Putative Hypoxia Response Elements Research Articles

IL‐20 is regulated by hypoxia‐inducible factor and upregulated after experimental ischemic stroke

IL‐20, an IL‐10 family member, is involved in various inflammatory diseases, such as psoriasis, rheumatoid arthritis, and atherosclerosis. We investigated whether hypoxia in vitro and an in vivo model of ischemic stroke would upregulate IL‐20 expression. In vitro, IL‐20 expression increased in hypoxic HaCaT , HEK293 cells, chondrocytes, monocytes and glioblastoma cells. Inhibiting HIF‐1alpha inhibited CoCl2‐induced IL‐20 expression. We identified two putative hypoxia response elements in human il20‐gene promoter. Promoter activity assays showed that CoCl2‐mimicked hypoxia activated luciferase reporter‐gene expression. In vivo, experimental ischemic stroke upregulated IL‐20 in the sera and brain tissue of rats. IL‐20 stained positively in glia‐like cells in peri‐infarcted lesions, but not in contralateral tissue. Administration of IL‐20 monoclonal antibody ameliorated ischemia‐induced brain infarction of rats after experimental ischemic stroke. In vitro, RT‐PCR analysis showed that glioblastoma cells GBM8901 cells expressed IL‐20 and its receptor subunits IL‐20R1, IL‐20R2, and IL‐22R1. IL‐20 also induced production of IL‐1beta, IL‐8, and MCP‐1 in GBM8901 cells. We conclude that IL‐20 was responsive to hypoxia in vitro and in the ischemic stroke model, and that upregulation of IL‐20 in the ischemic brain may contribute to brain injury.

Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

The serine/threonine kinase-15 (STK15) acts as a cell cycle regulator being overexpressed in various tumors. One mechanism that could contribute to overexpression of STK15 is tumor hypoxia where hypoxia-inducible factor-1 (HIF-1) is a major regulator of transcription. Therefore, we analyzed whether hypoxia and HIF-1 could contribute to overexpression of STK15. We found that hypoxia increased STK15 expression and STK15 promoter activity in HepG2 tumor cells. Overexpression of HIF-1 alpha induced STK15 gene transcription, whereas HIF-1 alpha siRNA and overexpression of prolyl hydroxylase 2 (PHD-2), a negative regulator of HIF-1 alpha, reversed this effect. In addition, site-directed mutagenesis experiments and chromatin immunoprecipitation revealed that from the three putative hypoxia responsive elements (HRE) within the STK15 promoter only HRE-2 was functional and bound HIF-1. Further, siRNA against STK15 inhibited proliferation of HepG2 cells induced by hypoxia. These results show that STK15 gene transcription can be regulated by hypoxia and HIF-1 via HRE-2 of the STK15 promoter. Thus, tumor hypoxia may trigger overexpression of STK15 observed in various tumors.

Regulation of Dual specificity protein phosphatase‐2 (DUSP2) by hypoxia

Constitutive activation of MAP kinases was found in many cancers, and it's still an enigma that needs to be revealed. DUSP2 is a member of dual specificity protein phosphatase that regulates the activity of MAP kinases. Until now, the regulation of DUSP2 expression has not yet investigated. Hypoxia occurs in many solid tumors and has been shown to be a factor of increasing the MAP kinase activity. Therefore, we hypothesize that hypoxia‐induced MAP kinase activity may be mediated via deregulation of DUSP2 expression. We predicted a putative hypoxia response element (HRE) in the DUSP2 promoter region by using bioinformatic tool. Expression of DUSP2 was decreased by hypoxia treatment in HeLa cancer cells and normal endometrial stroma cells. Knockdown of HIF‐1α or HIF‐2αby siRNA reversed hypoxia‐mediated inhibition of DUSP2 expression. Promoter activity assay and site‐direct mutagenesis showed that predicted putative HRE was essential for downregulation of DUSP2 expression under hypoxic condition. Chromatin immunoprecipitation (ChIP) assay further demonstrated the binding of HIF‐1α to the HRE of DUSP2 promoter. ERK1/2, the downstream target of DUSP2, was activated by hypoxia and was inversely correlated with the expression of DUSP2. Taken together, we demonstrate that hypoxia downregulates the expression of DUSP2 thus enhances the activity of ERK1/2, which may be an important factor for cells to survive in hypoxic environment.

Effect of hypoxia‐regulated CD151 expression on cancer cell adhesion

CD151 is a member of tetraspanin family that plays important roles in cell adhesion and motility. However, the regulation of CD151 expression in normal and cancerous cells has not yet revealed. In cancer tissue, it is suggested that CD151 acts as a metastasis‐suppressor while hypoxia has been shown to be a factor of causing metastasis. We hypothesize that hypoxia‐induced cancer cell metastasis may be mediated via deregulation of CD151. Indeed, colon cancer cells expressed less CD151 than its normal counterpart, which is inversely correlated with the expression of hypoxia‐inducible factor‐1α (HIF‐1α). By using bioinformatic approach, we identified a putative hypoxia response element (HRE) in the promoter region of human CD151 gene. Treatment of colon cancer cells with hypoxia or chemicals that are known to increase accumulation of HIF‐1α resulted in decreased CD151 expression. Promoter activity assay further demonstrated that the predicted putative HRE was pivotal for HIF‐1α‐dependent downregulation of CD151 expression. Furthermore, inhibition of CD151 expression by hypoxia resulted in decreased cancer cell adhesion to laminin‐coated matrix, a process that enables cancer cell to detach from its surrounding matrix and be ready for metastasis. In conclusion, we demonstrated that hypoxia downregulates the expression of CD151 thus enhances the detachment of cancer cells from its basement membrane protein, an important first step for metastasis.

Hypoxia regulates choline kinase expression through hypoxia-inducible factor-1 alpha signaling in a human prostate cancer model.

The intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using (1)H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5' of the translation start site of the human chk-alpha gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-alpha promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1 alpha showed that HIF-1 can directly bind this region of the endogenous chk-alpha promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-alpha promoter region can increase Chk-alpha expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments.

Hypoxia, via stabilization of the hypoxia-inducible factor HIF-1α, is a direct and sufficient stimulus for brain-type natriuretic peptide induction

BNP (brain-type natriuretic peptide) is a cardiac hormone with systemic haemodynamic effects as well as local cytoprotective and antiproliferative properties. It is induced under a variety of pathophysiological conditions, including decompensated heart failure and myocardial infarction. Since regional hypoxia is a potential common denominator of increased wall stretch and myocardial hypoperfusion, we investigated the direct effects of hypoxia on BNP expression, and the role of the HIF (hypoxia-inducible transcription factor) in BNP regulation. Using an RNase protection assay we found a strong hypoxic induction of BNP mRNA expression in different cell lines and in cultured adult rat cardiomyocytes. Systemic hypoxia and exposure to 0.1% CO induced BNP expression in the rodent myocardium in vivo, although this was at a lower amplitude. BNP promoter-driven luciferase expression increased 10-fold after hypoxic stimulation in transient transfections. Inactivation of four putative HREs (hypoxia-response elements) in the promoter by site-directed mutagenesis revealed that the HRE at -466 nt was responsible for hypoxic promoter activation. A functional CACAG motif was identified upstream of this HRE. The HIF-1 complex bound specifically and inducibly only to the HRE at -466 nt, as shown by EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation). siRNA (small interfering RNA)-mediated knockdown of HIF-1alpha, but not HIF-2alpha, interfered with hypoxic BNP mRNA induction and BNP promoter activation, confirming that BNP is a specific HIF-1alpha target gene. In conclusion, BNP appears to be part of the protective program steered by HIF-1 in response to oxygen deprivation. Induction of BNP may therefore contribute to the potential benefits of pharmacological HIF inducers in the treatment of ischaemic heart disease and heart failure.

Regulation of miR-210 Generation in Response to Hypoxia in Erythrocytic Cells.

EPO-EPOR-mediated intracellular signaling activates JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Alternatively, recent studies suggest that microRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes. Although recent reports showed the importance of miRNA functions in hematopoietic cells, the effect of EPO on miRNA expression and the role of miRNA in modulating EPO-mediated cell proliferation and differentiation are not reported yet. In this study, we investigate the function of miRNAs in factor-dependent human cell lines, EPO-dependent UT-7/EPO and GM-CSF-dependent UT-7/GM. We employed a human miRNA microarray to detect 470 miRNAs in order to evaluate the expression patterns of miRNA between UT-7/EPO and UT-7/GM. The expression of miR-210 in UT-7/EPO was 30-fold higher than that in UT-7/GM. The EPO-dependent cell lines TF-1 showed substantially increased levels of miR-210 by stimulation with EPO, compared to expression in TF-1 cells maintained by GM-CSF. Recently, miR-210 was identified as a group of miRNAs that were induced in a hypoxic environment in colon and breast cancer cell lines (Kulshreshtha et al., MCB. 2007). Enforced expression of miR-210 decrease proapoptotic signaling in a hypoxic environment in breast cancer cell line. To investigate the regulation of miR-210 in hematopoietic cells under hypoxic condition, expression level of miR-210 was determined and compared between hypoxia (1% O2) or normoxia (21% O2) in UT-7 cell lines. The expression of miR-210 increased 5- to 80-fold in UT-7, UT-7/GM and TF-1 under hypoxic condition. UT-7/EPO expressed high level of miR-210 in normoxia and maintained the same level in hypoxia. To examine whether the transcription of miR-210 primary transcripts (pri-miR-210) is regulated by hypoxia signaling, pri-miR-210 was detected by RT-PCR in hypoxia- or normoxia-treated cells. Higher expression of pri-miR-210 was detected in hypoxia-treated cells. Through sequence analysis, several putative hypoxia response elements that are highly conserved among different species were identified in the 5′-upstream region of pri-miR-210. These observations indicated that the transcription of pri-miR-210 was at least in part controlled by HIF-1 α protein. It is well known that hypoxic stress strongly enhances erythropoiesis, but the effect of hypoxia on erythroid progenitors has not been examined precisely. HIF mediates short-term adaptive responses to hypoxia, and several glucose transporters and glycolytic enzymes are transcriptionally regulated by HIF, as well as genes involved in cell growth and survival. In this view, we hypothesize that the physiological function of miR-210 is erythroid cell survival in the hypoxic stress. In silico analysis revealed candidate targets that are part of the apoptotic machinery and glucose metabolism. Therefore, we examined whether miR-210 is expressed in primary erythrocytic cells. These findings suggest a novel pathway that miR-210 contribute to the erythroid cell survival in hypoxic condition.

Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation

We have previously demonstrated increased fibroblast growth factor-2 (FGF-2) expression in a lamb model of increased pulmonary blood flow secondary to congenital heart disease, which may contribute to the associated increases in pulmonary arterial muscularization. However, the mechanisms underlying these increases in FGF-2 expression remain to be identified. Initially, we found that exogenous FGF-2 increased endogenous FGF-2 promoter activity and protein levels in ovine pulmonary arterial smooth muscle cells (PASMC). Furthermore, we found that these increases in FGF-2 expression were mediated by increases in superoxide levels via NADPH oxidase activation. In addition, FGF-2-mediated increases in FGF-2 expression and PASMC proliferation were attenuated by inhibition of phosphatidylinositol 3-kinase, Akt, and NADPH oxidase. Increases in FGF-2 expression could be stimulated by other factors known to increase reactive oxygen species (ROS) signaling in PASMC (endothelin-1 and transforming growth factor-beta1), whereas antioxidants attenuated these increases. Deletion constructs localized the growth factor- and ROS-sensitive region within the proximal 103 bp of the FGF-2 promoter, and sequence analysis identified a putative hypoxia response element (HRE), a DNA binding site for the ROS-sensitive transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). Stabilization of HIF-1alpha increased FGF-2 promoter activity, whereas mutation of the putative HRE attenuated FGF-2-induced FGF-2 promoter activity. Furthermore, FGF-2 increased HIF-1alpha protein levels and consensus HRE promoter activity in PASMC via antioxidant-sensitive mechanisms. Thus we conclude that FGF-2 can stimulate its own expression in PASMC via NADPH oxidase-mediated activation of ROS-sensitive transcription factors, including HIF-1alpha. This positive feedback mechanism may contribute to pulmonary vascular remodeling associated with increased pulmonary blood flow.

HIF-2α specifically activates the VE-cadherin promoter independently of hypoxia and in synergy with Ets-1 through two essential ETS-binding sites

The mechanisms that are responsible for the restricted pattern of expression of the VE-cadherin gene in endothelial cells are not clearly understood. Regulation of expression is under the control of an approximately 140 bp proximal promoter that provides basal, non-endothelial specific expression. A larger region contained within the 2.5 kb genomic DNA sequence located ahead of the transcription start is involved in the specific expression of the gene in endothelial cells. We show here that the VE-cadherin promoter contains several putative hypoxia response elements (HRE) which are able to bind endothelial nuclear factors under normoxia. The VE-cadherin gene is not responsive to hypoxia but hypoxia-inducible factor (HIF)-2alpha specifically activates the promoter while HIF-1alpha does not. The HRE, that are involved in this activity have been identified. Further, we show that HIF-2alpha cooperates with the Ets-1 transcription factor for activation of the VE-cadherin promoter and that this synergy is dependent on the binding of Ets-1 to DNA. This cooperative action of HIF-2alpha with Ets-1 most probably participates to the transcriptional regulation of expression of the gene in endothelial cells. This mechanism may also be involved in the expression of the VE-cadherin gene by tumor cells in the process of vascular mimicry.

Molecular regulation of the PAI‐1 gene by hypoxia: contributions of Egr‐1, HIF‐1 α, and C/EBPα

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.

Hypoxia Induces Hemoglobin Production in Human Erythroleukemia Cells by Increasing the Secretion of Transforming Growth Factor -β1 and the Expression of Mitoferrin.

Hypoxia induces erythropoiesis by stimulating erythropoietin secretion from kidney in vivo. Hypoxia also stimulates hemoglobin production in cultured murine erythroleukemia cells, but its mechanism is still unknown. We have found that hypoxia induces hemoglobin production in human erythroleukemia cell line, YN-1, and have analyzed the mRNA expression profile of YN-1 cells under hypoxic conditions (1% O2, 5% CO2 and 94% N2) using DNA microarrays, and compared it with that of normoxic conditions (5% CO2 and 95% room air). For DNA microarray analysis, total RNA was prepared from YN-1 cells, which were incubated under hypoxic or normoxic conditions for 6 hours. We thus found the increased mRNA expression for several genes involved in erythroid differentiation, including transforming growth factor β1 (TGF-β1) and mitoferrin (mitochondrial iron importer, previously known as SLC25A37). Among these candidates, we have focused on TGF-β1, since TGF-β1 is able to induce erythroid differentiation of several erythroleukemia cell lines, including YN-1 cells and K562 cells. We initially confirmed the increased mRNA expression of TGF-β1 under hypoxic conditions by Northern blotting analysis in YN-1 cells and YN-1-0A cells, which is a daughter cell line of YN-1 cells and is maintained in serum-free medium. Likewise, the hypoxic induction of mitoferrin mRNA in YN-1 cells was confirmed by Real Time PCR analysis. Then, using ELISA system, we measured the TGF-β1 concentration in the culture medium of YN-1-0A cells, and have found that hypoxia stimulates TGF-β1 secretion from the cells. Notably, the hypoxia-mediated increases in hemoglobin production and γ-globin mRNA expression were significantly suppressed in YN-1-0A cells, when TGF-β1 activity in the culture medium was neutralized with anti-TGF-β1 antibody. These results suggest that hypoxia stimulates TGF-β1 secretion from YN-1 cells and YN-1-0A cells, which in turn induces the terminal differentiation of erythroid cells in an autocrine manner. Interestingly, in contrast to the hemoglobin production, addition of TGF-β1 into the culture medium did not influence mitoferrin expression, suggesting that hypoxia induces mitoferrin expression by a mechanism independent of TGF-β1. Since several putative hypoxia responsive elements are present in the promoter region of the human mitoferrin gene, hypoxia inducible factor 1 (HIF-1) may be involved in inducible expression of mitoferrin under hypoxic conditions. The hypoxia-mediated induction of mitoferrin expression is important for effective supply of iron for heme biosynthesis in erythroid cells, as several missense mutations of the zebrafish mitoferrin gene are associated with hypochromic anemia and erythroid maturation arrest (Shaw G.C. et al., Nature, vol.440, p96–100, 2006). Taken together, we suggest that hypoxia stimulates hemoglobin production in erythroid cells by coordinately increasing the iron supply into mitochondria and the γ-globin expression, which may be achieved in part by the induction of mitoferrin and the enhanced secretion of TGF-β1, respectively.

Kaposi's Sarcoma-Associated Herpesvirus Latent Protein LANA Interacts with HIF-1α To Upregulate RTA Expression during Hypoxia: Latency Control under Low Oxygen Conditions

Hypoxia can induce lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in primary effusion lymphoma (PEL) cells. However, the molecular mechanism of lytic reactivation of KSHV by hypoxia remains unclear. Here we show that the latency-associated nuclear antigen (LANA), which plays a crucial role in modulating viral and cellular gene expression, directly associated with a low oxygen responder, hypoxia-inducible factor-1 alpha (HIF-1 alpha). LANA enhanced not only the transcriptional activities of HIF-1 alpha but also its mRNA level. Coimmunoprecipitation and immunofluorescence studies documented a physical interaction between LANA and HIF-1 alpha in transiently transfected 293T cells as well as in PEL cell lines during hypoxia. Through sequence analysis, several putative hypoxia response elements (HRE-1 to -6) were identified in the essential lytic gene Rta promoter. Reporter assays showed that HRE-2 (-1130 to -1123) and HRE-5 and HRE-6 (+234 to +241 and +812 to +820, respectively, within the intron sequence) were necessary and sufficient for the LANA-mediated HIF-1 alpha response. Electrophoretic mobility shift assays showed HIF-1 alpha-dependent binding of a LANA protein complex specifically to the HRE-2, -5, and -6 motifs within the promoter regulatory sequences. This study demonstrates that hypoxia-induced KSHV lytic replication is mediated at least in part through cooperation of HIF-1 alpha with LANA bound to the HRE motifs of the Rta promoter.

1141584386 Cell type specific regulation of human placenta growth factor

Background: Maternal serum levels of PlGF are significantly lower in preeclamptic patients than in normal pregnant women. PlGF mRNA expression is cell type specific with high levels in trophoblast but comparatively low levels in most nontrophoblast. Despite two consensus hypoxia response elements (HREs), PlGF expression is down regulated by hypoxia in trophoblast, but increased in other cell types. These observations suggest unique regulatory mechanisms control PlGF expression. We sought to characterize PlGF promoter regions which function to regulate transcription rates in different cell types and under hypoxia.Methods: Regions of human PlGF gene (−1521/+34), (−828/+34), (−698/+34) were cloned into a reporter vector and (−844/−677) was cloned into a minimal TK promoter containing expression vectors. Trophoblast (JEG3 and JAR), and nontrophoblast (HeLa and hEK‐293) were transiently transfected to determine functional responsiveness of the regions. A PlGF gene region containing two HREs was cloned upstream of a TK containing vector to determine hypoxia (1% O2) responsiveness.Results: PlGF (−1521/+34), (−828/+34) regions showed high promoter activity in trophoblast cells, but little activity in nontrophoblast cells. PlGF (−844/−677) cloned downstream of the TK reporter, augmented promoter activity in trophoblast but not nontrophoblast cells. When cloned upstream to the TK promoter, this region significantly decreased activity in nontrophoblast cells. Surprisingly, the PlGF region containing HREs produced little increase in minimal promoter activity during hypoxia in any of the cells.Conclusions: PlGF transcription is cell type specific and a region of PlGF (−828/−698) may act as an enhancer in trophoblast yet mediates repressor activity in nontrophoblast. The putative HREs in PlGF 5’UTR do not seem to increase PlGF transcription under hypoxia suggesting that they are not essential for governing PlGF expression. Further understanding of transcriptional regulation of PlGF in trophoblast may facilitate approaches to increase expression during preeclampsia.

Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Transcriptional repression of human cad gene by hypoxia inducible factor-1

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1α accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (−710/+122) and the −67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1α but abolished by overexpression of dominant-negative HIF-1α. A chromatin immunoprecipitation assay provides further evidence that HIF-1α binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1α, which represents a functional link between hypoxia and cell-cycle arrest.

Hypoxia Increases Group IIA Phospholipase A2 Expression under Inflammatory Conditions in Rat Renal Mesangial Cells

Hypoxia evokes a common mechanism of oxygen sensing mediated by hypoxia-inducible transcription factors (HIF) in many mammalian cells. This study investigated the effect of hypoxia on group-IIA secretory phospholipase A(2) (sPLA(2)-IIA) expression in renal mesangial cells. Stimulation of cells with IL-1beta under normoxic conditions (21% O(2)) is known to induce expression and secretion of the group sPLA(2)-IIA. This induction is further enhanced by constantly reducing the O(2) concentration to 1% O(2), and is accompanied by increased sPLA(2) activity. To see whether hypoxia potentiates IL-1beta-induced sPLA(2)-IIA gene expression, a 2.67-kb fragment of the rat sPLA(2)-IIA promoter was fused to a luciferase reporter construct and used to transfect mesangial cells. Hypoxia alone is not able to activate the sPLA(2) promoter, whereas it significantly enhances IL-1beta-stimulated promoter activity. A deletion mutant of the promoter that lacks the two putative hypoxia responsive elements (HRE) is devoid of the potentiating effect of hypoxia. Moreover, site-directed mutagenesis of either of the two HRE is sufficient to abolish the potentiating effect of hypoxia. Electrophoretic mobility shift assays show that HIF-2alpha, which is the only HIF subtype expressed in mesangial cells, binds to both HRE in the sPLA(2)-IIA promoter. In summary, the data show that in an inflammatory setting hypoxia is able to potentiate sPLA(2)-IIA expression and activity in renal mesangial cells, and thereby may critically contribute to enhanced formation of inflammatory lipid mediators seen in a diverse range of kidney diseases.

6-Phosphofructo-2-kinase (pfkfb3) Gene Promoter Contains Hypoxia-inducible Factor-1 Binding Sites Necessary for Transactivation in Response to Hypoxia

The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

Up-regulation of 94-kDa glucose-regulated protein by hypoxia-inducible factor-1 in human endothelial cells in response to hypoxia

Hypoxic environment in solid tumor is known to favor cell survival and to initiate the formation of new capillaries. In this work, we identified by 2D gel analysis 94-kDa glucose-regulated protein (GRP94) as being upregulated in human endothelial cells in response to hypoxia. Three putative hypoxia responsive elements (HRE) were found in the GRP94 promoter. Competition experiments of HIF-1 DNA binding using specific probes containing each HRE sequence of the GRP94 promoter clearly evidenced that HIF-1 binds these sequences with high affinity. The human GRP94 promoter was then cloned upstream of the luciferase gene and showed enhanced activity in hypoxic conditions. Mutation of two of the three HREs present in this promoter completely inhibited the hypoxia-induced increase in luciferase activity.

Prolonged Hypoxia Differentially Regulates Hypoxia-inducible Factor (HIF)-1α and HIF-2α Expression in Lung Epithelial Cells: IMPLICATION OF NATURAL ANTISENSE HIF-1α

Transcriptional adaptations to hypoxia are mediated by hypoxia-inducible factor (HIF)-1, a heterodimer of HIF-alpha and aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1alpha and HIF-2alpha subunits both undergo rapid hypoxia-induced protein stabilization and bind identical target DNA sequences. When coexpressed in similar cell types, discriminating control mechanisms may exist for their regulation, explaining why HIF-1alpha and HIF-2alpha do not substitute during embryogenesis. We report that, in a human lung epithelial cell line (A549), HIF-1alpha and HIF-2alpha proteins were similarly induced by acute hypoxia (4 h, 0.5% O(2)) at the translational or posttranslational level. However, HIF-1alpha and HIF-2alpha were differentially regulated by prolonged hypoxia (12 h, 0.5% O(2)) since HIF-1alpha protein stimulation disappeared because of a reduction in its mRNA stability, whereas HIF-2alpha protein stimulation remained high and stable. Prolonged hypoxia also induced an increase in the quantity of natural antisense HIF-1alpha (aHIF), whose gene promoter contains several putative hypoxia response elements to which (as we confirm here) the HIF-1alpha or HIF-2alpha protein can bind. Finally, transient transfection of A549 cells by dominant-negative HIF-2alpha, also acting as a dominant-negative for HIF-1alpha, prevented both the decrease in the HIF-1alpha protein and the increase in the aHIF transcript. Taken together, these data indicate that, during prolonged hypoxia, HIF-alpha proteins negatively regulate HIF-1alpha expression through an increase in aHIF and destabilization of HIF-1alpha mRNA. This trans-regulation between HIF-1alpha and HIF-2alpha during hypoxia likely conveys target gene specificity.

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding.

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.