HIF-1alpha Activity Research Articles

Abstract 5722: Retention of the radiosensitizing effect of gemcitabine and its main metabolite dFdU under reduced oxygen conditions is not influenced by HIF-1 functionality

Abstract Introduction: Regions within solid tumors often experience mild to severe oxygen deprivation, associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitizing effect of gemcitabine and its main metabolite dFdU under normal versus reduced oxygen conditions and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitizing mechanism. Materials & methods: The clonogenic assay was performed in three isogenic MDA-MB-231 breast cancer cell lines differing in HIF-1alpha proficiency (24h 0-8 nM gemcitabine or 0-4 microM dFdU, 0-8 Gy irradiation). Validation of the transfection with dominant negative HIF-1alpha was done by western blot and by assessment of HIF-1alpha activity. The relative expression of 84 genes related to the hypoxia signaling pathway was characterized by human hypoxia signaling pathway PCR array. Using radiosensitizing conditions, cells were collected for cell cycle analysis. Results: HIF-1 activity was significantly inhibited after transfection with a dominant negative protein. Furthermore, anoxia-induced VEGF secretion was significantly lower (p<0.05) in MDA-MB-231 cells stably expressing dnHIF (dominant negative HIF-1alpha) than in MDA-MB-231 EV (empty vector) cells. PCR array indicated that hypoxic conditions significantly influenced the expression of HIF-1alpha and ANGPTL4 (angiopoetin-like 4). A clear radiosensitizing effect under normoxic and anoxic conditions was observed for both gemcitabine and dFdU. The radiosensitizing effect was similar under normoxic and anoxic conditions (p=0.48 for gemcitabine, p=0.56 for dFdU) and the dose enhancement factor (DEF) was not significantly influenced by the cell line used. Under anoxia, DEFs for gemcitabine ranged from 1.26 to 1.59, from 1.11 to 1.77, and from 1.34 to 2.04 in MDA-MB-231 wt, EV and dnHIF cells, respectively. Statistical analysis revealed no significant differences in radiobiological parameters between HIF-1 proficient and HIF-1 deficient cells. Cell cycle analysis showed that, in contrast to previous findings in other cell lines, exposure of MDA-MB-231 cells to low oxygen conditions did not induce a significant increase in the percentage of G0/1 cells (p=0.21). Gemcitabine and dFdU caused a block of cells in the S phase of the cell cycle under both normoxic and anoxic conditions. Conclusion: For the first time, radiosensitization by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. As dFdU has a prolonged half-life, the sustained presence of dFdU in the blood might induce radiosensitization despite the short half-life of the parent drug, gemcitabine. This might be highly relevant, especially considering delivery of the drug to hypoxic tumor regions. No major role for HIF-1 in radiosensitization by gemcitabine or dFdU could be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5722. doi:1538-7445.AM2012-5722

Hypoxia suppresses E-cadherin and enhances matrix metalloproteinase-2 expression favoring esophageal carcinoma migration and invasion via hypoxia inducible factor-1 alpha activation

The goal of this study was to explore the molecular mechanism of hypoxia inducible factor-1 alpha (HIF-1 alpha) action on migration and invasion of esophageal carcinoma cells. We used cobalt chloride (CoCl(2) ) to mimic tumor hypoxic microenvironment and analyzed the expressions of E-cadherin, matrix metalloproteinase-2 (MMP-2), and HIF-1 alpha in esophageal carcinoma cells under hypoxia by reverse transcription polymerase chain reaction and Western blotting. To analyze the function of HIF-1 alpha in Eca109 and TE1 cells, we established stable HIF-1 alpha knockdown cells using small interfering RNA. Blocking effect was detected by Western blotting. The concentrations of MMP-2 protein in the conditioned medium were also determined by enzyme-linked immunosorbent assay. Wound-healing and cell invasion assay were used to evaluate the migration and invasion of esophageal carcinoma cells. After exposure to hypoxia, expressions of HIF-1 alpha protein in Eca109 and TE1 cells were upregulated, both mRNA and protein levels of E-cadherin were downregulated, and MMP-2 were upregulated (P < 0.05), whereas HIF-1 alpha mRNA had no significant change (P > 0.05). Small interfering RNA could block HIF-1 alpha effectively under hypoxia, then enhanced E-cadherin expression and inhibited MMP-2 expression, respectively. Furthermore, expression of HIF-1 alpha protein was stable even though MMP-2 repressed by BB2516. Compared with that in normoxia, Snail expression was enhanced when Eca109 or TE1 cells exposed to hypoxia. Once HIF-1 alpha blocked, Snail expressions were inhibited accordingly. Wound recovery and the number of invading cells decreased (P < 0.05) after HIF-1 alpha blocked. The hypoxia suppresses E-cadherin expression and enhances MMP-2 expression favoring esophageal carcinoma migration and invasion via HIF-1 alpha activation. Our observations suggest that HIF-1 alpha inhibition might be an effective strategy to weaken the migration and invasion of esophageal carcinoma cells.

Cis-regulatory functions of overlapping HIF-1alpha/E-box/AP-1-like sequences of CD164

BackgroundCD164 (also known as MGC-24v or endolyn) is a sialomucin which has been suggested to participate in regulating the proliferation, cell adhesion and differentiation of hematopoietic stem and progenitor cells. CD164 is also involved in the development of cancer. The functions of cis-regulatory elements of CD164 remain relatively unknown.MethodsIn this study, we investigated the function of cis-regulatory elements within the promoter of CD164. We fused the 5'-flanking region of CD164 to a luciferase reporter vector. The minimal promoter region was confirmed by luciferase reporter assay. Using in silico analysis, we found the presence of one HIF-1alpha (HIF-1A) motif (5_-RCGTG-3_) overlapping E-box (CACGTG) and two AP-1-like binding sites (CGCTGTCCC, GTCTGTTG), one of which is also overlapped with HIF-1alpha sequence. Dual-luciferase assay was performed to examine the transcriptional activity of AP-1 and HIF-1alpha of CD164 promoter. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to measure CD164 expression. Chromatin Immunoprecipitation was used to confirm the binding of HIF-1alpha and CD164.ResultsCo-transfection of c-jun, HIF-1alpha and minimal promoter region construct demonstrated that c-jun and HIF-1alpha bound the CD164 promoter and promoted CD164 expression. Hypoxia treatment also led to the up-regulation of CD164 expression. The mutation of overlapping sequences resulted in the reduced expression of CD164 induced by HIF-1alpha. Chromatin Immunoprecipitation demonstrated that the HIF-1alpha bound the minimal promoter region.ConclusionsDetermination of the optimal promoter region and transcription factors governing CD164 expression is useful in understanding CD164 functions. These results suggest that cis-regulatory elements of CD164 overlapping HIF-1alpha/E-box/AP-1-like sequences may play important regulatory roles.

A non-specific effect of orally administered Escherichia coli

A number of genetically modified bacteria able to deliver a therapeutic gene into target cells has already been tested. Apart from the expected effects of bacterial therapy, the therapeutic bacterial strain also mediates a non-specific effect independent of the gene to be delivered. In this regard, we have recently shown that oral administration of the bacterial strain Escherichia coli XL1-Blue via gastric gavage to rats leads to a non-specific decrease in expression of vascular endothelial growth factor (VEGF) in intestinal wall without corresponding changes in other parameters. We tried to adopt a model of intestinal ischemia and to treat the subsequent hypoxic condition using a strain carrying the effector plasmid encoding hypoxia-inducible factor 1 alpha (HIF-1alpha), as well as the helper plasmid encoding invasion and listeriolysin O. However, the model was ineffective, as obvious from macroscopic and molecular observations. We hypothesize that a competitive behavior of the administered strain in the intestinal microbiota leads to a decrease in activity of HIF-1alpha and reduction in expression of VEGF. Also, a functional disease model would be necessary for the invasion-expressing therapeutic strain to be effective. A different approach using bacterial protein delivery would possibly circumvent these bactofection-related problems.

Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration

Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.

Pharmacological Strategies that Affect HIF-1 in the Ischemic Brain: Focus on Hydroxylases Activity and Protein Kinase Pathways

Ischemic stroke is a leading cause of morbidity and mortality worldwide which incidence is increasing with society aging. Unfortunately, despite intensive research in treatments to reduce mortality and the severity of the cerebrovascular injury, there are not effective therapies available. Hypoxia-Inducible Factor-1 (HIF-1) triggers the overexpression of genes coding for proteins involved in the adaptative response of the cell to oxygen deprivation. The functional HIF unit is a heterodimeric protein composed of the two subunits, HIF-1alpha and HIF-1beta. HIF-1alpha activity is regulated through different mechanisms involving post-transcriptional and post-translational modifications. During normoxia, prolyl hydroxylases modify HIF-1alpha and marks it for proteasomal degradation. Under hypoxia, reduced activity of prolyl hydroxylases allows stabilization of HIF-1alpha and increases protein levels. Also, several transduction pathways have been proposed to act downstream of putative oxygen sensors and lead to HIF-1alpha phosphorylation by kinases. Collectively, these modifications regulate HIF-1alpha activity inducing gene expression associated with brain protection after ischemia. In this review, we summarize some of the latest compounds that have shown to regulate HIF function and induce protection in brain cells subjected to ischemia. We focus on compounds that target HIF hydroxylases and the PI-3K/Akt and the MAPK/ERK pathways. Increased HIF-1alpha expression holds great promise for the treatment of cerebral ischemia and makes HIF hydroxylases and kinases attractive therapeutic targets. The reduced number of reports associated to the study of kinases and HIF-1alpha in brain, points out the need of further investigation. Keywords: Cerebral ischemia, HIF-1alpha, MAPK/ERK, PI-3K/Akt, prolyl hydroxylases inhibitors, brain ischemia, Hypoxia-Inducible Factor-1 (HIF-1a), prolyl hydroxylases (PH), phosphoinositide 3-kinase (PI-3k), mitogen activated protein kinases (MAPK), Prolil hydroxylation inhibition, Asparaginil hydroxylation inhibition, TM6008, TM6089, Ethyl 3,4-dihydroxybenzoato (EDHB), Deferoxamine (DFO), Metal Ions, Cobalt chloride (CoCl2), Ginkgolides, Baicalein, Clioquinol, Zinc, Erytrhopoietin, hypoxia, HIF-1 phosphorylation, PH inhibitors, Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), MCAO, chemical hypoxia, ischemic stroke

Abstract 2047: Betulinic acid exerts anti-angiogenic activity via regulation of HIF-1 alpha and STAT3 in PC-3 prostate cancer cells under hypoxia

Abstract Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively overexpressed in several human cancer cells and related to various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined whether betulinic acid (BA), a triterpene from the bark of white birch, could inhibit hypoxia-mediated activation of STAT3 and HIF-1 alpha in androgen independent human prostate cancer PC-3 cells. Our results show that BA inhibited the transcriptional activities and nuclear accumulation of both HIF-1 alpha and STAT3 under hypoxia. In addition, BA significantly reduced cellular and secreted levels of hypoxia-induced vascular endothelial growth factor (VEGF), a critical angiogenic factor, in PC-3 cells. BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced PC-3 cells. Moreover, silencing HIF 1 alpha and STAT3 by siRNA transfection augmented the suppression of VEGF expression by BA under hypoxia. Mechanistically, BA inhibited the recruitment of HIF-1 alpha and STAT3 on the VEGF promoter. Taken together, these findings suggest that BA may exert angio-prevention via inactivation of hypoxia-induced STAT3 and HIF-1 alpha in PC-3 cells. The in vivo anti-angiogenic and anti-PCa efficacy are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2011-2047

Chromosome 14q imbalances and pathways associated with resistance to antiangiogenic therapy in clear cell renal cell carcinoma.

339 Background: Antiangiogenic agents are used to treat metastatic clear cell renal cell carcinoma (ccRCC). Currently there are no biomarkers of therapeutic efficacy for these agents. Hypoxia inducible factor (HIF) alpha ratios have been linked to phenotypically distinct ccRCC subpopulations. The HIF1 alpha gene is located on chromosome 14q. In this study, the goal was to determine whether chromosomal imbalances identified with SNP arrays are linked to HIF ratios, and to clinical outcome. Methods: We obtained archival FFPE tumor specimens from 56 patients with mRCC treated with sorafenib or bevacizumab. DNA from the FFPE blocks was analyzed with Affymetrix 250K Nsp SNP microarrays. We identified the presence of genomic imbalances and loss of heterozygosity (LOH) to obtain virtual karyotypes. We then evaluated candidate genes in gain/lost chromosomal regions by qPCR and immunohistochemistry (IHC) in the bevacizumab treated specimens. Results: In the bevacizumab cohort, HIF1-alpha containing14q loss showed a significant association with worse response to treatment (CR/ PR vs. SD/PD, Fisher exact test, p = 0.0473). In addition, HIF1A mRNA expression was significantly reduced in all samples with 14q loss and was associated with PFS (HR = 2.29, 95% CI = 1.01-5.16, p = 0.045). HIF-1alpha protein expression was also reduced in samples with 14q loss. Conclusions: Chromosomal imbalances are associated with outcomes in ccRCC patients treated with antiangiogenic agents, and can lead to changes in gene expression. Low HIF1A expression was strongly correlated with shorter PFS. We hypothesize that loss of 14q could lead to an imbalance in HIF-1alpha/HIF-2alpha activity, leading to increased HIF-2alpha and enhanced c-Myc expression, which improves tumor cell viability and engenders resistance to cellular stress induced by antiangiogenic therapy. No significant financial relationships to disclose.

HIF-1's Role in Estrogen-induced VEGF Expression: Implications for both Normal and Pathological Cell Proliferation.

Vascular endothelial growth factor (VEGF) is a potent inducer of vascular hyperpermeability and the key mediator of angiogenesis, both of which are involved in normal development and reproduction, as well as cancer and other diseases. VEGF is rapidly and strongly induced in endometrial epithelial cells by 17beta-estradiol (E2). We have proposed that this "immediate-early" response is the key to E2-induced cell proliferation. Recently, we linked E2-induced VEGF expression in the endometrium to the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), which regulates over 100 genes essential for cellular adaptations to low oxygen and other stresses. E2, via the PI3K/Akt pathway, induces the rapid recruitment of both HIF-1alpha and ERalpha to the VEGF gene promoter. In contrast to the endometrium, however, E2 has little or no effect on VEGF expression in breast and endometrial cancer cells in vitro. We recently resolved this inconsistency by showing that the lack of E2 induction in vitro is due to a deficiency of HIF-1alpha caused by the high oxygen level to which cultured cells are exposed. Restoration of HIF-1alpha in endometrial and breast cancer cells restores rapid and strong VEGF expression by E2. Thus, VEGF-induced vascular hyperpermeability, which floods tissues with plasma proteins, should now be considered a potential mediator of E2-induced cell proliferation in both normal target tissues and E2-dependent tumors. In summary, we have for the first time linked E2 action to HIF-1alpha activation and VEGF expression, thereby providing new insight into E2's effects in target tissues, and into possible treatments for E2-associated cancers. (platform)

The Distinct Metabolic Profile of Hematopoietic Stem Cells Reflects Their Location in a Hypoxic Niche

Bone marrow transplantation is the primary therapy for numerous hematopoietic disorders. The efficiency of bone marrow transplantation depends on the function of long-term hematopoietic stem cells (LT-HSCs), which is markedly influenced by their hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. Here, we show that LT-HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We used flow cytometry to identify a unique low mitochondrial activity/glycolysis-dependent subpopulation that houses the majority of hematopoietic progenitors and LT-HSCs. Finally, we demonstrate that Meis1 and Hif-1alpha are markedly enriched in LT-HSCs and that Meis1 regulates HSC metabolism through transcriptional activation of Hif-1alpha. These findings reveal an important transcriptional network that regulates HSC metabolism.

Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB

BackgroundReduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1α has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1α's role in determining chemosensitivity focussing on responsible molecular pathways.Methodology and Principal FindingsRNA interference was applied to inactivate HIF-1α or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-κB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1α in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1α-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1α markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1α-competent cells was identified as the molecular mechanism of HIF-1α-mediated inhibition of p53. Furthermore, loss of HIF-1α abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-κB and expression of anti-apoptotic NF-κB target genes. Accordingly, reconstitution of the NF-κB subunit p65 reversed the increased chemosensitivity of HIF-1α-deficient cells.Conclusion and SignificanceIn summary, we identified HIF-1α as a potent regulator of p53 and NF-κB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

Activation of hypoxia-inducible factor-1&amp;alpha; via nuclear factor-&amp;kappa;B in rats with chronic obstructive pulmonary disease

Accumulating data suggested that hypoxia inducible factor (HIF)-1alpha plays an important role in the evolution and propagation of the inflammatory process. To characterize the activation of HIF-1alpha in rats with chronic obstructive pulmonary disease (COPD) and examine the possible role of nuclear factor (NF)-kappaB in this process, rats were challenged by introtracheal instillation of lipopolysaccharide (LPS) and exposure to cigarette smoke. Pyrrolidine dithiocarbamate (PDTC) was administered via the oral route 1 h before LPS or cigarettes administration. Four weeks later, pulmonary function and histology were tested; bronchoalveolar lavage fluid (BALF) and arterial blood gases were assayed. Activation of pulmonary NF-kappaB was assessed by quantitative PCR, immunoblot analysis, and electrophoretic mobility shift assay, respectively. Results showed that LPS and smog induced the characteristics of COPD seen in human. PDTC alleviated the development of COPD and the levels of cytokines in BALF of PDTC+COPD group were significantly decreased compared with that of COPD group. The activation of pulmonary NF-kappaB was inhibited by PDTC and the accumulation of HIF-1alpha gene expression in the COPD group was attenuated by PDTC pretreatment. Furthermore, the mRNA levels of HIF-1alpha target genes heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were parallel to the attenuation of HIF-1alpha by PDTC. These findings indicated that the activation of HIF-1alpha pathway via NF-kappaB contributes to the development of COPD, and administration of NF-kappaB inhibitor may attenuate the development of COPD.

Placenta Growth Factor-induced Early Growth Response 1 (Egr-1) Regulates Hypoxia-inducible Factor-1α (HIF-1α) in Endothelial Cells

Leukotrienes, the lipid inflammatory products derived from arachidonic acid, are involved in the pathogenesis of respiratory and cardiovascular diseases and reactive airway disease in sickle cell disease. Placenta growth factor (PlGF), elaborated from erythroid cells, increased the mRNA expression of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) in human pulmonary microvascular endothelial cells. PlGF-induced both promoter activity and mRNA expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was abrogated by early growth response-1 (EGR-1) small interfering RNA. PlGF showed a temporal reciprocal relationship in the mRNA levels of EGR-1 and NAB2, the latter a repressor of Egr-1. Moreover, Nab2, but not mutant Nab2, significantly reduced promoter activity and mRNA expression of HIF-1alpha and also reduced expression of the HIF-1alpha target gene FLAP. Furthermore, overexpression of Egr-1 led to increased promoter activities for both HIF-1alpha and FLAP in the absence of PlGF. Additionally, the Egr-1-mediated induction of HIF-1alpha and FLAP promoters was reduced to basal levels by EGR-1 small interfering RNA. The binding of Egr-1 to HIF-1alpha promoter was corroborated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, which showed increased Egr-1 binding to the HIF-1alpha promoter in response to PlGF stimulation. These studies provide a novel mechanism for PlGF-mediated regulation of HIF-1alpha via Egr-1, which results in increased FLAP expression. This study provides a new therapeutic target, namely Egr-1, for attenuation of elevated leukotriene levels in patients with sickle cell disease and other inflammatory diseases.

Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1alpha in melanoma cells.

BackgroundThe endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.Principal FindingsHere we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.ConclusionsIn this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

BackgroundHypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.Methodology/Principal FindingsWe investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Conclusions/SignificanceBoth HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1α in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1α using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1α stabilization or activity through different mechanisms. Stabilization of HIF-1α protein by hypoxia or CoCl2 did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1α controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1α in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1α under normoxic conditions with physiological roles that differ from those induced by hypoxia.

Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α

To survive in hypoxic environments, organisms must be able to cope with redox imbalance and oxygen deficiency. The SIRT1 deacetylase and the HIF-1alpha transcription factor act as redox and oxygen sensors, respectively. Here, we found that SIRT1 binds to HIF-1alpha and deacetylates it at Lys674, which is acetylated by PCAF. By doing so, SIRT1 inactivated HIF-1alpha by blocking p300 recruitment and consequently repressed HIF-1 target genes. During hypoxia, SIRT1 was downregulated due to decreased NAD(+) levels, which allowed the acetylation and activation of HIF-1alpha. Conversely, when the redox change was attenuated by blocking glycolysis, SIRT1 was upregulated, leading to the deacetylation and inactivation of HIF-1alpha even in hypoxia. In addition, we confirmed the SIRT1-HIF-1alpha interaction in hypoxic mouse tissues and observed in vivo that SIRT1 has negative effects on tumor growth and angiogenesis. Our results suggest that crosstalk between oxygen- and redox-responsive signal transducers occurs through the SIRT1-HIF-1alpha interaction.

Validated analysis of HIF-1α expression in cancer cells using a controlled and comparative immunoassay

The immunoreactivity of hypoxia inducible factor 1alpha (HIF-1alpha) has been considered a reliable indicator of the HIF-1 pathway activation in tissue hypoxia. However, HIF-1alpha immunoreactivity has been evaluated with different antibodies and heterogeneous protocols. The need to interpret contradictory findings requires, among other things, a comparison of the antibodies. This could be accomplished by using identical, well characterized antigenic targets and by decreasing the influence of other variables. We applied most of the commercially available antibodies, and an antibody developed in our laboratories, to the human cervical cancer HeLa cell line and tissue sections from a renal cell carcinoma systematically, and to other tumors selectively. The expression of HIF-1alpha in HeLa cells was induced by the hypoxia-mimetic DFO. Non-induced HeLa cells were used as 'genuine' negative controls in addition to routine ones. HeLa cells (both induced and not induced) were also examined by immunofluorescence and Western blotting. We found that the antibodies showed immunostaining patterns with remarkable qualitative and quantitative differences, an observation not emphasized in previous literature. Certain antibodies require careful application to avoid specificity issues, and others to avoid low sensitivity problems. Pairing certain antibodies can optimize evaluation of HIF-1alpha expression. Most previous immunohistochemical studies of HIF-1alpha have attempted to map hypoxic neoplastic tissues or to demonstrate hypoxia in studies of neoangiogenesis, rather than 'measuring' HIF-1alpha expression or activation, because this requires a validated immunoassay. Our study thus allows for the development of a controlled and comparative HIF-1alpha immunoassay, which could be valuable if HIF-1alpha becomes a therapeutic target.

Hyperforin is a modulator of inducible nitric oxide synthase and phagocytosis in microglia and macrophages

Upon activation, microglia, the immunocompetent cells in the brain, get highly phagocytic and release pro-inflammatory mediators like nitric oxide (NO). Excessive NO production is pivotal in neurodegenerative disorders, and there is evidence that abnormalities in NO production and inflammatory responses may at least support a range of neuropsychiatric disorders, including depression. Although extracts of St. John's wort (Hypericum perforatum L.) have been used for centuries in traditional medicine, notably for the treatment of depression, there is still considerable lack in scientific knowledge about the impact on microglia. We used N11 and BV2 mouse microglia, as well as RAW 264.7 macrophages to investigate the effects of St. John's wort extract and constituents thereof on NO production Moreover, flow cytometry and fluorescence microscopy were employed to analyze the influence on phagocytosis, transcription factor activation states, and cell motility. We found that extracts of St. John's wort efficiently suppress lipopolysaccharide-induced NO release and identified hyperforin as the responsible compound, being effective at concentrations between 0.25 and 0.75 microM. The reduced NO production was mediated by diminished inducible nitric oxide synthase expression on the mRNA and protein level. In addition, at similar concentrations, hyperforin reduced zymosan phygocytosis to 20-40% and putatively acted by downregulating the CD206 macrophage mannose receptor and modulation of cell motility. We found that the observed effects correlate with a suppression of the activated state of Nf-kappaB and phospho-CREB, while c-JUN, STAT1, and HIF-1alpha activity and cyclooxygenase-2 expression remained unaffected by hyperforin. These results reveal that hyperforin influences pro-inflammatory and immunological responses of microglia that are involved in the progression of neuropathologic disorders.

Granulosa cell tumor with activated mTOR‐HIF‐1α‐VEGF pathway

The DNA-binding activity of hypoxia-inducible factor-1 alpha (HIF-1alpha) has been analyzed for various gynecological tumors. Among the tumors that were studied, there was a finding of a high level of DNA-binding HIF-1alpha activity, although it was limited to one case of adult type granulosa cell tumor (GCT). In this case a 60-year-old female had marked immunohistochemical expression of HIF-1alpha. The expressions of the mammalian target of rapamycin (mTOR) and phosphorylated-mTOR (p-mTOR) were also marked, and vascular endothelial growth factor (VEGF) was moderately expressed. To compare the expression profiles, 11 consecutive cases with adult type GCT were used. All cases showed marked expressions of HIF-1alpha and mTOR, but p-mTOR expression was moderately to markedly observed in four of the 12 cases. VEGF was expressed in all cases in varying degrees. Based on the evidence that downregulation of the mTOR pathway due to treatment with rapamycin (everolimus) would suppress tumor cell growth, an experimental study using the GCT cell line was designed to clarify whether HIF-1alpha and VEGF expressions decline. As a result, the expressions of p-mTOR, HIF-1alpha and VEGF were suppressed, but those of mTOR were not. It was concluded that mTOR-targeted therapy may represent a promising strategy for some GCT with an activated mTOR-HIF-1alpha-VEGF pathway.