted in a significant time-dependent increase in PHD3, but not PHD1 or PHD2, mRNA levels, which correlated with an increase in HIF-1 alpha protein expression. Overexpression studies revealed that PHD3 levels influence HIF-1 alpha stability in both normoxic and hypoxic conditions, suggesting that PHD3 may participate in a feedback loop controlling HIF activity.

Base Sequence↗
PubMed2003

Expression of proinflammatory cytokines via HIF-1alpha and NF-kappaB activation on desferrioxamine-stimulated HMC-1 cells.

We investigated the expression and the role of hypoxia-inducible factor 1alpha (HIF-1alpha) on the desferrioxamine (DFX)-induced cytokine production in human mast cells, HMC-1 cells. HIF-1alpha mRNA was constitutively expressed in mast cell lines including the P815, RBL-2H3, and HMC-1. DFX (100 microM) resulted in a great increase in protein levels of HIF-1alpha in HMC-1 cells, but it did not affect HIF-1alpha mRNA expression. Iron (HIF-1 inhibitor) inhibited increase of HIF-1alpha and NF-kappaB protein levels. Pyrriolidine-dithiocarbamate (PDTC, NF-kappaB inhibitor) inhibited increase of NF-kappaB protein levels, but it slightly increased HIF-1alpha protein levels. In addition, DFX significantly increased the production of IL-6, IL-8, and TNF-alpha in HMC-1 (P<0.05). These increased cytokine levels were significantly inhibited by treatment of iron or PDTC in a dose-dependent manner (P<0.05). We demonstrated the regulatory effects of HIF-1alpha on the DFX-induced proinflammatory cytokine production in human mast cells for the first time. These data indicate that inflammatory cytokines seem to be under HIF-1alpha or NF-kappaB transcriptional regulation in the hypoxic conditions on mast cells.

Blotting, Western↗
PubMed2002

Intramyocardial injection of naked DNA encoding HIF-1alpha/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat.

OBJECTIVES: The therapeutic utility of hypoxia-inducible factor-1 (HIF-1) transcriptional regulatory system for ischemic hindlimb has been demonstrated. It is not yet known whether this transcriptional regulatory system can be used as a therapeutic strategy to enhance collateral vessel formation in myocardial tissues, where acute hypoxia occurs due to inadequate perfusion. We aimed to test the hypothesis that exogenous administration of HIF-1alpha/VP16 could enhance collateral vessel formation in a rat acute myocardial infarction model. METHODS: Sprague-Dawley rats received ligation of the proximal left anterior descending coronary artery to induce acute myocardial infarction. Immediately after the ligation, 50 microg total plasmid DNA (control, plasmid encoding human vascular endothelial growth factor (pVEGF(165)), or pHIF-1alpha/VP16) was injected into the infarct area at three locations. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of HIF-1alpha and VEGF mRNA in the myocardium, but not in other organs at days 3 and 7. The infarct size significantly decreased from 37+/-4% (control) to 24+/-2% in the VEGF-treated group and 23+/-2% in the HIF-1alpha/VP16 treated group (P<0.05). Capillary density also significantly increased from 550+/-75/mm(2) (control) to 850+/-75/mm(2) in the VEGF group and 850+/-50/mm(2) in the HIF-1alpha/VP16-treated group (P<0.01). Combined therapy with HIF-1alpha/VP16 and VEGF resulted in higher capillary density (1230+/-50/mm(2)) than treatment with either therapy alone. Regional myocardial blood flow was also higher in the treated groups than in the control. Plasma levels of VEGF were also significantly higher in the HIF-1alpha/VP16 and VEGF-treated group than in the control group. CONCLUSIONS: The HIF-1alpha/VP16 hybrid transcription factor is able to reduce infarct size and enhance neovascularization in an acute ischemic myocardium. The potency of VEGF and HIF-1alpha/VP16 hybrid as therapeutic angiogenic factors in acute hypoxic myocardium is similar.

Animals↗
PubMed2003

S-nitrosation of Cys-800 of HIF-1alpha protein activates its interaction with p300 and stimulates its transcriptional activity.

Hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcriptional complex that plays pivotal role in the regulation of cellular utilization of oxygen as well as glucose and is an essential regulator of angiogenesis in solid tumor and ischemic disorders. Recently HIF-1alpha, a subunit of HIF-1 complex, was characterized as a potential target for S-nitrosation, providing no information about the impact of this posttranslational modification on the protein transactivation. Cys-800 of HIF-1alpha protein has reactive SH-group, which is critical for the recruitment of p300 co-activator that is necessary for transcriptional activity of HIF-1 complex. Here we report that S-nitrosation of Cys-800 activates HIF-1alpha-p300 interaction and stimulates protein transactivation. We have found that S-nitrosation of the HIF-1alpha C-terminal domain by nitric oxide derived from donors and nitric oxide synthase increases protein transcriptional activity. The increase of HIF-1 transcriptional activity was not observed in the case of Cys-800 substitution to Ala, though other protein thiol groups were nitrosated. Experiments with GST pull-down assay suggest that S-nitrosation of Cys-800 stimulates the recruitment of p300 co-activator protein to the HIF-1alpha C-terminal domain.

Binding Sites↗
PubMed2002

Positive expression of HIF-2alpha/EPAS1 in invasive bladder cancer.

OBJECTIVES: To investigate the expression of hypoxia-inducible factor (HIF)-2alpha/endothelial PAS domain protein 1 (EPAS1) in bladder cancer. METHODS: Two bladder cancer cell lines (T24, EJ-1) were examined for the expression of HIF-2alpha/EPAS1 mRNA by reverse transcriptase-polymerase chain reaction and HIF-2alpha/EPAS1 protein by Western blot analysis, respectively. Expression of HIF-2alpha/EPAS1 protein was also investigated immunohistochemically on paraffin-embedded sections from 24 patients with bladder cancer. RESULTS: HIF-2alpha/EPAS1 mRNA and protein could be detected in the two bladder cell lines under normoxia. The normal urothelium and all 7 superficial cases (pTis-pTa) were negative for HIF-2alpha/EPAS1. Five of the 8 pT1 cases (62.5%) and all 9 muscle-invasive cases were positive. The invasive compartment showed stronger HIF-2alpha/EPAS1 immunoreactivity compared with the superficial compartment. All the tumor clusters infiltrating to the interstitial tissue among muscle fibers showed moderate to strong positive staining. CONCLUSIONS: HIF-2alpha/EPAS1 is mostly expressed in invasive bladder cancer, which indicates a possible role for HIF-2alpha/EPAS1 in the invasion of bladder cancer.

Aged↗