HIF-1α Binding Research Articles

Selective HIF-1 Regulation under Nonhypoxic Conditions by the p42/p44 MAP Kinase Inhibitor PD184161

Hypoxia-inducible factor-1 (HIF-1) is a key gene regulator for cellular adaptation to low oxygen. In addition to hypoxia, several nonhypoxic stimuli, including hormones and growth factors, are essential for cell-specific HIF-1 regulation. Our studies have highlighted angiotensin II (AngII), a vasoactive hormone, as a potent HIF-1 activator in vascular smooth muscle cells (VSMC). AngII increases HIF-1 transcriptional activity by modulating specific signaling pathways. In VSMC, p42/p44 mitogen-activated protein kinase (MAPK) pathway activation is essential for HIF-1-mediated transcription during AngII treatment. The present study shows that PD184161, a potent MEK1/2 inhibitor, is an HIF-1 blocker in Ang II-treated VSMC. Unlike PD98059, a widely-used MEK1/2 inhibitor, we found that PD184161 blocked AngII-driven HIF-1α protein induction in a dose-dependent manner. Interestingly, the effect of PD184161 was specific to nonhypoxic activators, since HIF-1α induction by hypoxia (1% O2) was unaffected under similar conditions. VSMC treatment with MG132, a proteasome inhibitor, indicated that PD184161 influenced HIF-1α protein stability. PD184161 also increased HIF-1α binding to von Hippel-Lindau tumor suppressor protein, an E3 ligase component and an indication of HIF-1α hydroxylation. Finally, we show that PD184161 blocked mitochondrial ROS (mtROS) production and cellular ATP levels, at the same time enhancing ascorbate availability in AngII-treated VSMC. Taken together, our study indicates that, independently of p42/p44 MAPK activation, PD184161 blocks mtROS generation by AngII, leading to re-establishment of cellular ascorbate levels, increased VHL binding, and decreased HIF-1α stability. Therefore, this study reveals a previously unsuspected role for PD184161 as an HIF-1 inhibitor in VSMC under nonhypoxic conditions.

Abstract 5469: Low dose ionizing radiation induces persistent activation of NADPH oxidase pathway in mouse colon

Abstract Exposure to ionizing radiation has been linked to persistent oxidative stress. The purpose of the current study was to characterize the role of NADPH oxidase pathway in radiation-induced increased oxidant production in colon epithelial cells. Mice (C57BL/6J; 6-8 weeks, male) were exposed to either sham or 0.5 Gy γ radiation, and NADPH oxidase pathway and oxidative stress markers were assessed in colon samples 60 d after exposure. Radiation exposure led to higher elevation of 4-HNE and 8-oxo-dG staining relative to unirradiated control. We also observed increased staining for the proliferative marker phospho-histone H3 in colon sections of irradiated mice. PCR analysis showed increased expression of two NADPH oxidase isoforms, Nox1 and Nox3, in irradiated samples. Additionally, expression of NoxA1 and Noxo1 along with Hif1α were also increased after radiation. Immunoblot analysis confirmed the PCR results, and ChIP assay revealed greater binding of stress response factor GATA6 and Hif1α to Nox1 and Nox3, and Nox3 promoters respectively after radiation exposure relative to control. Co-immunoprecipitation experiments show enhanced binding of Rac1, an activator of NADPH oxidase, to Nox1 and Nox3. In summary, we demonstrated that exposure to a low dose of γ radiation caused long-term upregulation of NADPH oxidase isoforms as well as its regulators and activators such as NoxA1, Noxo1, Hif1α, TLR4, GATA6, Doux1, and Doux2. When considered along with our results on oxidative stress and proliferative markers, our observations on NADPH oxidase pathway provides new insight into molecular events contributing to radiation-induced persistent oxidative stress and cell proliferation in colon, and have implications for colorectal carcinogenesis. Note: This abstract was not presented at the meeting. Citation Format: Santosh Kumar, Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace, Kamal Datta. Low dose ionizing radiation induces persistent activation of NADPH oxidase pathway in mouse colon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5469. doi:10.1158/1538-7445.AM2017-5469

Methylation of BNIP3 in pancreatic cancer inhibits the induction of mitochondrial-mediated tumor cell apoptosis.

Bcl-2 interacting protein 3 (BNIP3) is involved in various cellular processes and is considered a key regulator of hypoxia-induced apoptosis. In the present study, the expression of BNIP3 in pancreatic cancer tissues, the correlation with clinicopathological characteristics and prognosis and the regulation of this protein in pancreatic cancer cell lines with regard to the induction of apoptosis were investigated. BNIP3 expression was significantly lower in pancreatic cancer tissues compared with normal epithelia and was associated with tumor size, clinical stage, and lymph node metastasis. The expression of BNIP3 correlated positively to the proapoptotic protein Bax and negatively to the antiapoptotic protein Bcl-2, whereas the induction of apoptosis by BNIP3 was independent of caspase 3 and 9 activation. The restoration of BNIP3 expression in pancreatic cancer cells in vitro, caused loss of ΔΨm, increase in ROS production, and apoptosis induction. The opposite effect was observed in pancreatic cancer cells, following BNIP3 silencing by RNAi. The absence of BNIP3 expression in pancreatic cancer cells was related to gene methylation that suppressed binding of HIF-1α to the BNIP3 promoter, whereas 5-Aza-2′-deoxycytidine (Aza-dC) treatment restored BNIP3 expression and sensitized pancreatic cancer cells to BNIP3-induced apoptosis. The findings indicated that BNIP3 was significantly downregulated in pancreatic cancer resulting in reduced apoptosis induction. Silencing of BNIP3 expression was associated with methylation of the hypoxia-responsive element (HRE) site that in turn inhibited the binding of HIF-1α to the BNIP3 promoter. The data suggest that BNIP3 reactivation is a potential target for therapeutic intervention against pancreatic cancer.

Abstract 548: Molecular Mechanism Behind Protein S Deficiency in Obese Patients

Introduction: Protein S is a vitamin K-dependent plasma protein, produced mainly in the liver; Protein S circulates in the blood at a concentration of 450 nM. Protein S is an anticoagulant, serving as a cofactor for APC and TFPI, and as an inhibitor of Factor IX (FIXa). Protein S deficiency causes deep vein thrombosis (DVT), increased risk of inflammation and, because DVT is a complication commonly observed in obese individuals, Protein S deficiency might be associated with obesity. Aim: To identify a correlation between Protein S deficiency and obesity, and identify the probable molecular mechanism behind the Protein S deficiency in the obese subjects. Methods: Immunoblots, ELISA, EMSA, CHIP, aPTT assay, and thrombin generation assay. Results: By ELISA, we measured a decrease in Protein S level in obese mice compared with wild type mice. In obesity, the liver becomes hypoxic, thus, we hypothesized that hypoxia and hypoxia inducible factor 1 alpha (HIF1α) may regulate Protein S expression in obesity. We found that a high fat diet induced HIF1α stability in mice. HIF1α levels were inversely proportional to Protein S levels, suggesting that HIF1α is a negative regulator of Protein S expression. We further identified a putative HIF1α binding site in the Protein S promoter, and, by using in vitro and in vivo assays, we demonstrated that HIF1α binds directly to the Protein S promoter and suppresses transcription. We further confirmed HIF1α-mediated Protein S transcriptional regulation in vivo, Plasma Protein S levels are increased in the liver-specific HIF1α knockout mouse whereas, liver-specific overexpression of HIF1α reduced the concentration of Protein S in the plasma. Conclusion: We conclude that HIF1α regulates Protein S expression in mouse liver and in obesity. Inhibition of HIF1α or intravenous injection of Protein S may reduce the occurrence of DVT in obese individuals.

Hypoxia-inducible microRNA-218 inhibits trophoblast invasion by targeting LASP1: Implications for preeclampsia development

Preeclampsia (PE) is a major contributor to maternal morbidity and mortality. However, the molecular mechanisms underlying PE progression are not well characterized. Here, we investigated the role of miR-218 in PE development. The expression of miR-218 and its host genes SLIT2 and SLIT3 was up-regulated in preeclamptic placentae compared to normal placentae. miR-218 expression was induced by hypoxia and decreased after knockdown of HIF-1α in an extravillous trophoblast cell line (HTR-8/SVneo). Chromatin immunoprecipitation assays showed direct binding of HIF-1α to the promoters of SLIT2 and SLIT3. Bioinformatics analysis identified LASP1 as a direct target of miR-218. Overexpression of miR-218 repressed the expression of LASP1 at both the mRNA and protein level. Meanwhile, miR-218 repressed the activity of a luciferase reporter containing the 3′-untranslated region of the LASP1 gene. Furthermore, expression of LASP1 rescued the inhibitory effect of miR-218 on HTR-8/SVneo cell invasion. Together, these results indicated that miR-218 contributes to PE by targeting LASP1 to inhibit trophoblast invasion.

Synovial cell death is regulated by TNF-\u03b1-induced expression of B-cell activating factor through an ERK-dependent increase in hypoxia-inducible factor-1\u03b1

B-cell activating factor (BAFF) has a role in the maturation and maintenance of B cells and is associated with rheumatoid arthritis (RA). Here, we investigated whether tumor necrosis factor (TNF)-α-induced BAFF expression controls the survival of fibroblast-like synoviocytes (FLS) and whether their survival can be regulated by TNF-α-mediated upregulation of hypoxia-inducible factor (HIF)-1α using MH7A synovial cells transfected with the SV40 T antigen. More TNF-α-treated cells died compared with the control. Survival was increased by incubation with Z-VAD but inhibited after transfection with BAFF-siRNA. Both BAFF and HIF-1α expression were enhanced when MH7A cells were treated with TNF-α. TNF-α-induced BAFF expression decreased in response to HIF-1α-siRNA, whereas it increased under hypoxia or by overexpressing HIF-1α. The HIF-1α binding site on the BAFF promoter (−693 to −688 bp) was confirmed by chromatin immunoprecipitation assay to detect the −750 to −501 bp and −800 to −601 bp regions. The BAFF promoter increased in response to TNF-α treatment or overexpression of HIF-1α. However, TNF-α-induced BAFF expression and promoter activity decreased after treatment with the ERK inhibitor PD98059. Cell death was enhanced by PD98059 but was inhibited by overexpression of HIF-1α. Taken together, our results demonstrate that BAFF expression to control synovial cell survival was regulated by HIF-1α binding to the BAFF promoter, and suggest for the first time that HIF-1α might be involved in the production of inflammatory cytokines to regulate the physiological function of rheumatic FLS.

Reduced Placental Regulator of G‐Protein Signaling‐2 (RGS2) and Preeclampsia

Though mid‐ and late‐gestational mechanisms of the late‐pregnancy cardiovascular disorder preeclampsia (PreE) are relatively well characterized, genetic risk factors and mechanisms driving the early gestational pathogenesis of PreE remain largely unclear. Various hormone activators of Gαq second‐messenger signaling pathways (eg ‐ vasopressin, endothelin, and angiotensin) have been implicated in preeclampsia. Regulator of G‐protein Signaling 2 (RGS2) acts as an endogenous terminator of Gαq signaling, and previous association studies have identified an increased risk for PreE and its sequelae in women carrying a single nucleotide polymorphism that results in reduced RGS2. We hypothesized that RGS2 is expressed in placental trophoblasts, and that reduced expression of RGS2 in placental tissue may represent a risk factor for the development of PreE due to disinhibited Gαq signaling. Placental tissue samples from PreE and clinically‐matched control pregnancies were obtained from the University of Iowa Maternal‐Fetal Tissue Bank and examined for mRNA of the B/R4 family of RGS proteins, including RGS2. Of the members examined (RGS2, ‐3, ‐4.2, ‐4.3, ‐4.4, ‐4.5, and ‐ 5) in control placentas (n=9), only RGS2 (Ct 28.8±0.7 vs 18S Ct 12.1±0.4) and RGS4.3 (Ct 23.0±0.4 vs 18S Ct 13.3±0.3) transcripts were detected above background levels. In PreE placenta (n=11), RGS2 mRNA may be suppressed (1.0±0.4 vs 0.2±0.3‐fold, p=0.1) while RGS4.3 remains unchanged (1.0±0.4 vs 1.1±0.4 fold, p=0.8). Immunohistochemical (IHC) detection similarly confirms cytoplasmic localization of RGS2 protein in trophoblasts of placentas from control and PreE human pregnancies. RGS2 positive trophoblasts are found in the junctional zone and labyrinth of placentas from wildtype C57BL/6J mice. We next examined the cardiovascular consequences of reducing feto‐placental RGS2 within a wildtype mother (wildtype C57BL/6J dam bred with either an RGS2‐knockout (KO) or littermate control sire). Reduced RGS2 in the feto‐placental unit caused increased HIF1α binding to chromatin (control 0.144±0.004 vs RGS2‐KO 0.155±0.004 AU, p=0.05, n=5 each), supporting placental hypoxia. Further, dams exhibited isolated diastolic hypertension by telemetry (24 hr mean: pre‐pregnancy control n=3, 92±2 vs RGS2‐KO n=5, 92±2; late‐pregnancy control 92±2 vs RGS2‐KO 98.2±2 mmHg; p<0.05 vs pre‐pregnancy RGS2‐KO, and pregnant control), consistent with microvascular dysfunction. We conclude that RGS2 is present in the cytoplasm of trophoblasts, that placental RGS2 is suppressed in PreE, and that loss of feto‐placental RGS2 is sufficient to cause placental hypoxia and maternal diastolic hypertension. Collectively these studies provide evidence supporting the novel concepts that disinhibited Gαq signaling in the placenta may contribute to PreE, and that a father's genetics may contribute to the risk for PreE.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma.

Role of cyclin dependent kinase 9(CDK9) as a potential target in esophageal adenocarcinoma (EAC) is unknown. We investigated CDK9 protein expression in EAC and Barrett's esophagus and role of CDK9 in oncogenic processes of EAC in vitro and in murine xenografts. The CDK9 expression was significantly higher in EAC as compared to Barrett's esophagus in patient samples. Stable shCDK9 in SKGT4 reduced proliferation by 37% at day 4, increased apoptosis at 48 hours and induced G1 cell cycle arrest at 48 hours (58.4% vs. 45.8%) compared to controls SKGT4 cells. SKGT4-shCDK9 cell-derived tumors were significantly smaller than control SKGT4-derived tumors in xenografts (72.89mm3 vs. 270mm3). Pharmaceutical inhibition of CDK9 by Flavopiridol (0.1μm for 48 hours) and CAN508 (20 and 40μm for 72 hours) induced significant reduction in proliferation and 2-fold increase in apoptosis in SKGT4, FLO1 and OE33 cells. In xenograft models, CAN508 (60 mg/kg/dayx10 days) and Flavopiridol (4mg/kg/dayx10 days) caused 50.8% and 63.1% reduction in xenograft tumors as compared to control on post-treatment day 21. Reduction of MCL-1 and phosphorylated RNA polymerase II was observed with transient shCDK9 in SKGT4 cells but not with stable shCDK9. CAN508 (20 and 40 μm) and Flavopiridol (0.1, 0.2 and 0.3 μm) for 4 hours showed reduction in MCL-1 mRNA (84% and 96%) and protein. Mcl-1 overexpression conferred resistance to Flavopiridol (0.2 μm or 0.4 μm for 48 hours) and CAN 508 (20 or 40μm for 72 hours). Chromatin immunoprecipitation demonstrated significant reduction of binding of transcriptional factor HIF-1α to MCL-1 promoter in FLO-1 cells by CDK9 inhibitors.

Canonical hedgehog signalling regulates hepatic stellate cell-mediated angiogenesis in liver fibrosis.

Hepatic stellate cells (HSCs) are liver-specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications. Rats and mice were treated with carbon tetrachloride for in vivo evaluation of hepatic angiogenesis and fibrotic injury. Diversified molecular approaches including real-time PCR, Western blot, luciferase reporter assay, chromatin immunoprecipitation, electrophoretic mobility shift assay and co-immunoprecipitation were used to investigate the underlying mechanisms in vitro. Angiogenesis was concomitant with up-regulation of Smoothened (SMO) and hypoxia inducible factor-1α (HIF-1α) in rat fibrotic liver. The SMO inhibitor cyclopamine and Gli1 inhibitor GANT-58 reduced expression of VEGF and angiopoietin 1 in HSCs and suppressed HSC tubulogenesis capacity. HIF-1α inhibitor PX-478 suppressed HSC angiogenic behaviour, and inhibition of hedgehog decreased HIF-1α expression. Furthermore, heat shock protein 90 (HSP90) was characterized as a direct target gene of canonical hedgehog signalling in HSCs. HSP90 inhibitor 17-AAG reduced HSP90 binding to HIF-1α, down-regulated HIF-1α protein abundance and decreased HIF-1α binding to DNA. 17-AAG also abolished 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (a SMO agonist)-enhanced HSC angiogenic properties. Finally, the natural compound ligustrazine was found to inhibit canonical hedgehog signalling leading to suppressed angiogenic properties of HSCs in vitro and ameliorated liver fibrosis and sinusoidal angiogenesis in mice. We have provided evidence that the canonical hedgehog pathway controlled HSC-mediated liver angiogenesis. Selective inhibition of HSC hedgehog signalling could be a promising therapeutic approach for hepatic fibrosis.

Hypoxia sensing through β-adrenergic receptors.

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the β-adrenergic receptor (β-AR) and hypoxia responses, we hypothesized that the β-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in β-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation-deficient β-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a hypoxia-mediated β-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the β-AR is fundamental to the molecular and physiological responses to hypoxia.

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and dictates H3K56 acetylation promoting hypoxia-induced tumour progression.

Intratumoural hypoxia induces HIF-1α and promotes tumour progression, metastasis and treatment resistance. HIF-1α stability is regulated by VHL-E3 ligase-mediated ubiquitin-dependent degradation; however, the hypoxia-regulated deubiquitinase that stabilizes HIF-1α has not been identified. Here we report that HAUSP (USP7) deubiquitinase deubiquitinates HIF-1α to increase its stability, induce epithelial-mesenchymal transition and promote metastasis. Hypoxia induces K63-linked polyubiquitinated HAUSP at lysine 443 to enhance its functions. Knockdown of HAUSP decreases acetylation of histone 3 lysine 56 (H3K56Ac). K63-polyubiquitinated HAUSP interacts with a ubiquitin receptor CBP to specifically mediate H3K56 acetylation. ChIP-seq analysis of HAUSP and HIF-1α binding reveals two motifs responsive to hypoxia. HectH9 is the E3 ligase for HAUSP and a prognostic marker together with HIF-1α. This report demonstrates that hypoxia-induced K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and causes CBP-mediated H3K56 acetylation on HIF-1α target gene promoters to promote EMT/metastasis, further defining HAUSP as a therapeutic target in hypoxia-induced tumour progression.

Hypoxia-Inducible Factor-1α Mediates Increased Sympathoexcitation via Glutamatergic N-Methyl-d-Aspartate Receptors in the Paraventricular Nucleus of Rats With Chronic Heart Failure.

Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the upregulation of NMDA-NR1 remains unexplored. We hypothesized that hypoxia via hypoxia-inducible factor 1α (HIF-1α) might contribute to the augmentation of the NMDA-NR1-mediated sympathoexcitatory responses from the PVN in CHF. Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible factor 1α were upregulated within the PVN of left coronary artery-ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA and protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR1 mRNA, protein levels, and glutamate-induced Ca+ influx. Chromatin immunoprecipitation assay identified HIF-1α binding to NMDA-NR1 promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR1, suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR1. Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone and sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF. These results uncover a critical role for HIF-1 in the upregulation of NMDA-NR1 to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF.

Reactive oxygen species-driven HIF1α triggers accelerated glycolysis in endothelial cells exposed to low oxygen tension

Endothelial cells and their metabolic state regulate glucose transport into underlying tissues. Here, we show that low oxygen tension stimulates human umbilical vein endothelial cell 18F–fluorodeoxyglucose (18F–FDG) uptake and lactate production. This was accompanied by augmented hexokinase activity and membrane Glut-1, and increased accumulation of hypoxia-inducible factor-1α (HIF1α). Restoration of oxygen reversed the metabolic effect, but this was blocked by HIF1α stabilization. Hypoxia-stimulated 18F–FDG uptake was completely abrogated by silencing of HIF1α expression or by a specific inhibitor. There was a rapid and marked increase of reactive oxygen species (ROS) by hypoxia, and ROS scavenging or NADPH oxidase inhibition completely abolished hypoxia-stimulated HIF1α and 18F–FDG accumulation, placing ROS production upstream of HIF1α signaling. Hypoxia-stimulated HIF1α and 18F–FDG accumulation was blocked by the protein kinase C (PKC) inhibitor, staurosporine. The phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, blocked hypoxia-stimulated 18F–FDG uptake and attenuated hypoxia-responsive element binding of HIF1α without influencing its accumulation. Thus, ROS-driven HIF1α accumulation, along with PKC and PI3K signaling, play a key role in triggering accelerated glycolysis in endothelial cells under hypoxia, thereby contributing to 18F–FDG transport.

Role of HIF-1α in the hypoxia inducible expression of the thiamine transporter, SLC19A3

Ensuring continuous intracellular supply of thiamine is essential to maintain metabolism. Cellular homeostasis requires the function of the membrane bound thiamine transporters THTR1 and THTR2. In the absence of increased dietary intake of thiamine, varying intracellular levels to meet metabolic demands during pathophysiological stressors, such as hypoxia, requires adaptive regulatory mechanisms to increase thiamine transport capacity. Previous work has established the up-regulation of SLC19A3 (THTR2) gene expression and activity during hypoxic stress through the activity of the hypoxia inducible transcription factor 1 alpha (HIF-1α). However, it is unknown whether HIF-1α acts directly or indirectly to trans-activate expression of SLC19A3. This work utilized the breast cancer cell line BT-474 treated with 1% O2 or a hypoxia chemical mimetic deferoxamine to determine the minimal promoter region of SLC19A3 responsible for hypoxia responsiveness. In silico sequence analysis determined two contiguous hypoxia responsive elements in close proximity to the transcriptional start site of the SLC19A3 gene. Using a HIF-1α transcriptional factor ELISA assay, HIF-1α was capable of binding to a dsDNA construct of the SLC19A3 minimal promoter. Chromatin immunoprecipitation assay established that SP1 was bound to the SLC19A3 minimal promoter region under normoxic conditions. However, HIF-1α binding to the minimal promoter region occurred during hypoxic treatments, while no SP1 binding was observed under these conditions. This work demonstrates the direct binding and activation of SLC19A3 expression by HIF-1α during hypoxic stress, suggesting an important adaptive regulatory role for HIF-1α in maintaining thiamine homeostasis.

Long noncoding RNA ANRIL is activated by hypoxia-inducible factor-1α and promotes osteosarcoma cell invasion and suppresses cell apoptosis upon hypoxia.

BackgroundOsteosarcoma is the most common malignancy of bone. Intratumoral hypoxia occurs in many solid tumors, where it is associated with the development of aggressive phenotype. ANRIL has been shown to be a long noncoding RNA that facilitates the progression of a number of malignancies. Yet, few studies have explored the expression pattern of ANRIL in osteosarcoma and the effect of hypoxia on ANRIL.MethodsWe evaluated the expression levels of ANRIL in osteosarcoma tissues, adjacent normal tissues and cells with quantitative real-time polymerase chain reaction. Multiple approaches including luciferase reporter assay with nucleotide substitutions, chromatin immunoprecipitation assay and electrophoretic mobility shift assay were used to confirm the direct binding of HIF-1α to the ANRIL promoter region. SiRNA-based knockdown and other molecular biology techniques were employed to measure the effect of HIF-1α on the expression of ANRIL.ResultsWe found that the expression of ANRIL was upregulated in 15 pairs of osteosarcoma compared with adjacent normal tissues. We found that hypoxia is sufficient to upregulate ANRIL expression in osteosarcoma cells (MNNG and U2OS). HIF-1α directly binds to the putative hypoxia response element in the upstream region of ANRIL. What’s more, siRNA and small molecular inhibitors-mediated HIF-1α suppression attenuated ANRIL upregulation under hypoxic conditions. Upon hypoxia, ANRIL promoted cancer cell invasion and suppressed cell apoptosis.ConclusionTaken together, these data suggest that HIF-1α may contribute to the upregulation of ANRIL in osteosarcoma under hypoxic conditions. ANRIL is involved in hypoxia-induced aggressive phenotype in osteosarcoma.

Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

The function of hypoxia-inducible factor-1α (HIF-1α) in chronic kidney disease is disputed. Here we report that interactions of HIF-1α with transforming growth factor-β (TGF-β) signaling may promote its fibrotic effects. Knockout of HIF-1α is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model. Transcriptional analysis of cultured renal cells showed that α2(I) collagen expression is directly regulated by HIF-1α binding to a functional hypoxia-responsive element in its promoter at -335 relative to the transcription start site. Activation of COL1A2 transcription by HIF-1α occurred in the absence of hypoxia and is strongly enhanced by TGF-β signaling. TGF-β, in addition to increasing HIF-1α levels, increased both HIF-1α binding to the COL1A2 promoter and HIF-1α N-terminal transactivation domain activity. These effects of TGF-β on HIF-1α were inhibited in Smad3-null mouse embryonic fibroblasts, suggesting a requirement for Smad3. Phosphorylated Smad3 also associated with the -335 hypoxia-responsive element of the COL1A2 promoter independent of a Smad DNA binding sequence. Smad3 binding to the -335 hypoxia-responsive element required HIF-1α both invitro and in kidney lysate from the disease model, suggesting formation of an HIF-1α-Smad3 transcriptional complex. Thus, HIF-1α-Smad3 has a novel interaction in glomerulosclerosis.

Abstract C35: Epigenetic repression of hexokinase 2 is responsible for the heterogeneity of 18F-FDG uptake in hepatocellular carcinoma

Abstract The clinical usefulness of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET), based on aerobic glycolysis, has been recently challenged in hepatocellular carcinoma (HCC) due to the heterogeneity of 18F-FDG uptake. Although inconsistent 18F-FDG uptake in HCCs is explained by the heterogeneous expression of hexokinase 2 (HK2), the underlying molecular mechanism remains elusive. Here, we examined the methylation status of the HK2 promoter and its influence on hypoxia-inducible factor-1α (HIF-1α) binding. We first confirmed inconsistent 18F-FDG uptake and correlative HK2 expression. Utilizing the HumanMethylation450 array, we discovered hypermethylation of the HK2 promoter CpG island (CGI) region in HK2negative HCCs. Studies using HK2 promoter luciferase constructs showed that a newly defined hypoxia responsive element (HRE) regulates HK2 expression. We also found that differential glucose uptake is caused by the differential HK2 expression due to differential methylation of the HK2 promoter CGI region in HCC cell lines. Finally, treatment with 5-Aza-2-deoxycytidine and hypoxic stimuli significantly increased HK2 expression due to increased HIF-1α binding to the HRE, resulting in increased glucose uptake in HK2negative SNU449 cells. Conclusion: These data indicate that methylation changes in the HK2 promoter CGI region in HCCs influence HK2 expression by regulating HIF-1α binding to the HRE in the CGI region. Hypermethylation of the HK2 promoter CGI region in HK2negative tissues inhibits the binding of HIF-1α to the HK2 promoter, resulting in the suppression of HK2 expression, which contributes to the heterogeneity of 18F-FDG uptake. Citation Format: Kim Hyemi, Lee Hyun Gyu, Lee Jae Myun, Park Jeon Han. Epigenetic repression of hexokinase 2 is responsible for the heterogeneity of 18F-FDG uptake in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C35.

Thrombopoietin induces hematopoiesis from mouse ES cells via HIF-1α-dependent activation of a BMP4 autoregulatory loop.

Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the conditions for the in vitro generation of hematopoietic cells. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-MPL. This knowledge, together with observations in vitro and in vivo, suggests that TPO/c-MPL signaling promotes early hematopoiesis. However, the mechanisms underlying TPO signaling are not fully elucidated. Here, we describe a direct connection between TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in determining the hematopoietic fate of ES cells. Morphogen BMP4 is known to induce early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 with concomitant upregulation of the BMP receptor BMPR1A, phosphorylation of SMAD1, 5, 8, and activation of specific BMP4 target genes; this was mediated by TPO-dependent binding of transcription factor HIF-1α to the BMP4 gene promoter. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cells. Thus, our findings contribute to the establishment of techniques for generating hematopoietic cells from ES cells.

Oxygen-dependent regulation of aquaporin-3 expression.

The purpose of this study was to investigate whether aquaporin-3 (AQP3) expression is altered in hypoxia and whether hypoxia-inducible transcription factor (HIF)-1 regulates the hypoxic expression. AQP3 mRNA expression was studied in L929 fibrosarcoma cells and in several tissues derived from mice that were subjected to hypoxia. Computational analysis of the AQP3 promoter revealed conserved HIF binding sites within close proximity to the translational start site, and chromatin immunoprecipitation assays confirmed binding of HIF-1α to the endogenous hypoxia response elements. Furthermore, hypoxia resulted in increased expression of AQP3 mRNA in L929 fibrosarcoma cells. Consistently, shRNA-mediated knockdown of HIF-1α greatly reduced the hypoxic induction of AQP3. In addition, mRNA analysis of organs from mice exposed to inspiratory hypoxia demonstrated pronounced hypoxia-inducible expression of AQP3 in the kidney. Overall, our findings suggest that AQP3 expression can be regulated at the transcriptional level and that AQP3 represents a novel HIF-1 target gene.