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Sample records for agmatine inhibits hypoxia-induced

  1. Galectin-3 inhibition ameliorates hypoxia-induced pulmonary artery hypertension

    PubMed Central

    Hao, Mingwen; Li, Miaomiao; Li, Wenjun

    2016-01-01

    Galectin-3 (Gal-3) is a β-galactoside-binding lectin, which is important in inflammation, fibrosis and heart failure. The present study aimed to investigate the role and mechanism of Gal-3 in hypoxia-induced pulmonary arterial hypertension (PAH). Male C57BL/6J and Gal-3−/− mice were exposed to hypoxia, then the right ventricular systolic pressure (RVSP) and Fulton's index were measured, and Gal-3 mRNA and protein expression in the pulmonary arteries was analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. Compared with the control, hypoxia increased the mRNA and protein expression levels of Gal-3 in wild type murine pulmonary arteries. Gal-3 deletion reduced the hypoxia-induced upregulation of RVSP and Fulton's index. Furthermore, human pulmonary arterial endothelial cells (HPAECs) and human pulmonary arterial smooth muscle cells (HPASMCs) were stimulated by hypoxia in vitro, and Gal-3 expression was inhibited by small interfering RNA. The inflammatory response of HPAECs, and the proliferation and cell cycle distribution of HPASMCs was also analyzed. Gal-3 inhibition alleviated the hypoxia-induced inflammatory response in HPAECs, including tumor necrosis factor-α and interleukin-1 secretion, expression of intercellular adhesion molecule-1 and adhesion of THP-1 monocytes. Gal-3 inhibition also reduced hypoxia-induced proliferation of HPASMCs, partially by reducing cyclin D1 expression and increasing p27 expression. Furthermore, Gal-3 inhibition suppressed HPASMC switching from a ‘contractile’ to a ‘synthetic’ phenotype. In conclusion, Gal-3 serves a fundamental role in hypoxia-induced PAH, and inhibition of Gal-3 may represent a novel therapeutic target for the treatment of hypoxia-induced PAH. PMID:27959409

  2. Identification of approved and investigational drugs that inhibit hypoxia-inducible factor-1 signaling

    PubMed Central

    Hsu, Chia-Wen; Huang, Ruili; Khuc, Thai; Shou, David; Bullock, Joshua; Grooby, Suzanne; Griffin, Sue; Zou, Chaozhong; Little, Annette; Astley, Holly; Xia, Menghang

    2016-01-01

    One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling. We generated an endogenous HIF-1α–NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1α-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1α accumulation and HIF-1α transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1α. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies. PMID:26882567

  3. Agmatine: identification and inhibition of methamphetamine, kappa opioid, and cannabinoid withdrawal in planarians.

    PubMed

    Rawls, Scott M; Gerber, Kristin; Ding, Zhe; Roth, Christopher; Raffa, Robert B

    2008-12-01

    Agmatine blocks morphine physical dependence in mammals, but its effects on withdrawal signs caused by other abused drugs have been less studied. One of the reasons is that withdrawal to some of these drugs is difficult to quantify in mammals. An alternative to mammals is planarians, a type of flatworm. Planarians possess mammalian-like neurotransmitters and display withdrawal from amphetamines, benzodiazepines, cannabinoids, cocaine, and opioids. The withdrawal is manifested as a reduction in locomotor behavior following discontinuation of drug exposure. In the present study, our goal was to identify agmatine in planarians and to determine if planarians exposed to agmatine display withdrawal to methamphetamine, a cannabinoid receptor agonist (WIN 55,212-2), or a kappa-opioid receptor agonist (U-50,488H). Neurochemical experiments revealed that the concentration of agmatine in planarians was 185 +/- 33.7 pmol per mg of planarian weight (dry weight). In behavioral experiments, withdrawal (i.e., reduced locomotor activity) was observed when planarians exposed to each drug (10 microM) for 60 min were placed into water. The withdrawal was attenuated when methamphetamine- or U-50,488H-exposed planarians were tested in agmatine (100 microM). Withdrawal was inhibited similarly when planarians coexposed to agmatine (100 microM) plus methamphetamine (10 microM), WIN 55,212-2 (10 microM), or U-50,488H (10 microM) were tested in water. Arginine, the metabolic precursor to agmatine, was ineffective. Our results identify endogenous agmatine in planarians and demonstrate that agmatine exposure blocks withdrawal to three different drugs in planarians. This suggests that a change in agmatine signaling is a common mechanism in the withdrawal caused by these drugs, at least in planarians.

  4. Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism.

    PubMed

    Ansó, Elena; Zuazo, Alicia; Irigoyen, Marta; Urdaci, María C; Rouzaut, Ana; Martínez-Irujo, Juan J

    2010-06-01

    Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure-activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4' position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4',7-dihydroxiflavone, reduced the expression of HIF-1alpha under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1alpha expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1alpha translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process.

  5. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    PubMed Central

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J.

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway. PMID:25649293

  6. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1 α /VEGF-A Pathway in Colorectal Cancer.

    PubMed

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J; Peng, Jun

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

  7. Isorhamnetin Inhibits Reactive Oxygen Species-Dependent Hypoxia Inducible Factor (HIF)-1α Accumulation.

    PubMed

    Seo, Suho; Seo, Kyuhwa; Ki, Sung Hwan; Shin, Sang Mi

    2016-01-01

    Isorhamnetin is a flavonoid metabolite of quercetin and isolated from water dropwort (Oenanthe javanica, Umbelliferae). It has been reported that isorhamnetin exerts beneficial effects including antioxidant, anti-inflammatory, and anti-proliferative activities. The present study investigated whether the antioxidant activity of isorhamnetin is correlated with its anti-cancer effects on colorectal cancer cells. Isorhamnetin significantly repressed cobalt chloride (CoCl2)- or hypoxia-induced hypoxia inducible factor-1α (HIF-1α) accumulation in HCT116 and HT29 cells. When compared with quercetin, isorhamnetin showed potent inhibition of HIF-1α. Moreover, it inhibited CoCl2-induced activity of hypoxia response element reporter gene and HIF-1α-dependent transcription of genes such as glucose transporter 1, lactate dehydrogenase A, carbonic anhydrase-IX, and pyruvate dehydrogenase kinase 1. Isorhamnetin also blocked hydrogen peroxide (H2O2)-induced HIF-1α accumulation. The antioxidant effects of isorhamnetin were confirmed by observation of CoCl2- or H2O2-induced reactive oxygen species (ROS) production. Consistently, overexpressed HIF-1α was decreased by isorhamnetin or N-acetyl-L-cysteine in HEK293 cells. In vitro migration and invasion assay further confirmed the inhibitory effects of isorhamnetin on cancer cells. Collectively, these results demonstrate that isorhamnetin inhibits ROS-mediated HIF-1α accumulation, which contributes to its anti-metastatic efficacy.

  8. [Inhibition of NHE1 promotes hypoxia-induced differentiation of K562 leukemic cells].

    PubMed

    Jin, Wei-Na; Wang, Jian; Chang, Guo-Qiang; Lin, Ya-Ni; Wang, Li-Hong; Li, Hua-Wen; Gao, Wei; Li, Qing-Hua; Pang, Tian-Xiang

    2011-06-01

    This study was purposed to investigate the effect of hypoxia microenvironment on K562 leukemic cell differentiation, and characteristics of NHE1 involvement in this process. The K562 cells were treated with hypoxia-mimical agent CoCl₂ or under actual hypoxia culture, and the specific NHE1 inhibitor Cariporide was used to inhibit NHE1 activity. The fluorescent probe BCECF was used for pH(i) measurements. Gene expression was analyzed by RT-PCR. The morphological characteristics was determined by Wright's staining. Signaling pathways were detected by Western blot using phosphospecific antibodies. The results indicated that the hypoxia or mimetic hypoxia favored K562 cells differentiation with up-regulation of C/EBPα. Moreover, treatment with Cariporide under hypoxia synergistically enhanced leukemia cell differentiation. Treatment with Cariporide increased levels of phosphorylated ERK5 and P38 mitogen-activated protein kinase (MAPK). It is concluded that the hypoxia or mimetic hypoxia can induce the differentiation of K562 cells, the inhibition of NHE1 activity can promote the hypoxia-induced K562 cell differentiation. The enhancement of hypoxia-induced K562 differentiation by Cariporide via MAPK signal pathway suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukemias.

  9. Curcumin inhibits hypoxia-induced migration in K1 papillary thyroid cancer cells

    PubMed Central

    Tan, Cheng; Zhang, Li; Cheng, Xian; Lin, Xiu-Feng; Lu, Rong-Rong; Bao, Jian-Dong

    2014-01-01

    Curcumin, traditionally used as food and medicinal purposes, has recently been reported to have protective efficacy against hypoxia. Hypoxia is one of the important reactive factors in tumor metastasis, which is a key problem in clinical thyroid cancer therapy. In present study, we investigate the anti-metastatic effect of curcumin on the K1 papillary thyroid cancer cells as well as its potential mechanisms. The results show that curcumin effectively inhibits hypoxia-induced reactive oxygen species (ROS) upregulation and significantly decreases the mRNA and protein expression levels of hypoxia-inducible factor-1α (HIF-1α) in K1 cells. Curcumin also decreases the DNA binding ability of HIF-1α to hypoxia response element (HRE). Furthermore, curcumin enhances E-cadherin expression, inhibits metalloproteinase-9 (MMP-9) enzyme activity, and weakens K1 cells migration under hypoxic conditions. In summary, these results indicate that curcumin possesses a potent anti-metastatic effect and might be an effective tumoristatic agent for the treatment of aggressive papillary thyroid cancers. PMID:25349216

  10. A synthetic manassantin a derivative inhibits hypoxia-inducible factor 1 and tumor growth.

    PubMed

    Lang, Liwei; Liu, Xiaoyu; Li, Yan; Zhou, Qing; Xie, Ping; Yan, Chunhong; Chen, Xiaoguang

    2014-01-01

    The dineolignan manassantin A from Saururaceae was recently identified as a hypoxia-inducible factor 1 (HIF-1) inhibitor, but its in-vivo anti-tumor effect has not been explored. We synthesized a series of manassantin A derivatives, and found that replacing the central tetrahydrofuran moiety with a cyclopentane ring yielded a compound (LXY6006) with increased HIF-1-inhibitory activity yet decreased stereochemically complexity amenable to a simplified synthesis scheme. LXY6006 inhibited HIF-1α nuclear accumulation induced by hypoxia, and inhibited cancer cell growth as a consequence of G2/M arrest. Oral administration of LXY6006 significantly inhibited growth of breast, lung, and pancreatic tumors implanted in nude mice. These results indicate that LXY6006 represents a novel class of agents targeting a broad range of human cancers.

  11. A Synthetic Manassantin A Derivative Inhibits Hypoxia-Inducible Factor 1 and Tumor Growth

    PubMed Central

    Li, Yan; Zhou, Qing; Xie, Ping; Yan, Chunhong; Chen, Xiaoguang

    2014-01-01

    The dineolignan manassantin A from Saururaceae was recently identified as a hypoxia-inducible factor 1 (HIF-1) inhibitor, but its in-vivo anti-tumor effect has not been explored. We synthesized a series of manassantin A derivatives, and found that replacing the central tetrahydrofuran moiety with a cyclopentane ring yielded a compound (LXY6006) with increased HIF-1-inhibitory activity yet decreased stereochemically complexity amenable to a simplified synthesis scheme. LXY6006 inhibited HIF-1α nuclear accumulation induced by hypoxia, and inhibited cancer cell growth as a consequence of G2/M arrest. Oral administration of LXY6006 significantly inhibited growth of breast, lung, and pancreatic tumors implanted in nude mice. These results indicate that LXY6006 represents a novel class of agents targeting a broad range of human cancers. PMID:24925080

  12. Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible Factor 1α Inhibition.

    PubMed

    Kwon, Do-Yeon; Lee, Hye Eun; Weitzel, Douglas H; Park, Kyunghye; Lee, Sun Hee; Lee, Chen-Ting; Stephenson, Tesia N; Park, Hyeri; Fitzgerald, Michael C; Chi, Jen-Tsan; Mook, Robert A; Dewhirst, Mark W; Lee, You Mie; Hong, Jiyong

    2015-10-08

    To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin's structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers.

  13. Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible Factor 1α Inhibition

    PubMed Central

    2015-01-01

    To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin’s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers. PMID:26394152

  14. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

    SciTech Connect

    Park, Choa; Lee, YoungJoo

    2014-07-18

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression.

  15. Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Kim, So Yeon; Yang, Eun Gyeong

    2016-04-01

    Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 μM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

  16. TIPE2 Inhibits Hypoxia-Induced Wnt/β-Catenin Pathway Activation and EMT in Glioma Cells.

    PubMed

    Liu, Zhi-Jun; Liu, Hong-Lin; Zhou, Hai-Cun; Wang, Gui-Cong

    2016-01-01

    Hypoxia-induced epithelial-to-mesenchymal transition (EMT) could facilitate tumor progression. TIPE2, the tumor necrosis factor-α (TNF-α)-induced protein 8-like 2 (also known as TNFAIP8L2), is a member of the TNF-α-induced protein 8 (TNFAIP8, TIPE) family and has been involved in the development and progression of several tumors. However, the effects of TIPE2 on the EMT process in glioma cells and the underlying mechanisms of these effects have not been previously reported. In our study, we assessed the roles of TIPE2 in the EMT process in glioma cells in response to hypoxia. Our results indicated that TIPE2 expression was significantly decreased in human glioma cell lines. TIPE2 overexpression significantly inhibited hypoxia-induced migration and invasion, as well as suppressed the EMT process in glioma cells. Furthermore, TIPE2 overexpression prevented hypoxia-induced expression of β-catenin, cyclin D1, and c-myc in human glioma cells. In summary, these data suggest that TIPE2 overexpression inhibited hypoxia-induced Wnt/β-catenin pathway activation and EMT in glioma cells.

  17. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    PubMed

    Olivares-González, Lorena; Martínez-Fernández de la Cámara, Cristina; Hervás, David; Marín, María Pilar; Lahoz, Agustin; Millán, José María; Rodrigo, Regina

    2016-01-01

    Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  18. Inhibition of hypoxia-inducible factors limits tumor progression in a mouse model of colorectal cancer

    PubMed Central

    Simon, M.Celeste

    2014-01-01

    Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression. PMID:24408928

  19. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants

    PubMed Central

    Olivares-González, Lorena; Martínez-Fernández de la Cámara, Cristina; Hervás, David; Marín, María Pilar; Lahoz, Agustin; Millán, José María

    2016-01-01

    Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions. PMID:27861632

  20. Noggin inhibits hypoxia-induced proliferation by targeting store-operated calcium entry and transient receptor potential cation channels.

    PubMed

    Yang, Kai; Lu, Wenju; Jia, Jing; Zhang, Jie; Zhao, Mingming; Wang, Sabrina; Jiang, Haiyang; Xu, Lei; Wang, Jian

    2015-06-01

    Abnormally elevated bone morphogenetic protein 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla protein (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (10% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin protein, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression.

  1. Suppression of Akt-HIF-1α signaling axis by diacetyl atractylodiol inhibits hypoxia-induced angiogenesis

    PubMed Central

    Choi, Sik-Won; Lee, Kwang-Sik; Lee, Jin Hwan; Kang, Hyeon Jung; Lee, Mi Ja; Kim, Hyun Young; Park, Kie-In; Kim, Sun-Lim; Shin, Hye Kyoung; Seo, Woo Duck

    2016-01-01

    Hypoxia-inducible factor (HIF)-1α is a key regulator associated with tumorigenesis, angiogenesis, and metastasis. HIF-1α regulation under hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. Here, we demonstrate that diacetyl atractylodiol (DAA) from Atractylodes japonica (A. japonica) is a potent HIF-1α inhibitor that inhibits the Akt signaling pathway. DAA dose-dependently inhibited hypoxia-induced HIF-1α and downregulated Akt signaling without affecting the stability of HIF-1α protein. Furthermore, DAA prevented hypoxia-mediated angiogenesis based on in vitro tube formation and in vivo chorioallantoic membrane (CAM) assays. Therefore, DAA might be useful for treatment of hypoxia-related tumorigenesis, including angiogenesis. [BMB Reports 2016; 49(9): 508-513] PMID:27439603

  2. Emodin Decreases Hepatic Hypoxia-Inducible Factor-1[Formula: see text] by Inhibiting its Biosynthesis.

    PubMed

    Ma, Feifei; Hu, Lijuan; Yu, Ming; Wang, Feng

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) is an [Formula: see text] dimeric transcription factor. Because HIF-1[Formula: see text] is instable with oxygen, HIF-1 is scarce in normal mammalian cells. However, HIF-1[Formula: see text] is expressed in pathological conditions such as cancer and obesity. Inhibiting HIF-1[Formula: see text] may be of therapeutic value for these pathologies. Here, we investigated whether emodin, derived from the herb of Rheum palmatum L, which is also known as Chinese rhubarb, and is native to China, regulates HIF-1[Formula: see text] expression. Male C57BL/6 mice without or with diet-induced obesity were treated with emodin for two weeks, while control mice were treated with vehicle. HIF-1[Formula: see text] expression was determined by Western blot. We found that emodin inhibited obesity-induced HIF-1[Formula: see text] expression in liver and skeletal muscle but did not regulate HIF-1[Formula: see text] expression in the kidneys or in intra-abdominal fat. In vitro, emodin inhibited HIF-1[Formula: see text] expression in human HepG2 hepatic cells and Y1 adrenocortical cells. Further, we investigated the mechanisms of HIF-1[Formula: see text] expression in emodin-treated HepG2 cells. First, we found that HIF-1[Formula: see text] had normal stability in the presence of emodin. Thus, emodin did not decrease HIF-1[Formula: see text] by stimulating its degradation. Importantly, emodin decreased the activity of the signaling pathways that led to HIF-1[Formula: see text] biosynthesis. Interestingly, emodin increased HIF-1[Formula: see text] mRNA in HepG2 cells. This may be a result of feedback in response to the emodin-induced decrease in the protein of HIF-1[Formula: see text]. In conclusion, emodin decreases hepatic HIF-1[Formula: see text] by inhibiting its biosynthesis.

  3. TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition.

    PubMed

    Raninga, Prahlad V; Di Trapani, Giovanna; Vuckovic, Slavica; Tonissen, Kathryn F

    2016-01-01

    Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-кβ subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-кβ p65 protein levels in the nucleus and reduced the expression of NF-кβ-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кβ-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-кβ signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.

  4. Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1

    PubMed Central

    Liang, Ke; Ding, Xue-qin; Lin, Chen

    2015-01-01

    The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus. PMID:25687434

  5. Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1.

    PubMed

    Liang, Ke; Ding, Xue-Qin; Lin, Chen; Kang, Y James

    2015-11-01

    The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus.

  6. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

    PubMed Central

    Krekorian, Massis; Alles, Lindy K.; van Wijk, Albert C.; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C.; van Gulik, Thomas M.; Storm, Gert; Heger, Michal

    2016-01-01

    Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. Results: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. Methods: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1β dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. Conclusions: HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas. PMID:26657503

  7. Daxx inhibits hypoxia-induced lung cancer cell metastasis by suppressing the HIF-1α/HDAC1/Slug axis

    PubMed Central

    Lin, Ching-Wen; Wang, Lu-Kai; Wang, Shu-Ping; Chang, Yi-Liang; Wu, Yi-Ying; Chen, Hsuan-Yu; Hsiao, Tzu-Hung; Lai, Wei-Yun; Lu, Hsuan-Hsuan; Chang, Ya-Hsuan; Yang, Shuenn-Chen; Lin, Ming-Wei; Chen, Chi-Yuan; Hong, Tse-Ming; Yang, Pan-Chyr

    2016-01-01

    Hypoxia is a major driving force of cancer invasion and metastasis. Here we show that death domain-associated protein (Daxx) acts to negatively regulate hypoxia-induced cell dissemination and invasion by inhibiting the HIF-1α/HDAC1/Slug pathway. Daxx directly binds to the DNA-binding domain of Slug, impeding histone deacetylase 1 (HDAC1) recruitment and antagonizing Slug E-box binding. This, in turn, stimulates E-cadherin and occludin expression and suppresses Slug-mediated epithelial–mesenchymal transition (EMT) and cell invasiveness. Under hypoxic conditions, stabilized hypoxia-inducible factor (HIF)-1α downregulates Daxx expression and promotes cancer invasion, whereas re-expression of Daxx represses hypoxia-induced cancer invasion. Daxx also suppresses Slug-mediated lung cancer metastasis in an orthotopic lung metastasis mouse model. Using clinical tumour samples, we confirmed that the HIF-1α/Daxx/Slug pathway is an outcome predictor. Our results support that Daxx can act as a repressor in controlling HIF-1α/HDAC1/Slug-mediated cancer cell invasion and is a potential therapeutic target for inhibition of cancer metastasis. PMID:28004751

  8. Noggin inhibits hypoxia-induced proliferation by targeting store-operated calcium entry and transient receptor potential cation channels

    PubMed Central

    Yang, Kai; Lu, Wenju; Jia, Jing; Zhang, Jie; Zhao, Mingming; Wang, Sabrina; Jiang, Haiyang; Xu, Lei

    2015-01-01

    Abnormally elevated bone morphogenetic protein 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla protein (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (10% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin protein, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression. PMID:25740156

  9. Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20)

    PubMed Central

    Burrows, Natalie; Cane, Gaelle; Robson, Mathew; Gaude, Edoardo; J. Howat, William; Szlosarek, Peter W.; Pedley, R. Barbara; Frezza, Christian; Ashcroft, Margaret; Maxwell, Patrick H.

    2016-01-01

    The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours. PMID:26972697

  10. Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

    PubMed

    Burrows, Natalie; Cane, Gaelle; Robson, Mathew; Gaude, Edoardo; Howat, William J; Szlosarek, Peter W; Pedley, R Barbara; Frezza, Christian; Ashcroft, Margaret; Maxwell, Patrick H

    2016-03-14

    The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.

  11. [Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

    PubMed

    Liu, Yanhua; Li, Bingong; Wang, Yuqin; Wang, Delong; Zou, Jin; Ke, Xuan; Hao, Yanqin

    2016-12-01

    Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

  12. Hypoxia-inducible factor prolyl hydroxylase inhibition: robust new target or another big bust for stroke therapeutics?

    PubMed Central

    Karuppagounder, Saravanan S; Ratan, Rajiv R

    2012-01-01

    A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative ‘antioxidant' iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke. PMID:22415525

  13. Hypoxia-inducible factor prolyl hydroxylase inhibition: robust new target or another big bust for stroke therapeutics?

    PubMed

    Karuppagounder, Saravanan S; Ratan, Rajiv R

    2012-07-01

    A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative 'antioxidant' iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke.

  14. Aloe-emodin suppresses hypoxia-induced retinal angiogenesis via inhibition of HIF-1α/VEGF pathway.

    PubMed

    Wu, Jianming; Ke, Xiao; Wang, Wei; Zhang, Hongcheng; Ma, Na; Fu, Wei; Zhao, Manxi; Gao, Xiaoping; Hao, Xiaofeng; Zhang, Zhirong

    2016-01-01

    Background: Aloe-emodin (AE) has been reported to possess the antiangiogenic effect on laser induced choroidal neovascularization. AE inhibits the vessel formation in the zebrafish embryos. However, it is still unclear whether AE can alleviate neovascularization. Here, we investigated the inhibitory effect of AE on the hypoxia-induced retinal neovascularization and the possible mechanisms. Methods: We established a vascular endothelial growth factor (VEGF) secretion model under chemical induced hypoxia by exposure of 150 µM CoCl2 to the ARPE-19 cells, then treated the cells with different concentrations of AE (0.2, 1.0 and 5.0 µg/mL) or a special hypoxia-inducible factor 1α (HIF-1α) inhibitor [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, YC-1, 1.0 µg/mL]. The cellular supernatants were collected 48 h later to measure the VEGFA concentrations by human VEGFA enzyme-linked immunosorbent assay (ELISA) kits, the mRNA expressions of VEGFA, HIF-1α and prolyl hydroxylase-2 (PHD-2) by quantitative reverse transcription-PCR (qRT-PCR) and the protein expressions of HIF-1α and PHD-2 by Western blots. For in vivo study, the rat pups with oxygen-induced retinopathy were treated with Conbercept ophthalmic injection (1.0 mg/kg) or AE (5.0 and 10.0 mg/kg) for five days, then the retinal avascular areas were assessed via visualization of the retinal vasculature with ADPase and hematoxylin & eosin (H&E) stains. Results: AE inhibits the VEGFA secretion of ARPE-19 cells under hypoxia condition, decreases the mRNA expressions of VEGFA and PHD-2 and the protein expressions of VEGFA, HIF-1α and PHD-2 in vitro and prevents hypoxia-induced retinal neovascularization in vivo.Conclusions: AE ameliorates retinal neovascularization throuth inhibition of the HIF-1α/VEGF signaling pathway. AE may be developed as a potential drug for the prevention and treatment of diabetic retinopathy.

  15. Aloe-emodin suppresses hypoxia-induced retinal angiogenesis via inhibition of HIF-1α/VEGF pathway

    PubMed Central

    Wu, Jianming; Ke, Xiao; Wang, Wei; Zhang, Hongcheng; Ma, Na; Fu, Wei; Zhao, Manxi; Gao, Xiaoping; Hao, Xiaofeng; Zhang, Zhirong

    2016-01-01

    Background: Aloe-emodin (AE) has been reported to possess the antiangiogenic effect on laser induced choroidal neovascularization. AE inhibits the vessel formation in the zebrafish embryos. However, it is still unclear whether AE can alleviate neovascularization. Here, we investigated the inhibitory effect of AE on the hypoxia-induced retinal neovascularization and the possible mechanisms. Methods: We established a vascular endothelial growth factor (VEGF) secretion model under chemical induced hypoxia by exposure of 150 µM CoCl2 to the ARPE-19 cells, then treated the cells with different concentrations of AE (0.2, 1.0 and 5.0 µg/mL) or a special hypoxia-inducible factor 1α (HIF-1α) inhibitor [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, YC-1, 1.0 µg/mL]. The cellular supernatants were collected 48 h later to measure the VEGFA concentrations by human VEGFA enzyme-linked immunosorbent assay (ELISA) kits, the mRNA expressions of VEGFA, HIF-1α and prolyl hydroxylase-2 (PHD-2) by quantitative reverse transcription-PCR (qRT-PCR) and the protein expressions of HIF-1α and PHD-2 by Western blots. For in vivo study, the rat pups with oxygen-induced retinopathy were treated with Conbercept ophthalmic injection (1.0 mg/kg) or AE (5.0 and 10.0 mg/kg) for five days, then the retinal avascular areas were assessed via visualization of the retinal vasculature with ADPase and hematoxylin & eosin (H&E) stains. Results: AE inhibits the VEGFA secretion of ARPE-19 cells under hypoxia condition, decreases the mRNA expressions of VEGFA and PHD-2 and the protein expressions of VEGFA, HIF-1α and PHD-2 in vitro and prevents hypoxia-induced retinal neovascularization in vivo. Conclusions: AE ameliorates retinal neovascularization throuth inhibition of the HIF-1α/VEGF signaling pathway. AE may be developed as a potential drug for the prevention and treatment of diabetic retinopathy. PMID:27877088

  16. A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

    PubMed Central

    Chai, Xiqing; Kong, Weina; Liu, Lingyun; Yu, Wenguo; Zhang, Zhenqing; Sun, Yimin

    2014-01-01

    Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we constructed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1α gene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1α represses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results confirmed that rAAV-HIF-1α significantly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1α administration also induced robust and prolonged HIF-1α production in rat hippocampus. Single rAAV-HIF-1α administration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer's disease rat model established by intracerebroventricular injection of aggregated amyloid-beta protein (25–35). Our in vitro and in vivo findings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurodegenerative diseases using gene therapy. PMID:25206774

  17. Leptin Counteracts the Hypoxia-Induced Inhibition of Spontaneously Firing Hippocampal Neurons: A Microelectrode Array Study

    PubMed Central

    Gavello, Daniela; Rojo-Ruiz, Jonathan; Marcantoni, Andrea; Franchino, Claudio; Carbone, Emilio; Carabelli, Valentina

    2012-01-01

    Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O2). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca2+-activated K+ channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 µM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons. PMID:22848520

  18. The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis*

    PubMed Central

    DiStefano, Marina T.; Danai, Laura V.; Roth Flach, Rachel J.; Chawla, Anil; Pedersen, David J.; Guilherme, Adilson; Czech, Michael P.

    2015-01-01

    The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation. PMID:25922078

  19. The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis.

    PubMed

    DiStefano, Marina T; Danai, Laura V; Roth Flach, Rachel J; Chawla, Anil; Pedersen, David J; Guilherme, Adilson; Czech, Michael P

    2015-06-12

    The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.

  20. Asparagine and Aspartate Hydroxylation of the Cytoskeletal Ankyrin Family Is Catalyzed by Factor-inhibiting Hypoxia-inducible Factor*

    PubMed Central

    Yang, Ming; Ge, Wei; Chowdhury, Rasheduzzaman; Claridge, Timothy D. W.; Kramer, Holger B.; Schmierer, Bernhard; McDonough, Michael A.; Gong, Lingzhi; Kessler, Benedikt M.; Ratcliffe, Peter J.; Coleman, Mathew L.; Schofield, Christopher J.

    2011-01-01

    Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13–24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification. PMID:21177872

  1. RUNX3 inhibits hypoxia-inducible factor-1α protein stability by interacting with prolyl hydroxylases in gastric cancer cells.

    PubMed

    Lee, S H; Bae, S C; Kim, K W; Lee, Y M

    2014-03-13

    RUNX3 is silenced by histone modification and hypoxia-inducible factor (HIF)-1α is stabilized under hypoxia, but little is known of cross-talk between RUNX3 and HIF-1α under hypoxia. In the present study, the authors investigated the effect of RUNX3 on HIF-1α stability in gastric cancer cells. RUNX3 overexpression was found to downregulate HIF-1α stability under normoxic and hypoxic conditions. Furthermore, the activity of a luciferase reporter containing five copies of vascular endothelial growth factor (VEGF) promoter hypoxia-responsive element (5 × HRE) and the amount of secreted VEGF, were diminished in RUNX3-expressing but increased in RUNX3-knockdown cells. When expression of RUNX3 was recovered using epigenetic reagents the expressions of HIF-1α and VEGF were clearly suppressed under hypoxic conditions. RUNX3 also significantly attenuated the half-life of HIF-1α protein, and induced the cytosolic localization and ubiquitination of HIF-1α. In addition, RUNX3 directly interacted with the C-terminal activation domain of HIF-1α and prolyl hydroxylase (PHD) 2 and enhanced the interaction between HIF-1α and PHD2, which potentiated proline hydroxylation and promoted the degradation of HIF-1α. Furthermore, RUNX3 overexpression significantly inhibited hypoxia-induced angiogenesis in vitro and in vivo. Taken together, these results suggest that RUNX3 destabilizes HIF-1α protein by promoting the proline hydroxylation of HIF-1α through binding to HIF-1α/PHD2. RUNX3 appears to be a novel suppressor of HIF-1α and of hypoxia-mediated angiogenesis in gastric cancer cells.

  2. Inhibition of hypoxia-induced relaxation of rabbit isolated coronary arteries by NG-monomethyl-L-arginine but not glibenclamide.

    PubMed Central

    Jiang, C.; Collins, P.

    1994-01-01

    1. The effects of NG-monomethyl-L-arginine, tetrodotoxin and glibenclamide on hypoxia-induced coronary artery relaxation, induced by bubbling Krebs solution with 95% N2 and 5% CO2 instead of 95% O2 and 5% CO2, were assessed by measuring the changes in isometric tension in isolated epicardial coronary artery rings of the rabbit. In addition, the effects of glibenclamide on the relaxation induced by adenosine were investigated. 2. Hypoxia caused a transient relaxation of 38 +/- 3% (P < 0.01) and 17 +/- 2% (P < 0.01) in endothelium-intact or -denuded arteries respectively. NG-monomethyl-L-arginine (30 and 100 microM) inhibited the relaxation in endothelium-intact rings to 31 +/- 2% (P < 0.05) and 16 +/- 2% (P < 0.01) respectively and slightly but significantly attenuated the relaxation in endothelium-denuded rings to 15 +/- 1% and 13 +/- 1% (P < 0.05) respectively. 3. Glibenclamide, a potassium channel inhibitor, did not significantly after the hypoxia-induced relaxation. 4. Incubation with tetrodotoxin (3 and 10 microM) for 30 min reduced the relaxation to 31 +/- 3% (P < 0.05) and 14 +/- 2% (P < 0.01), and 14 +/- 2% (P < 0.05) and 11 +/- 1% (P < 0.05) in endothelium-intact and -denuded rings respectively. However, indomethacin (10 microM), atropine (1 microM), propranolol (10 microM) and phentolamine (10 microM) did not significantly affect the relaxation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8019749

  3. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    PubMed Central

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  4. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Dhar, Deepanshi; Panigrahi, Gati K; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P; Oberlies, Nicholas H; Agarwal, Rajesh

    2016-03-16

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

  5. Agmatine protects Müller cells from high-concentration glucose-induced cell damage via N-methyl-D-aspartic acid receptor inhibition.

    PubMed

    Han, Ning; Yu, Li; Song, Zhidu; Luo, Lifu; Wu, Yazhen

    2015-07-01

    Neural injury is associated with the development of diabetic retinopathy. Müller cells provide structural and metabolic support for retinal neurons. High glucose concentrations are known to induce Müller cell activity. Agmatine is an endogenous polyamine, which is enzymatically formed in the mammalian brain and has exhibited neuroprotective effects in a number of experimental models. The aims of the present study were to investigate whether agmatine protects Müller cells from glucose-induced damage and to explore the mechanisms underlying this process. Lactate dehydrogenase activity and tumor necrosis factor-α mRNA expression were significantly reduced in Müller cells exposed to a high glucose concentration, following agmatine treatment, compared with cells not treated with agmatine. In addition, agmatine treatment inhibited glucose-induced Müller cell apoptosis, which was associated with the regulation of Bax and Bcl-2 expression. Agmatine treatment suppressed glucose-induced phosphorylation of mitogen-activated protein kinase (MAPK) protein in Müller cells. The present study demonstrated that the protective effects of agmatine on Müller cells were inhibited by N-methyl-D-aspartic acid (NMDA). The results of the present study suggested that agmatine treatment protects Müller cells from high-concentration glucose-induced cell damage. The underlying mechanisms may relate to the anti-inflammatory and antiapoptotic effects of agmatine, as well as to the inhibition of the MAPK pathway, via NMDA receptor suppression. Agmatine may be of use in the development of novel therapeutic approaches for patients with diabetic retinopathy.

  6. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    PubMed Central

    Li, Jie; Zhang, Chao; Jiang, Hongchuan; Cheng, Jiao

    2015-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF) gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future. PMID:25709476

  7. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: insight into the cytotoxicity and antiangiogenesis.

    PubMed

    Yang, Tieshan; Yao, Qian; Cao, Fei; Liu, Qianqian; Liu, Binlei; Wang, Xiu-Hong

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is activated upon exposure to hypoxic stress. It modulates a number of cellular responses including proliferation, apoptosis, angiogenesis, and metabolism by activating a panel of target genes in response to hypoxia. The HIF-1 level is often upregulated in the hypoxic microenvironment of solid tumors, which contributes to cancer treatment failure. Here we report that silver nanoparticles (AgNPs), which are widely used as an antimicrobial agent, are an effective inhibitor of HIF-1. AgNPs inhibited the activation of a HIF-dependent reporter construct after the cells were exposed to hypoxic conditions or treated with cobalt chloride, a hypoxia mimetic agent. The AgNPs also interfered with the accumulation of HIF-1α protein and the induction of the endogenous HIF target genes, VEGF-A and GLUT1. Since both HIF-1 and vascular endothelial growth factor-A play an important role in angiogenesis, AgNPs also inhibited angiogenesis in vitro. Our data reveal a new mechanism of how AgNPs act on cellular function, that is, they disrupt HIF signaling pathway. This finding provides a novel insight into how AgNPs can inhibit cancer cell growth and angiogenesis.

  8. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: insight into the cytotoxicity and antiangiogenesis

    PubMed Central

    Yang, Tieshan; Yao, Qian; Cao, Fei; Liu, Qianqian; Liu, Binlei; Wang, Xiu-Hong

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is activated upon exposure to hypoxic stress. It modulates a number of cellular responses including proliferation, apoptosis, angiogenesis, and metabolism by activating a panel of target genes in response to hypoxia. The HIF-1 level is often upregulated in the hypoxic microenvironment of solid tumors, which contributes to cancer treatment failure. Here we report that silver nanoparticles (AgNPs), which are widely used as an antimicrobial agent, are an effective inhibitor of HIF-1. AgNPs inhibited the activation of a HIF-dependent reporter construct after the cells were exposed to hypoxic conditions or treated with cobalt chloride, a hypoxia mimetic agent. The AgNPs also interfered with the accumulation of HIF-1α protein and the induction of the endogenous HIF target genes, VEGF-A and GLUT1. Since both HIF-1 and vascular endothelial growth factor-A play an important role in angiogenesis, AgNPs also inhibited angiogenesis in vitro. Our data reveal a new mechanism of how AgNPs act on cellular function, that is, they disrupt HIF signaling pathway. This finding provides a novel insight into how AgNPs can inhibit cancer cell growth and angiogenesis. PMID:27994464

  9. PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation via PEDF-R.

    PubMed

    Wang, Xiaoyu; Zhang, Yiqian; Lu, Peng; Zhang, Hao; Li, Yufeng; Dong, Hongyan; Zhang, Zhongming

    2015-09-25

    Pigment epithelial-derived factor (PEDF) is a multifunctional secreted glycoprotein, which could protect against hypoxia-induced cell death related to its anti-oxidative effect in cultured cardiomyocytes. However, the pathway mediating this cytoprotective process has not been fully established. Here we confirmed that PEDF bound to pigment epithelial-derived factor receptor (PEDF-R) expressed on the membrane of H9c2 cells. Under hypoxic condition, PEDF increased the ratio of MDM2:p53, so as to inhibited p53 mitochondrial translocation via PEDF-R. As a result, mitochondrial outer membrane permeabilization (MOMP) and mitochondrial permeability transition pore (MPTP) opening were inhibited, meanwhile cleaved caspase-3, PARP and the release of HMGB1 were reduced. Accordingly, apoptosis and necrosis were attenuated simultaneously. We conclude that PEDF-R mediates PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation.

  10. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases*

    PubMed Central

    Chan, Mun Chiang; Ilott, Nicholas E.; Schödel, Johannes; Sims, David; Tumber, Anthony; Lippl, Kerstin; Mole, David R.; Pugh, Christopher W.; Ratcliffe, Peter J.; Ponting, Chris P.; Schofield, Christopher J.

    2016-01-01

    The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable. PMID:27502280

  11. Wheatgrass extract inhibits hypoxia-inducible factor-1-mediated epithelial-mesenchymal transition in A549 cells

    PubMed Central

    Do, Nam Yong; Shin, Hyun-Jae

    2017-01-01

    BACKGROUND/OBJECTIVES Epithelial-mesenchymal transition (EMT) is involved in not only cancer development and metastasis but also non-cancerous conditions. Hypoxia is one of the proposed critical factors contributing to formation of chronic rhinosinusitis or nasal polyposis. Wheatgrass (Triticum aestivum) has antioxidant, anti-aging, and anti-inflammatory effects. In this study, we analyzed whether wheatgrass has an inhibitory effect on the EMT process in airway epithelial cells. MATERIALS/METHODS A549 human lung adenocarcinoma cells were incubated in hypoxic conditions (CO2 5%/O2 1%) for 24 h in the presence of different concentrations of wheatgrass extract (50, 75, 100, and 150 µg/mL) and changes in expression of epithelial or mesenchymal markers were evaluated by immunoblotting and immunofluorescence. Accordingly, associated EMT-related transcriptional factors, Snail and Smad, were also evaluated. RESULTS Hypoxia increased expression of N-cadherin and reduced expression of E-cadherin. Mechanistically, E-cadherin levels were recovered during hypoxia by silencing hypoxia inducible factor (HIF)-1α or administering wheatgrass extract. Wheatgrass inhibited the hypoxia-mediated EMT by reducing the expression of phosphorylated Smad3 (pSmad3) and Snail. It suppressed the hypoxia-mediated EMT processes of airway epithelial cells via HIF-1α and the pSmad3 signaling pathway. CONCLUSION These results suggest that wheatgrass has potential as a therapeutic or supplementary agent for HIF-1-related diseases. PMID:28386380

  12. Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells.

    PubMed

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Acierno, Giovanni; Andolfo, Immacolata; Capasso, Mario; Iolascon, Achille

    2015-06-09

    Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

  13. LXY6090 – a novel manassantin A derivative – limits breast cancer growth through hypoxia-inducible factor-1 inhibition

    PubMed Central

    Lai, Fangfang; Liu, Qian; Liu, Xiaoyu; Ji, Ming; Xie, Ping; Chen, Xiaoguang

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents a novel antitumor target owing to its involvement in vital processes considered hallmarks of cancer phenotypes. Manassantin A (MA) derived from Saururus cernuus has been reported as a selective HIF-1 inhibitor. Herein, the structure of MA was optimized to achieve new derivatives with simple chemical properties while retaining its activity. LXY6090 was designed to replace the central tetrahydrofuran moiety of MA with a cyclopentane ring and was identified as a potent HIF-1 inhibitor with an IC50 value of 4.11 nM. It not only inhibited the activity of HIF-1 in breast cancer cells but also downregulated the protein level of HIF-1α, which depended on von Hippel–Lindau for proteasome degradation. The related biological evaluation showed that the activity of HIF-1 target genes, VEGF and IGF-2, was decreased by LXY6090 in breast cancer cell lines. LXY6090 presented potent antitumor activity in vitro. Furthermore, LXY6090 showed in vivo anticancer efficacy by decreasing the HIF-1α expression in nude mice bearing MX-1 tumor xenografts. In conclusion, our data provide a basis for the future development of the novel compound LXY6090 as a potential therapeutic agent for breast cancer. PMID:27445487

  14. LXY6090 - a novel manassantin A derivative - limits breast cancer growth through hypoxia-inducible factor-1 inhibition.

    PubMed

    Lai, Fangfang; Liu, Qian; Liu, Xiaoyu; Ji, Ming; Xie, Ping; Chen, Xiaoguang

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents a novel antitumor target owing to its involvement in vital processes considered hallmarks of cancer phenotypes. Manassantin A (MA) derived from Saururus cernuus has been reported as a selective HIF-1 inhibitor. Herein, the structure of MA was optimized to achieve new derivatives with simple chemical properties while retaining its activity. LXY6090 was designed to replace the central tetrahydrofuran moiety of MA with a cyclopentane ring and was identified as a potent HIF-1 inhibitor with an IC50 value of 4.11 nM. It not only inhibited the activity of HIF-1 in breast cancer cells but also downregulated the protein level of HIF-1α, which depended on von Hippel-Lindau for proteasome degradation. The related biological evaluation showed that the activity of HIF-1 target genes, VEGF and IGF-2, was decreased by LXY6090 in breast cancer cell lines. LXY6090 presented potent antitumor activity in vitro. Furthermore, LXY6090 showed in vivo anticancer efficacy by decreasing the HIF-1α expression in nude mice bearing MX-1 tumor xenografts. In conclusion, our data provide a basis for the future development of the novel compound LXY6090 as a potential therapeutic agent for breast cancer.

  15. Pien Tze Huang inhibits hypoxia-induced epithelial-mesenchymal transition in human colon carcinoma cells through suppression of the HIF-1 pathway.

    PubMed

    Chen, Hongwei; Shen, Aling; Zhang, Yuchen; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas; Peng, Jun

    2014-05-01

    Hypoxia-induced activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway is frequently observed in solid tumors and is strongly associated with numerous pathophysiological processes, including the induction of epithelial-mesenchymal transition (EMT), which result in cancer progression and metastasis. Thus, inhibiting EMT through the suppression of the HIF-1 pathway may be a promising strategy for anticancer chemotherapy. Pien Tze Huang (PZH), a well-established traditional Chinese medicine has been prescribed for >450 years and has been used for centuries to clinically treat various types of human cancer. We previously reported that PZH suppresses multiple intracellular signaling pathways and thereby promotes the apoptosis of cancer cells and the inhibition of cell proliferation and tumor angiogenesis. In the present study, to further explore the mechanisms underlying the antitumor action of PZH, HCT-8 human colon carcinoma cells were cultured under hypoxic conditions and the effect of PZH on hypoxia-induced EMT was assessed. Hypoxia was found to induce EMT-associated morphological changes in HCT-8 cells, including loss of cell adhesion and the development of spindle-shaped fibroblastoid-like morphology. In addition, hypoxia was observed to reduce the expression of the epithelial marker E-cadherin, but increase that of the mesenchymal marker N-cadherin. In addition, hypoxia significantly enhanced HCT-8 cell migration and invasion and induced the activation of the HIF-1 pathway. However, treatment of the HCT-8 cells with PZH significantly inhibited the hypoxia-mediated EMT and HIF-1 signaling. These findings suggest that PZH inhibits hypoxia-induced cancer EMT through the suppression of the HIF-1 pathway, which may be one of the molecular mechanisms by which PZH exerts its antitumor activity.

  16. Pien Tze Huang inhibits hypoxia-induced epithelial-mesenchymal transition in human colon carcinoma cells through suppression of the HIF-1 pathway

    PubMed Central

    CHEN, HONGWEI; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; LIN, JIUMAO; LIN, WEI; SFERRA, THOMAS; PENG, JUN

    2014-01-01

    Hypoxia-induced activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway is frequently observed in solid tumors and is strongly associated with numerous pathophysiological processes, including the induction of epithelial-mesenchymal transition (EMT), which result in cancer progression and metastasis. Thus, inhibiting EMT through the suppression of the HIF-1 pathway may be a promising strategy for anticancer chemotherapy. Pien Tze Huang (PZH), a well-established traditional Chinese medicine has been prescribed for >450 years and has been used for centuries to clinically treat various types of human cancer. We previously reported that PZH suppresses multiple intracellular signaling pathways and thereby promotes the apoptosis of cancer cells and the inhibition of cell proliferation and tumor angiogenesis. In the present study, to further explore the mechanisms underlying the antitumor action of PZH, HCT-8 human colon carcinoma cells were cultured under hypoxic conditions and the effect of PZH on hypoxia-induced EMT was assessed. Hypoxia was found to induce EMT-associated morphological changes in HCT-8 cells, including loss of cell adhesion and the development of spindle-shaped fibroblastoid-like morphology. In addition, hypoxia was observed to reduce the expression of the epithelial marker E-cadherin, but increase that of the mesenchymal marker N-cadherin. In addition, hypoxia significantly enhanced HCT-8 cell migration and invasion and induced the activation of the HIF-1 pathway. However, treatment of the HCT-8 cells with PZH significantly inhibited the hypoxia-mediated EMT and HIF-1 signaling. These findings suggest that PZH inhibits hypoxia-induced cancer EMT through the suppression of the HIF-1 pathway, which may be one of the molecular mechanisms by which PZH exerts its antitumor activity. PMID:24940418

  17. MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells

    PubMed Central

    Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

    2015-01-01

    Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells. PMID:26536662

  18. Anthrax lethal toxin inhibits translation of hypoxia-inducible factor 1α and causes decreased tolerance to hypoxic stress.

    PubMed

    Ouyang, Weiming; Torigoe, Chikako; Fang, Hui; Xie, Tao; Frucht, David M

    2014-02-14

    Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

  19. Inhibition of Breast Cancer by Repression of Angiogenic Hypoxia-Inducible Transcription Factors

    DTIC Science & Technology

    2003-09-01

    cancer cells to death receptor-induced apoptosis by inhibition ofNF-KB: Synergistic action of Apo2L/TRAIL, Interferon-y, Aspirin and Apigenin . (Abstract...of !KK0 (with ::leety! ,~81iCy!iC ::H~irl" ASA), and CK2 (with the plant flavonoid, apigenin ), results in loss of NF-KB-dependent expression of BcI...reduction of NF-KS-induced survival proteins by ASA and apigenin synergizes with interferon-y-mediated elevation of death signaling proteins to

  20. Inhibition of hypoxia-induced miR-155 radiosensitizes hypoxic lung cancer cells.

    PubMed

    Babar, Imran A; Czochor, Jennifer; Steinmetz, Allison; Weidhaas, Joanne B; Glazer, Peter M; Slack, Frank J

    2011-11-15

    miR-155 is a prominent microRNA (miRNA) that regulates genes involved in immunity and cancer-related pathways. miR-155 is overexpressed in lung cancer, which correlates with poor patient prognosis. It is unclear how miR-155 becomes increased in lung cancers and how this increase contributes to reduced patient survival. Here, we show that hypoxic conditions induce miR-155 expression in lung cancer cells and trigger a corresponding decrease in a validated target, FOXO3A. Furthermore, we find that increased levels of miR-155 radioprotects lung cancer cells, while inhibition of miR-155 radiosensitizes these cells. Moreover, we reveal a therapeutically important link between miR-155 expression, hypoxia, and irradiation by demonstrating that anti-miR-155 molecules also sensitize hypoxic lung cancer cells to irradiation. Our study helps explain how miR-155 becomes elevated in lung cancers, which contain extensive hypoxic microenvironments, and demonstrates that inhibition of miR-155 may have important therapeutic potential as a means to radiosensitize hypoxic lung cancer cells.

  1. A(1) and A(3) adenosine receptors inhibit LPS-induced hypoxia-inducible factor-1 accumulation in murine astrocytes.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Fazzi, Debora; Varani, Katia; Borea, Pier Andrea

    2013-10-01

    Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia and inflammation and plays an important role in brain injury. HIF-1 is expressed by astrocytes, however the regulatory role played by Ado on HIF-1α modulation induced by inflammatory and hypoxic conditions has not been investigated. Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without Ado, Ado receptor agonists, antagonists and receptor silencing, before exposure to normoxia or hypoxia. HIF-1α accumulation and downstream genes regulation were determined. Ado inhibited LPS-increased HIF-1α accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with the blockers of p44/42 MAPK and Akt, LPS-induced HIF-1α accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect and Ado inhibited kinases phosphorylation. A series of angiogenesis and metabolism related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, with the exception of GLUT-1 and hexochinase II that were elevated by LPS only in normoxia and inhibited by Ado receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia and inhibited by Ado, through A1 and A3 receptor subtypes. In conclusion A1 and A3 receptors reduce the LPS-mediated HIF-1α accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, like iNOS and A2B receptors, in both normoxic and hypoxic conditions.

  2. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    SciTech Connect

    Luo, Chunxia; Yi, Bin; Bai, Li; Xia, Yongzhi; Wang, Guansong; Qian, Guisheng; Feng, Hua

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  3. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    SciTech Connect

    Lee, Hae-June; Yoon, Changhwan; Park, Do Joong; Kim, Yeo-Jung; Schmidt, Benjamin; Lee, Yoon-Jin; Tap, William D.; Eisinger-Mathason, T.S. Karin; Choy, Edwin; Kirsch, David G.; Simon, M. Celeste; and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  4. Fucosterol protects cobalt chloride induced inflammation by the inhibition of hypoxia-inducible factor through PI3K/Akt pathway.

    PubMed

    Sun, Zhengwang; Mohamed, Mohamed Antar Aziz; Park, Sang Yong; Yi, Tae Hoo

    2015-12-01

    Fucosterol is a phytosterol commonly extracted from algae. It has been proved that fucosterol possesses antioxidant activity that is capable of scavenging the free radicals causing skin damages. In this study, we investigated the protective mechanisms of fucosterol on cobalt chloride (CoCl2) induced hypoxia damages to keratinocytes (HaCaT). We found that fucosterol inhibited CoCl2 induced cytotoxicity and inflammation in a dose-dependent manner. Furthermore, fucosterol attenuated CoCl2 induced excess expression of IL-6, IL-1β and TNF-α in HaCaT cells. In addition, fucosterol surpressed the phosphorylation of PI3K and Akt and accumulation of HIF1-α simulated by CoCl2. Taken together, these results suggested that fucosterol executed its protective effects against CoCl2 induced cytotoxicity and inflammation by the inhibition of hypoxia inducible factor through PI3K/Akt pathway.

  5. Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

    PubMed

    Liu, Maoxi; Fu, Zhongxue; Wu, Xingye; Du, Kunli; Zhang, Shouru; Zeng, Li

    2016-05-01

    Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

  6. Canstatin inhibits hypoxia-induced apoptosis through activation of integrin/focal adhesion kinase/Akt signaling pathway in H9c2 cardiomyoblasts

    PubMed Central

    Yamawaki, Hideyuki

    2017-01-01

    A hypoxic stress which causes apoptosis of cardiomyocytes is the main problem in the ischemic heart disease. Canstatin, a non-collagenous fragment of type IV collagen α2 chain, is an endogenous anti-angiogenic factor. We have previously reported that canstatin has a cytoprotective effect on cardiomyoblasts. In the present study, we examined the effects of canstatin on hypoxia-induced apoptosis in H9c2 cardiomyoblasts. Cell counting assay was performed to determine a cell viability. Western blotting was performed to detect expression of cleaved casepase-3 and phosphorylation of focal adhesion kinase (FAK) and Akt. Immunocytochemical staining was performed to observe a distribution of αv integrin. Hypoxia (1% O2, 48 h) significantly decreased cell viability and increased cleaved caspase-3 expression. Canstatin (10–250 ng/ml) significantly inhibited these changes in a concentration-dependent manner. Cilengitide (1 μM), an αvβ3 and αvβ5 integrin inhibitor, significantly prevented the protective effects of canstatin on cell viability. Canstatin significantly increased phosphorylation of FAK and Akt under hypoxic condition, which were inhibited by cilengitide. LY294002, an inhibitor of phosphatidylinositol-3 kinase/Akt pathway, suppressed the canstatin-induced Akt phosphorylation and reversed the protective effects of canstatin. It was observed that hypoxia caused a localization of αv integrin to focal adhesion. In summary, we for the first time clarified that canstatin inhibits hypoxia-induced apoptosis via FAK and Akt pathways through activating integrins in H9c2 cardiomyoblasts. PMID:28235037

  7. Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM*

    PubMed Central

    Siddiq, Ambreena; Ayoub, Issam A.; Chavez, Juan C.; Aminova, Leila; Shah, Sapan; LaManna, Joseph C.; Patton, Stephanie M.; Connor, James R.; Cherny, Robert A.; Volitakis, Irene; Bush, Ashley I.; Langsetmo, Ingrid; Seeley, Todd; Gunzler, Volkmar; Ratan, Rajiv R.

    2008-01-01

    Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress. PMID:16227210

  8. Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization.

    PubMed

    Takei, Anna; Ekström, Malena; Mammadzada, Parviz; Aronsson, Monica; Yu, Ma; Kvanta, Anders; André, Helder

    2017-02-10

    Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis.

  9. Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization

    PubMed Central

    Takei, Anna; Ekström, Malena; Mammadzada, Parviz; Aronsson, Monica; Yu, Ma; Kvanta, Anders; André, Helder

    2017-01-01

    Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis. PMID:28186209

  10. In vitro Ischemia Suppresses Hypoxic Induction of Hypoxia Inducible Factor-1α by Inhibition of Synthesis and Not Enhanced Degradation

    PubMed Central

    Karuppagounder, Saravanan S.; Basso, Manuela; Sleiman, Sama F.; Ma, Thong C.; Speer, Rachel E.; Smirnova, Natalya A.; Gazaryan, Irina G.; Ratan, Rajiv R.

    2015-01-01

    Hypoxia Inducible Factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. In this study, we investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation, OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to quantitatively monitor distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2 hour hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolyl hydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Further, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD versus hypoxia, and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia. PMID:23456821

  11. Hypoxia-Inducible Hydrogels

    PubMed Central

    Park, Kyung Min; Gerecht, Sharon

    2014-01-01

    Oxygen is vital for the existence of all multicellular organisms, acting as a signaling molecule regulating cellular activities. Specifically, hypoxia, which occurs when the partial pressure of oxygen falls below 5%, plays a pivotal role during development, regeneration, and cancer. Here we report a novel hypoxia-inducible (HI) hydrogel composed of gelatin and ferulic acid that can form hydrogel networks via oxygen consumption in a laccase-mediated reaction. Oxygen levels and gradients within the hydrogels can be accurately controlled and precisely predicted. We demonstrate that HI hydrogels guide vascular morphogenesis in vitro via hypoxia-inducible factors activation of matrix metalloproteinases and promote rapid neovascularization from the host tissue during subcutaneous wound healing. The HI hydrogel is a new class of biomaterials that may prove useful in many applications, ranging from fundamental studies of developmental, regenerative and disease processes through the engineering of healthy and diseased tissue models towards the treatment of hypoxia-regulated disorders. PMID:24909742

  12. Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces “angiogenic switch off” in a hepatoma mouse model

    PubMed Central

    Iwamoto, Hideki; Nakamura, Toru; Koga, Hironori; Izaguirre-Carbonell, Jesus; Kamisuki, Shinji; Sugawara, Fumio; Abe, Mitsuhiko; Iwabata, Kazuki; Ikezono, Yu; Sakaue, Takahiko; Masuda, Atsutaka; Yano, Hirohisa; Ohta, Keisuke; Nakano, Masahito; Shimose, Shigeo; Shirono, Tomotake; Torimura, Takuji

    2015-01-01

    “Angiogenic switch off” is one of the ideal therapeutic concepts in the treatment of cancer. However, the specific molecules which can induce “angiogenic switch off” in tumor have not been identified yet. In this study, we focused on von Hippel-Lindau protein (pVHL) in hepatocellular carcinoma (HCC) and investigated the effects of sulfoquinovosyl-acylpropanediol (SQAP), a novel synthetic sulfoglycolipid, for HCC. We examined mutation ratio of VHL gene in HCC using 30 HCC samples and we treated the HCC-implanted mice with SQAP. Thirty clinical samples showed no VHL genetic mutation in HCC. SQAP significantly inhibited tumor growth by inhibiting angiogenesis in a hepatoma mouse model. SQAP induced tumor “angiogenic switch off” by decreasing hypoxia-inducible factor (HIF)-1, 2α protein via pVHL upregulation. pVHL upregulation decreased HIFα protein levels through different multiple mechanisms: (i) increasing pVHL-dependent HIFα protein degradation; (ii) decreasing HIFα synthesis with decrease of NF-κB expression; and (iii) decrease of tumor hypoxia by vascular normalization. We confirmed these antitumor effects of SQAP by the loss-of-function experiments. We found that SQAP directly bound to and inhibited transglutaminase 2. This study provides evidence that upregulation of tumor pVHL is a promising target, which can induce “angiogenic switch off” in HCC. PMID:27119112

  13. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    SciTech Connect

    Kim, Hyung Gyun; Han, Eun Hee; Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo; Jeong, Hye Gwang

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  14. Novel agmatine analogue, {gamma}-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels

    SciTech Connect

    Singh, Sushma; Jhingran, Anupam; Sharma, Ankur; Simonian, Alina R.; Soininen, Pasi; Vepsalainen, Jouko; Khomutov, Alex R.; Madhubala, Rentala

    2008-10-10

    The efficacy of {gamma}-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K{sub i} value of {approx}60 {mu}M. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK{sub a} 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels.

  15. Hypoxia-Inducible Factor Pathway Inhibition Resolves Tumor Hypoxia and Improves Local Tumor Control After Single-Dose Irradiation

    SciTech Connect

    Helbig, Linda; Koi, Lydia; Brüchner, Kerstin; Gurtner, Kristin; Hess-Stumpp, Holger; Unterschemmann, Kerstin; Pruschy, Martin; and others

    2014-01-01

    Purpose: To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. Methods and Materials: UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD{sub 50}) was calculated. Results: BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P<.0001) and in UT-SCC-14 (0.3% vs 19%, P<.0001). This decrease was accompanied by a significant increase in fraction of perfused vessels in UT-SCC-14 but not in UT-SCC-5. Bromodeoxyuridine and Ki67 labeling indices were significantly reduced only in UT-SCC-5. No significant changes were observed in vascular area or necrosis. BAY-84-7296 before single-dose irradiation significantly decreased TCD{sub 50}, with an enhancement ratio of 1.37 (95% confidence interval [CI] 1.13-1.72) in UT-SCC-5 and of 1.55 (95% CI 1.26-1.94) in UT-SCC-14. BAY-84-7296 maintenance after irradiation did not further decrease TCD{sub 50}. Conclusions: BAY-84-7296 resulted in a marked decrease in tumor hypoxia and substantially reduced radioresistance of tumor cells with the capacity to cause a local recurrence after irradiation. The data suggest that reduction of cellular hypoxia tolerance by BAY-84-7296 may represent the primary biological mechanism underlying the observed enhancement of

  16. Formononetin, an active compound of Astragalus membranaceus (Fisch) Bunge, inhibits hypoxia-induced retinal neovascularization via the HIF-1α/VEGF signaling pathway

    PubMed Central

    Wu, Jianming; Ke, Xiao; Ma, Na; Wang, Wei; Fu, Wei; Zhang, Hongcheng; Zhao, Manxi; Gao, Xiaoping; Hao, Xiaofeng; Zhang, Zhirong

    2016-01-01

    Background It has been reported that formononetin (FMN), one of the main ingredients from famous traditional Chinese medicine “Huang-qi” (Astragalus membranaceus [Fisch] Bunge) for Qi-tonifying, exhibits the effects of immunomodulation and tumor growth inhibition via antiangiogenesis. Furthermore, A. membranaceus may alleviate the retinal neovascularization (NV) of diabetic retinopathy. However, the information of FMN on retinal NV is limited so far. In the present study, we investigated the effects of FMN on the hypoxia-induced retinal NV and the possible related mechanisms. Materials and methods The VEGF secretion model of acute retinal pigment epithelial-19 (ARPE-19) cells under chemical hypoxia was established by the exposure of cells to 150 μM CoCl2 and then cells were treated with 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1, a potent HIF-1α inhibitor, 1.0 μg/mL) or different concentrations of FMN (0.2 μg/mL, 1.0 μg/mL, and 5.0 μg/mL). The supernatants of cells were collected 48 hours later to measure the VEGF concentrations, following the manufacturer’s instruction. The mRNA expressions of VEGF, HIF-1α, PHD-2, and β-actin were analyzed by quantitative reverse transcription polymerase chain reaction, and the protein expressions of HIF-1α and PHD-2 were determined by Western blot analysis. Furthermore, the rats with retinopathy were treated by intraperitoneal administration of conbercept injection (1.0 mg/kg) or FMN (5.0 mg/kg and 10.0 mg/kg) in an 80% oxygen atmosphere. The retinal avascular areas were assessed through visualization of the retinal vasculature by adenosine diphosphatase staining and hematoxylin and eosin staining. Results FMN can indeed inhibit the VEGF secretion of ARPE-19 cells under hypoxia, downregulate the mRNA expression of VEGFA and PHD-2, and decrease the protein expression of VEGF, HIF-1α, and PHD-2 in vitro. Furthermore, FMN can prevent hypoxia-induced retinal NV in vivo. Conclusion FMN can ameliorate

  17. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α.

    PubMed

    Kim, Hyung Gyun; Han, Eun Hee; Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo; Jeong, Hye Gwang

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction.

  18. Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function.

    PubMed

    Escuin, Daniel; Kline, Erik R; Giannakakou, Paraskevi

    2005-10-01

    We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.

  19. Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

    PubMed Central

    Tang, Xu-dong; Zhou, Xin; Zhou, Ke-yuan

    2009-01-01

    Aim: To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7). Methods: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed. Results: Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs. Conclusion: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer. PMID:19349962

  20. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF.

    PubMed

    Koivunen, Peppi; Hirsilä, Maija; Remes, Anne M; Hassinen, Ilmo E; Kivirikko, Kari I; Myllyharju, Johanna

    2007-02-16

    The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by two oxygen-dependent events that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH. Fumarate and succinate were identified as in vitro inhibitors of all three HIF-P4Hs, fumarate having K(i) values of 50-80 microM and succinate 350-460 microM, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with K(i) values of 400-1000 microM and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a K(i) of 110 microM. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1alpha and increased production of vascular endothelial growth factor and erythropoietin. Similar, although much smaller, changes were found in cultured fibroblasts from a patient with fumarate hydratase (FH) deficiency and upon silencing FH using small interfering RNA. No such effects were seen upon culturing of cells with succinate diethyl or dimethyl ester. As FIH was not inhibited by fumarate, our data indicate that the transcriptional activity of HIF is quite high even when binding of the coactivator p300 is prevented. Our data also support recent suggestions that the increased fumarate and succinate levels present in the FH and succinate dehydrogenase-deficient tumors, respectively, can inhibit the HIF-P4Hs with consequent stabilization of HIF-alphas and effects on tumor pathology.

  1. A possible therapeutic potential of quercetin through inhibition of μ-calpain in hypoxia induced neuronal injury: a molecular dynamics simulation study

    PubMed Central

    Pandey, Anand Kumar; Shukla, Swet Chand; Bhattacharya, Pallab; Patnaik, Ranjana

    2016-01-01

    The neuroprotective property of quercetin is well reported against hypoxia and ischemia in past studies. This property of quercetin lies in its antioxidant property with blood-brain barrier permeability and anti-inflammatory capabilities. µ-Calpain, a calcium ion activated intracellular cysteine protease causes serious cellular insult, leading to cell death in various pathological conditions including hypoxia and ischemic stroke. Hence, it may be considered as a potential drug target for the treatment of hypoxia induced neuronal injury. As the inhibitory property of µ-calpain is yet to be explored in details, hence, in the present study, we investigated the interaction of quercetin with µ-calpain through a molecular dynamics simulation study as a tool through clarifying the molecular mechanism of such inhibition and determining the probable sites and modes of quercetin interaction with the µ-calpain catalytic domain. In addition, we also investigated the structure-activity relationship of quercetin with μ-calpain. Affinity binding of quercetin with µ-calpain had a value of –28.73 kJ/mol and a Ki value of 35.87 µM that may be a probable reason to lead to altered functioning of µ-calpain. Hence, quercetin was found to be an inhibitor of µ-calpain which might have a possible therapeutic role in hypoxic injury. PMID:27651771

  2. A new anti-angiogenic small molecule, G0811, inhibits angiogenesis via targeting hypoxia inducible factor (HIF)-1α signal transduction

    SciTech Connect

    Kim, Ki Hyun; Jung, Hye Jin; Kwon, Ho Jeong

    2013-11-15

    Highlights: •G0811 suppresses HIF-1α expression without cell toxicity. •G0811 exhibits anti-angiogenic activity both in vitro and in vivo. •G0811 provides a new molecular scaffold for the development of therapeutics targeting angiogenesis. -- Abstract: Regulation of hypoxia inducible factor (HIF)-1α stabilization, which in turn contributes to adaptation of tumor cells to hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. We have identified a new small molecule, G0811, as a potent angiogenesis inhibitor that targets HIF-1α signal transduction. G0811 suppressed HIF-1α stability in cancer cells and inhibited in vitro and in vivo angiogenesis, as validated by tube formation, chemoinvasion, and chorioallantoic membrane (CAM) assays. In addition, G0811 effectively decreased the expression of vascular endothelial growth factor (VEGF), which is one of target genes of HIF-1α. However, G0811 did not exhibit anti-proliferative activities or toxicity in human umbilical vein endothelial cells (HUVECs) at effective doses. These results demonstrate that G0811 could be a new angiogenesis inhibitor that acts by targeting HIF-1α signal transduction pathway.

  3. Curcumin inhibits hypoxia-induced proliferation and invasion of MG-63 osteosarcoma cells via downregulating Notch1.

    PubMed

    Wang, Zhan; Zhang, Kun; Zhu, Yangjun; Wang, Dengfeng; Shao, Yuxiong; Zhang, Jun

    2017-04-01

    Curcumin is a biologically active ingredient abundantly present in the ground rhizomes of Curcuma longa with a wide range of bioactive properties, including antitumor effects. Hypoxia is a common characteristic of solid tumors, including osteosarcoma. However, whether curcumin has antitumor effects on osteosarcoma under hypoxic conditions, and its underlying molecular mechanisms, remain unclear. The present study demonstrated that the MG‑63 osteosarcoma cell line exhibited increased proliferation and enhanced invasiveness upon exposure to hypoxic conditions. However, these effects were prevented by curcumin treatment. Further investigation revealed that curcumin may inhibit Notch1 upregulation induced by hypoxia. Overexpression of Notch1 via Notch1 cDNA transfection ameliorated curcumin‑inhibited MG‑63 cell growth under hypoxic conditions. Taken together, these data revealed that curcumin may suppress the growth of osteosarcoma cells in hypoxia via inhibiting Notch1 signaling.

  4. The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1{alpha} targeted gene expression

    SciTech Connect

    Miyake, Kotaro; Nishioka, Masanori; Imura, Satoru; Batmunkh, Erdenebulgan; Uto, Yoshihiro; Nagasawa, Hideko; Hori, Hitoshi; Shimada, Mitsuo

    2012-08-01

    Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of a novel hypoxic cytotoxin, 3-[2-hydroxyethyl(methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098) in inhibiting the expression of hypoxia inducible factor-1{alpha} (HIF-1{alpha}), and consequently vascular endothelial cell growth factor (VEGF) expression in pancreatic cancer. The antitumor effects of TX-2098 under hypoxia were tested against various human pancreatic cancer cell lines using WST-8 assay. VEGF protein induced pancreatic cancer was determined on cell-free supernatant by ELISA. Moreover, nude mice bearing subcutaneously (s.c.) or orthotopically implanted human SUIT-2 were treated with TX-2098. Tumor volume, survival and expression of HIF-1 and associated molecules were evaluated in treatment versus control groups. In vitro, TX-2098 inhibited the proliferation of various pancreatic cancer cell lines. In s.c model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-2098 showed significant reductions in volume (P < 0.01 versus control). Quantitative real-time reverse transcription-PCR analysis revealed that TX-2098 significantly inhibited mRNA expression of the HIF-1 associated molecules, VEGF, glucose transporter 1 and Aldolase A (P < 0.01 versus control). These treatments also prolong the survival in orthotopic models. These results suggest that the effect of TX-2098 in pancreatic cancer might be correlated with the expression of VEGF and HIF-1 targeted molecules. -- Highlights: Black-Right-Pointing-Pointer We designed and synthesized novel hypoxic cytoxin, TX-2098. Black-Right-Pointing-Pointer TX-2098 inhibited the proliferation of human pancreatic cancer cells than TPZ. Black-Right-Pointing-Pointer TX-2098 reduced VEGF protein level than TPZ. Black-Right-Pointing-Pointer TX-2098

  5. Agmatine : metabolic pathway and spectrum of activity in brain.

    PubMed

    Halaris, Angelos; Plietz, John

    2007-01-01

    Agmatine is an endogenous neuromodulator that, based on animal studies, has the potential for new drug development. As an endogenous aminoguanidine compound (1-amino-4-guanidinobutane), it is structurally unique compared with other monoamines. Agmatine was long thought to be synthesised only in lower life forms, until its biosynthetic pathway (decarboxylation of arginine) was described in the mammalian brain in 1994. Human arginine decarboxylase has been cloned and shown to have 48% identity to ornithine decarboxylase. In neurons of the brain and spinal cord, agmatine is packaged into synaptic vesicles and released upon neuronal depolarisation. Other evidence of a neuromodulation role for agmatine is the presence of a specific cellular uptake mechanism and a specific metabolic enzyme (agmatinase; which forms putrescine).Initially, agmatine was conceptualised as an endogenous clonidine-displacing substance of imidazoline receptors; however, it has now been established to have affinity for several transmembrane receptors, such as alpha(2)-adrenergic, imidazoline I(1) and glutamatergic NMDA receptors. In addition to activity at these receptors, agmatine irreversibly inhibits neuronal nitric oxide synthase and downregulates inducible nitric oxide synthase. Endogenous agmatine is induced in response to stress and/or inflammation. Stressful conditions that induce agmatine include hypoxic-ischaemia and cold-restraint stress of ulcerogenic proportion. Induction of agmatine in the brain seems to occur in astrocytes, although neurons also synthesise agmatine. The effects of injected agmatine in animals include anticonvulsant-, antineurotoxic- and antidepressant-like actions. Intraperitoneal or intracerebroventricular injections of agmatine rapidly elicit antidepressant-like behavioural changes in the rodent forced swim test and tail suspension test. Intraperitoneal injections of agmatine into rats and mice also elicit acute anxiolytic-like behavioural changes in the elevated

  6. Sodwanone and Yardenone Triterpenes from a South African Species of the Marine Sponge Axinella Inhibit Hypoxia-Inducible Factor-1 (HIF-1) Activation in both Breast and Prostate Tumor Cells

    PubMed Central

    Dai, Jingqiu; Fishback, James A.; Zhou, Yu-Dong; Nagle, Dale G.

    2010-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that promotes tumor cell adaptation and survival under hypoxic conditions. HIF-1 is currently recognized as an important molecular target for anti-cancer drug discovery. A T47D breast tumor cell-based reporter assay was used to evaluate the NCI Open Repository of marine invertebrates and algae lipid extracts for HIF-1 inhibitory activity. Bioassay-guided fractionation and isolation of an active extract from Axinella sp. yielded seven new sodwanone triterpenoids [3-epi-sodwanone K (1), 3-epi-sodwanone K 3-acetate (2), 10,11-dihydrosodwanone B (4), sodwanones T–W (3, 7, 8, 9), the new yardenone triterpene 12R-hydroxyyardenone (10), and the previously reported compounds sodwanone A (5), sodwanone B (6), and yardenone (11). The structures and relative configurations of these Axinella metabolites were determined spectroscopically. The absolute configuration of 1 was determined by the modified Mosher ester procedure. Sodwanone V (8) inhibited both hypoxia-induced and iron chelator (1,10-phenanthroline)-induced HIF-1 activation in T47D breast tumor cells (IC50 15 μM) and 8 was the only sodwanone that inhibited HIF-1 activation in PC-3 prostate tumor cells (IC50 15 μM). Compounds 1, 3, 4, and 5 inhibited hypoxia-induced HIF-1 activation in T47D cells (IC50 values 20-25 μM). Compound 2 was cytotoxic to T47D cells (IC50 22 μM) and 8 showed cytotoxicity to MDA-MB-231 breast tumor cells (IC50 23 μM). PMID:17190448

  7. Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α*S⃞

    PubMed Central

    Bove, Peter F.; Hristova, Milena; Wesley, Umadevi V.; Olson, Nels; Lounsbury, Karen M.; van der Vliet, Albert

    2008-01-01

    Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1α and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1α stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1α activation. Activation of HIF-1α by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1α mutant construct inhibited epithelial wound repair, implicating HIF-1α in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1α. Finally, NO-mediated inhibition of cell migration was associated with HIF-1α-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1α and p53, with potential consequences for epithelial repair and remodeling during airway inflammation. PMID

  8. Menadione and ethacrynic acid inhibit the hypoxia-inducible factor (HIF) pathway by disrupting HIF-1α interaction with p300.

    PubMed

    Na, Yu-Ran; Han, Ki-Cheol; Park, Hyunsung; Yang, Eun Gyeong

    2013-05-17

    Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation.

  9. Characterization and inactivation of an agmatine deiminase from Helicobacter pylori

    SciTech Connect

    Jones, Justin E.; Causey, Corey P.; Lovelace, Leslie; Knuckley, Bryan; Flick, Heather; Lebioda, Lukasz; Thompson, Paul R.

    2010-11-12

    Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10-30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1 {angstrom} resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine-based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described.

  10. Characterization and Inactivation of an Agmatine Deiminase from Helicobacter Pylori†||

    PubMed Central

    Jones, Justin E.; Causey, Corey P.; Lovelace, Leslie; Knuckley, Bryan; Flick, Heather; Lebioda, Lukasz; Thompson, Paul R.

    2010-01-01

    Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10–30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1 Å resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described. PMID:20036411

  11. The antiproliferative effects of agmatine correlate with the rate of cellular proliferation.

    PubMed

    Isome, Masato; Lortie, Mark J; Murakami, Yasuko; Parisi, Eva; Matsufuji, Senya; Satriano, Joseph

    2007-08-01

    Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times.

  12. Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1.

    PubMed

    Petry, Andreas; BelAiba, Rachida S; Weitnauer, Michae; Görlach, Agnes

    2012-11-01

    Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

  13. β-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α.

    PubMed

    Kim, Yoo-Sun; Lee, Hyun-Ah; Lim, Ji Ye; Kim, Yang; Jung, Chang-Hwan; Yoo, Sang-Ho; Kim, Yuri

    2014-06-01

    Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While β-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of β-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of β-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with β-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following β-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered β-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following β-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following β-carotene treatment. In conclusion, the present study provides the first evidence that β-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.

  14. Cromolyn sodium does not prevent hypoxia-induced pulmonary hypertension in newborn and young lambs.

    PubMed

    Frantz, E G; Schreiber, M D; Soifer, S J

    1988-12-01

    Hypoxia-induced pulmonary hypertension may be mediated by leukotrienes. Pulmonary mast cells produce leukotrienes, histamine and prostaglandin D2, and degranulate in response to hypoxia. Cromolyn sodium, a mast cell membrane stabilizing agent, may prevent hypoxia-induced mast cell degranulation. To investigate the role of mast cell products in hypoxia-induced pulmonary hypertension, we studied the haemodynamic responses to alveolar hypoxia before and during an intravenous infusion of 3-5 mg/min per kg of cromolyn sodium in 6 chronically instrumented, spontaneously breathing lambs. Since there are age-dependent differences in the response of the pulmonary circulation to some mast cell products, we studied the effects of cromolyn sodium on hypoxia-induced pulmonary hypertension in newborn (4-7 days) and young lambs (15-18 days). During alveolar hypoxia, mean pulmonary arterial pressure increased by 68% (P less than 0.05) and 59% (P less than 0.05) in the newborn and young lambs, respectively. With alveolar hypoxia during cromolyn sodium infusion, mean pulmonary arterial pressure increased by 71% (P less than 0.05) and 42% (P less than 0.05) in the newborn and young lambs, respectively. Cromolyn sodium did blunt the hypoxia-induced release of histamine into the circulation. Because hypoxia-induced pulmonary hypertension was not inhibited by cromolyn sodium in either age group, mast cell products are not important mediators of hypoxia-induced pulmonary hypertension.

  15. Apolipoprotein A-I mimetic peptides inhibit expression and activity of hypoxia-inducible factor-1α in human ovarian cancer cell lines and a mouse ovarian cancer model.

    PubMed

    Gao, Feng; Chattopadhyay, Arnab; Navab, Mohamad; Grijalva, Victor; Su, Feng; Fogelman, Alan M; Reddy, Srinivasa T; Farias-Eisner, Robin

    2012-08-01

    Our previous results demonstrated that the apolipoprotein A-I (apoA-I) mimetic peptides L-4F and L-5F inhibit vascular endothelial growth factor production and tumor angiogenesis. The present study was designed to test whether apoA-I mimetic peptides inhibit the expression and activity of hypoxia-inducible factor-1α (HIF-1α), which plays a critical role in the production of angiogenic factors and angiogenesis. Immunohistochemistry staining was used to examine the expression of HIF-1α in tumor tissues. Immunoblotting, real-time polymerase chain reaction, immunofluorescence, and luciferase activity assays were used to determine the expression and activity of HIF-1α in human ovarian cancer cell lines. Immunohistochemistry staining demonstrated that L-4F treatment dramatically decreased HIF-1α expression in mouse ovarian tumor tissues. L-4F inhibited the expression and activity of HIF-1α induced by low oxygen concentration, cobalt chloride (CoCl(2), a hypoxia-mimic compound), lysophosphatidic acid, and insulin in two human ovarian cancer cell lines, OV2008 and CAOV-3. L-4F had no effect on the insulin-induced phosphorylation of Akt, but inhibited the activation of extracellular signal-regulated kinase and p70s6 kinase, leading to the inhibition of HIF-1α synthesis. Pretreatment with L-4F dramatically accelerated the proteasome-dependent protein degradation of HIF-1α in both insulin- and CoCl(2)-treated cells. The inhibitory effect of L-4F on HIF-1α expression is in part mediated by the reactive oxygen species-scavenging effect of L-4F. ApoA-I mimetic peptides inhibit the expression and activity of HIF-1α in both in vivo and in vitro models, suggesting the inhibition of HIF-1α may be a critical mechanism responsible for the suppression of tumor progression by apoA-I mimetic peptides.

  16. Suppression of the expression of hypoxia-inducible factor-1α by RNA interference alleviates hypoxia-induced pulmonary hypertension in adult rats

    PubMed Central

    Li, Ying; Shi, Bo; Huang, Liping; Wang, Xin; Yu, Xiaona; Guo, Baosheng; Ren, Weidong

    2016-01-01

    Hypoxia-inducible factor-1α (HIF-1α) has been implicated in the pathogenesis of hypoxic pulmonary hypertension (PH). However, the potential clinical value of HIF-1α as a therapeutic target in the treatment of PH has not yet been evaluated. In this study, an animal model of hypoxia-induced PH was established by exposing adult rats to 10% O2 for 3 weeks, and the effects of the lentivirus-mediated delivery of HIF-1α short hairpin RNA (shRNA) by intratracheal instillation prior to exposure to hypoxia on the manifestations of hypoxia-induced PH were assessed. The successful delivery of HIF-1α shRNA into the pulmonary arteries effectively suppressed the hypoxia-induced upregulation of HIF-1α, accompanied by the prominent attenuation the symptoms associated with hypoxia-induced PH, including the elevation of pulmonary arterial pressure, hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs), as well as the muscularization of pulmonary arterioles. In addition, the knockdown of HIF-1α in cultured rat primary PASMCs significantly inhibited the hypoxia-induced acceleration of the cell cycle and the proliferation of the PASMCs, suggesting that HIF-1α may be a direct mediator of PASMC hyperplasia in hypoxia-induced PH. In conclusion, this study demonstrates the potent suppressive effects of HIF-1α shRNA on hypoxia-induced PH and PASMC hyperplasia, providing evidence for the potential application of HIF-1α shRNA in the treatment of hypoxic PH. PMID:27748831

  17. Agmatine ameliorates adjuvant induced arthritis and inflammatory cachexia in rats.

    PubMed

    Taksande, Brijesh G; Gawande, Dinesh Y; Chopde, Chandrabhan T; Umekar, Milind J; Kotagale, Nandkishor R

    2017-02-01

    The present study investigated the pharmacological effect of agmatine in Complete Freud Adjuvant (CFA) induced arthritis and cachexia in rats. The rats were injected with CFA (0.1ml/rat) to induced symptoms of arthritis. Day 8 onwards of CFA administration, rats were injected daily with agmatine for next 7days, and arthritis score, body weights and food intake were monitored daily (g). Since cachexia is known to produce severe inflammation, malnutrition and inhibition of albumin gene expression, we have also monitored the total proteins, albumin, TNF-α and IL-6 levels in arthritic rats and its modulation by agmatine. In the present study, CFA treated rats showed a progressive reduction in both food intake and body weight. In addition analysis of blood serum of arthritis animals showed a significant reduction in proteins and albumin and significant elevation in tumor necrosis factor (TNF)-α and Interleukins (IL)-6. Chronic agmatine (20-40mg/kg, ip) treatment not only attenuated the signs of arthritis but also reverses anorexia and body weight loss in CFA treated rats. In addition, agmatine restored total protein and albumin and reduces TNF-α and IL-6 levels in arthritis rats. These results suggest that agmatine administration can prevent the body weights loss and symptoms of arthritis via inhibition of inflammatory cytokines.

  18. The connectivity map links iron regulatory protein-1-mediated inhibition of hypoxia-inducible factor-2a translation to the anti-inflammatory 15-deoxy-delta12,14-prostaglandin J2.

    PubMed

    Zimmer, Michael; Lamb, Justin; Ebert, Benjamin L; Lynch, Mary; Neil, Christopher; Schmidt, Emmett; Golub, Todd R; Iliopoulos, Othon

    2010-04-15

    Hypoxia-inducible factors 1 and 2 (HIF1 and HIF2) are heterodimeric transcription factors consisting of alpha regulatory subunits and a constitutively expressed beta subunit. The expression of alpha regulatory subunits is promoted by hypoxia, cancer-associated mutations, and inflammatory cytokines. Thus, HIF1 and HIF2 provide a molecular link between cancer and inflammation. We have recently identified novel small molecules that selectively inhibit translation of the HIF2a message and thereby powerfully inhibit the expression of HIF2a target genes. We report here that Connectivity Map analysis links three of these compounds to the anti-inflammatory cytokine 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)). As with our identified compounds, PGJ(2) inhibits translation of the HIF2a message in a mammalian target of rapamycin-independent manner by promoting the binding of iron regulatory protein-1 (IRP1) to a noncanonical iron responsive element (IRE) embedded within the 5'-untranslated region of the HIF2a message. The IRE is necessary and sufficient for mediating the effect. Mutation of the IRE sequence, or downregulation of IRP1 expression, blocks the effect of PGJ(2) on HIF2a translation. This is the first report of an endogenous natural molecule regulating HIF2a translation, and it suggests that part of the anti-inflammatory and putative antineoplastic effects of PGJ(2) may be mediated through inhibition of HIF2a within tumor epithelial cells themselves and/or mesenchymal cells of the tumor microenvironment.

  19. The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells.

    PubMed

    Tadakawa, Mari; Takeda, Takashi; Li, Bin; Tsuiji, Kenji; Yaegashi, Nobuo

    2015-01-05

    The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas.

  20. Regulation of hypoxia-inducible genes by ETS1 transcription factor.

    PubMed

    Salnikow, Konstantin; Aprelikova, Olga; Ivanov, Sergey; Tackett, Sean; Kaczmarek, Monika; Karaczyn, Aldona; Yee, Herman; Kasprzak, Kazimierz S; Niederhuber, John

    2008-08-01

    Hypoxia-inducible factor (HIF-1) regulates the expression of genes that facilitate tumor cell survival by making them more resistant to therapeutic intervention. Recent evidence suggests that the activation of other transcription factors, in cooperation with HIF-1 or acting alone, is involved in the upregulation of hypoxia-inducible genes. Here we report that high cell density, a condition that might mimic the physiologic situation in growing tumor and most probably representing nutritional starvation, upregulates hypoxia-inducible genes. This upregulation can occur in HIF-independent manner since hypoxia-inducible genes carbonic anhydrase 9 (CA9), lysyloxidase like 2 (LOXL2) and n-myc-down regulated 1 (NDRG1)/calcium activated protein (Cap43) can be upregulated by increased cell density under both normoxic and hypoxic conditions in both HIF-1 alpha-proficient and -deficient mouse fibroblasts. Moreover, cell density upregulates the same genes in 1HAEo- and A549 human lung epithelial cells. Searching for other transcription factors involved in the regulation of hypoxia-inducible genes by cell density, we focused our attention on ETS1. As reported previously, members of v-ets erythroblastosis virus E26 oncogene homolog (ETS) family transcription factors participate in the upregulation of hypoxia-inducible genes. Here, we provide evidence that ETS1 protein is upregulated at high cell density in both human and mouse cells. The involvement of ETS1 in the upregulation of hypoxia-inducible genes was further confirmed in a luciferase reporter assay using cotransfection of ETS1 expression vector with NDRG1/Cap43 promoter construct. The downregulation of ETS1 expression with small interfering RNA (siRNA) inhibited the upregulation of CA9 and NDRG1/Cap43 caused by increased cell density. Collectively, our data indicate the involvement of ETS1 along with HIF-1 in regulating hypoxia-inducible genes.

  1. Environmental Enrichment Prevent the Juvenile Hypoxia-Induced Developmental Loss of Parvalbumin-Immunoreactive Cells in the Prefrontal Cortex and Neurobehavioral Alterations Through Inhibition of NADPH Oxidase-2-Derived Oxidative Stress.

    PubMed

    Zhang, Mingqiang; Wu, Jing; Huo, Lan; Luo, Liang; Song, Xi; Fan, Fei; Lu, Yiming; Liang, Dong

    2016-12-01

    We compared the expression of phenotype of parvalbumin (PV)-immunoreactive cells in the prefrontal cortex (PFC) of juvenile rats reared in enriched environment (EE) after daily intermittent hypoxia (IH) exposure to those reared in standard environment (SE) and investigated the involvement of NADPH oxidase-2 (NOX2)-derived oxidative stress in the IH-induced neurodevelopmental and neurobehavioral consequences in a juvenile rat model of obstructive sleep apnea. Postnatal day 21 (P21) rats were exposed to IH or room air 8 h daily for 14 consecutive days. After the daily exposure, the rats were raised in SE or EE. In the PFC of P34 rats, we determined the impact (i) of IH exposures on NOX2-derived oxidative stress and PV immunoreactivity, (ii) of pharmacological NOX2 inhibition on IH-induced oxidative stress and PV immunoreactivity, and (iii) of EE on the IH-induced oxidative stress and PV immunoreactivity. Behavioral testing of psychiatric anxiety was carried out consecutively in the open-field test and elevated plus maze at P35 and P36. The results showed IH exposures increased NOX2 expression in the PFC of P34 rats, which was accompanied with elevation of NOX activity and indirect markers of oxidative stress (4-HNE). IH exposures increased 4-HNE immunoreactivity in cortical PV cells, which was accompanied with reduction of PV immunoreactivity. Treatment of IH rats with the antioxidant/NOX inhibitor apocynin prevented the PV cells loss in the PFC and reversed the IH-induced psychiatric anxiety. EE attenuated the NOX2-derived oxidative stress and reversed the PV-immunoreactivity reduction in the PFC induced by IH. Our data suggest that EE might prevent the juvenile hypoxia-induced developmental loss of PV cells in the PFC and attenuate the neurobehavioral alterations through inhibition of NOX2-derived oxidative stress.

  2. Sodium tanshinone IIA sulfonate inhibits hypoxia-induced enhancement of SOCE in pulmonary arterial smooth muscle cells via the PKG-PPAR-γ signaling axis.

    PubMed

    Jiang, Qian; Lu, Wenju; Yang, Kai; Hadadi, Cyrus; Fu, Xin; Chen, Yuqin; Yun, Xin; Zhang, Jie; Li, Meichan; Xu, Lei; Tang, Haiyang; Yuan, Jason X-J; Wang, Jian; Sun, Dejun

    2016-07-01

    Our laboratory previously showed that sodium tanshinone IIA sulfonate (STS) inhibited store-operated Ca(2+) entry (SOCE) through store-operated Ca(2+) channels (SOCC) via downregulating the expression of transient receptor potential canonical proteins (TRPC), which contribute to the formation of SOCC (Wang J, Jiang Q, Wan L, Yang K, Zhang Y, Chen Y, Wang E, Lai N, Zhao L, Jiang H, Sun Y, Zhong N, Ran P, Lu W. Am J Respir Cell Mol Biol 48: 125-134, 2013). The detailed molecular mechanisms by which STS inhibits SOCE and downregulates TRPC, however, remain largely unknown. We have previously shown that, under hypoxic conditions, inhibition of protein kinase G (PKG) and peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling axis results in the upregulation of TRPC (Wang J, Yang K, Xu L, Zhang Y, Lai N, Jiang H, Zhang Y, Zhong N, Ran P, Lu W. Am J Respir Cell Mol Biol 49: 231-240, 2013). This suggests that strategies targeting the restoration of this signaling pathway may be an effective treatment strategy for pulmonary hypertension. In this study, our results demonstrated that STS treatment can effectively prevent the hypoxia-mediated inhibition of the PKG-PPAR-γ signaling axis in rat distal pulmonary arterial smooth muscle cells (PASMCs) and distal pulmonary arteries. These effects of STS treatment were blocked by pharmacological inhibition or specific small interfering RNA knockdown of either PKG or PPAR-γ. Moreover, targeted PPAR-γ agonist markedly enhanced the beneficial effects of STS. These results comprehensively suggest that STS treatment can prevent hypoxia-mediated increases in intracellular calcium homeostasis and cell proliferation, by targeting and restoring the hypoxia-inhibited PKG-PPAR-γ signaling pathway in PASMCs.

  3. Inhibition of prolyl hydroxylase protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity: model for the potential involvement of the hypoxia-inducible factor pathway in Parkinson disease.

    PubMed

    Lee, Donna W; Rajagopalan, Subramanian; Siddiq, Ambreena; Gwiazda, Roberto; Yang, Lichuan; Beal, M Flint; Ratan, Rajiv R; Andersen, Julie K

    2009-10-16

    Hypoxia-inducible factor (HIF) plays an important role in cell survival by regulating iron, antioxidant defense, and mitochondrial function. Pharmacological inhibitors of the iron-dependent enzyme class prolyl hydroxylases (PHD), which target alpha subunits of HIF proteins for degradation, have recently been demonstrated to alleviate neurodegeneration associated with stroke and hypoxic-ischemic injuries. Here we report that inhibition of PHD by 3,4-dihydroxybenzoate (DHB) protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic cell loss and up-regulates HIF-1alpha within these neurons. Elevations in mRNA and protein levels of HIF-dependent genes heme oxygenase-1 (Ho-1) and manganese superoxide dismutase (Mnsod) following DHB pretreatment alone are also maintained in the presence of MPTP. MPTP-induced reductions in ferroportin and elevations in nigral and striatal iron levels were reverted to levels comparable with that of untreated controls with DHB pretreatment. Reductions in pyruvate dehydrogenase mRNA and activity resulting from MPTP were also found to be attenuated by DHB. In vitro, the HIF pathway was activated in N27 cells grown at 3% oxygen treated with either PHD inhibitors or an iron chelator. Concordant with our in vivo data, the MPP(+)-elicited increase in total iron as well as decreases in cell viability were attenuated in the presence of DHB. Taken together, these data suggest that protection against MPTP neurotoxicity may be mediated by alterations in iron homeostasis and defense against oxidative stress and mitochondrial dysfunction brought about by cellular HIF-1alpha induction. This study provides novel data extending the possible therapeutic utility of HIF induction to a Parkinson disease model of neurodegeneration, which may prove beneficial not only in this disorder itself but also in other diseases associated with metal-induced oxidative stress.

  4. Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Prevents Steroid-Associated Osteonecrosis of the Femoral Head in Rabbits by Promoting Angiogenesis and Inhibiting Apoptosis

    PubMed Central

    Fan, Lihong; Li, Jia; Yu, Zefeng; Dang, Xiaoqian; Wang, Kunzheng

    2014-01-01

    The purpose of this study was to investigate the preventive effect of ethyl 3,4-dihydroxybenzoate(EDHB) on steroid-associated femoral head osteonecrosis(ONFH) in a rabbit model. New Zealand white rabbits were randomly divided into two groups (prevention group and model group), each containing 24 rabbits. Osteonecrosis was induced by lipopolysaccharide(LPS) combined with methylprednisolone(MPS). The prevention group received an intraperitoneal injection of EDHB at 50 mg/kg body weight every other day starting three days before establishing rabbit models of osteonecrosis, for a total of nine doses. Osteonecrosis was verified by haematoxylin-eosin (HE) staining. The expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Angiogenesis, apoptosis and microstructural parameters were also analyzed. The rabbit models of osteonecrosis were successfully established and observed by HE staining. Histopathological observations indicated that EDHB reduced the rate of empty lacunae and the incidence of osteonecrosis. Immunohistochemical staining for HIF-1α and VEGF suggested that EDHB therapy inhibited degradation of HIF-1α and promoted expression of VEGF. Ink artery infusion angiography and microvessel density analysis revealed that there were more microvessels in the prevention group than in the model group. The TUNEL apoptosis assay suggested that EDHB intervention could reduce the number of apoptotic cells in avascular osteonecrosis of the femoral head. Micro-CT scanning indicated that the treatment group had better microstructural parameters than the model group. EDHB prevents steroid-associated osteonecrosis of the femoral head in rabbits by promoting angiogenesis and inhibiting apoptosis of bone cells and hematopoietic tissue. PMID:25244080

  5. Agmatine Prevents Adaptation of the Hippocampal Glutamate System in Chronic Morphine-Treated Rats.

    PubMed

    Wang, Xiao-Fei; Zhao, Tai-Yun; Su, Rui-Bin; Wu, Ning; Li, Jin

    2016-12-01

    Chronic exposure to opioids induces adaptation of glutamate neurotransmission, which plays a crucial role in addiction. Our previous studies revealed that agmatine attenuates opioid addiction and prevents the adaptation of glutamate neurotransmission in the nucleus accumbens of chronic morphine-treated rats. The hippocampus is important for drug addiction; however, whether adaptation of glutamate neurotransmission is modulated by agmatine in the hippocampus remains unknown. Here, we found that continuous pretreatment of rats with ascending doses of morphine for 5 days resulted in an increase in the hippocampal extracellular glutamate level induced by naloxone (2 mg/kg, i.p.) precipitation. Agmatine (20 mg/kg, s.c.) administered concurrently with morphine for 5 days attenuated the elevation of extracellular glutamate levels induced by naloxone precipitation. Furthermore, in the hippocampal synaptosome model, agmatine decreased the release and increased the uptake of glutamate in synaptosomes from chronic morphine-treated rats, which might contribute to the reduced elevation of glutamate levels induced by agmatine. We also found that expression of the hippocampal NR2B subunit, rather than the NR1 subunit, of N-methyl-D-aspartate receptors (NMDARs) was down-regulated after chronic morphine treatment, and agmatine inhibited this reduction. Taken together, agmatine prevented the adaptation of the hippocampal glutamate system caused by chronic exposure to morphine, including modulating extracellular glutamate concentration and NMDAR expression, which might be one of the mechanisms underlying the attenuation of opioid addiction by agmatine.

  6. Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector.

    PubMed

    Liu, Xueping; Wang, Guansong; You, Zaichun; Qian, Pin; Chen, Huaping; Dou, Yin; Wei, Zhenghua; Chen, Yan; Mao, Chengde; Zhang, Jianxiang

    2014-05-01

    The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics

  7. Synthesis and biological evaluation of diaryl-substituted carboranes as inhibitors of hypoxia inducible factor (HIF)-1 transcriptional activity.

    PubMed

    Minegishi, Hidemitsu; Matsukawa, Takuya; Nakamura, Hiroyuki

    2013-02-01

    Diaromatic-substituted ortho- and meta-carboranes were synthesized as mimics of manassantin A. Among the carboranes synthesized, compounds 1 and 2 showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity, with IC(50) values of 3.2 and 2.2 μM, respectively. Compounds 1 and 2 similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of HIF-1α mRNA. The hypoxia-induced accumulation and translocation of HIF-1α into nuclei were not observed in HeLa cells treated with compounds 1 and 2 by immunofluorescence analysis, revealing that the inhibition of hypoxia-induced HIF-1 transcriptional activity is induced by compounds 1 and 2 through a degradation pathway of the HIF-1α protein under hypoxic conditions.

  8. Transcriptional Regulation by Hypoxia Inducible Factors

    PubMed Central

    Espinosa, Joaquín M.

    2015-01-01

    The cellular response to oxygen deprivation is governed largely by a family of transcription factors known as Hypoxia Inducible Factors (HIFs). This review focuses on the molecular mechanisms by which HIFs regulate the transcriptional apparatus to enable the cellular and organismal response to hypoxia. We discuss here how the various HIF polypeptides, their post-translational modifications, binding partners and transcriptional cofactors affect RNA polymerase II activity to drive context-dependent transcriptional programs during hypoxia. PMID:24099156

  9. Lysyl oxidase is essential for hypoxia-induced metastasis.

    PubMed

    Erler, Janine T; Bennewith, Kevin L; Nicolau, Monica; Dornhöfer, Nadja; Kong, Christina; Le, Quynh-Thu; Chi, Jen-Tsan Ashley; Jeffrey, Stefanie S; Giaccia, Amato J

    2006-04-27

    Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.

  10. Involvement of α₂-adrenoceptors, imidazoline, and endothelin-A receptors in the effect of agmatine on morphine and oxycodone-induced hypothermia in mice.

    PubMed

    Bhalla, Shaifali; Andurkar, Shridhar V; Gulati, Anil

    2013-10-01

    Potentiation of opioid analgesia by endothelin-A (ET(A)) receptor antagonist, BMS182874, and imidazoline receptor/α₂-adrenoceptor agonists such as clonidine and agmatine are well known. It is also known that agmatine blocks morphine hyperthermia in rats. However, the effect of agmatine on morphine or oxycodone hypothermia in mice is unknown. The present study was carried out to study the role of α₂-adrenoceptors, imidazoline, and ET(A) receptors in morphine and oxycodone hypothermia in mice. Body temperature was determined over 6 h in male Swiss Webster mice treated with morphine, oxycodone, agmatine, and combination of agmatine with morphine or oxycodone. Yohimbine, idazoxan, and BMS182874 were used to determine involvement of α₂-adrenoceptors, imidazoline, and ET(A) receptors, respectively. Morphine and oxycodone produced significant hypothermia that was not affected by α₂-adrenoceptor antagonist yohimbine, imidazoline receptor/α₂ adrenoceptor antagonist idazoxan, or ET(A) receptor antagonist, BMS182874. Agmatine did not produce hypothermia; however, it blocked oxycodone but not morphine-induced hypothermia. Agmatine-induced blockade of oxycodone hypothermia was inhibited by idazoxan and yohimbine. The blockade by idazoxan was more pronounced compared with yohimbine. Combined administration of BMS182874 and agmatine did not produce changes in body temperature in mice. However, when BMS182874 was administered along with agmatine and oxycodone, it blocked agmatine-induced reversal of oxycodone hypothermia. This is the first report demonstrating that agmatine does not affect morphine hypothermia in mice, but reverses oxycodone hypothermia. Imidazoline receptors and α₂-adrenoceptors are involved in agmatine-induced reversal of oxycodone hypothermia. Our findings also suggest that ET(A) receptors may be involved in blockade of oxycodone hypothermia by agmatine.

  11. Lysyl oxidase mediates hypoxia-induced radioresistance in non-small cell lung cancer A549 cells

    PubMed Central

    Gong, Chongwen; Gu, Runxia; Jin, Honglin; Sun, Yao; Li, Zhenyu; Wu, Gang

    2016-01-01

    Hypoxia-induced radioresistance has been well known as the main obstacle in cancer radiotherapy. Lysyl oxidase (LOX) was previously demonstrated to play an important role in hypoxia-induced biological behaviors, such as metastasis and angiogenesis, through hypoxia-inducible factor-1α (HIF-1α), which is an important contributing factor to radioresistance in tumor cells. However, how LOX plays a role in hypoxia-induced radioresistance has yet to be determined. Here, we found that LOX expression was in accordance with HIF-1α expression, and LOX expression at the mRNA and protein level, and enzymatic activity were remarkably upregulated in the hypoxic A549 cells, compared with normoxic A549 cells. Inhibition of LOX resulted in the reduction of the ability to repair double-stranded breaks (DSBs), promotion of apoptosis, relief of G2/M cycle arrest, and eventually reduction of hypoxia-induced radioresistance in the hypoxic A549 cells. This suggests that LOX may play an important role in hypoxia-induced radioresistance. Together, our results might suggest a novel potential therapeutic target in the management of non-small cell lung cancer (NSCLC). PMID:26515140

  12. Hypoxia inducible factors and the response to hypoxic stress

    PubMed Central

    Majmundar, Amar J.; Wong, Waihay J.; Simon, M. Celeste

    2011-01-01

    Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the Hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors including Factor Inhibiting HIF-1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway. PMID:20965423

  13. Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells.

    PubMed

    Peng, Wan-Xin; Xiong, Er-Meng; Ge, Lu; Wan, Yan-Ya; Zhang, Chun-Li; Du, Feng-Yi; Xu, Min; Bhat, Reyaz Ahmed; Jin, Jie; Gong, Ai-Hua

    2016-01-01

    Previous studies suggest that early growth response gene-1 (Egr-1) plays an important role in hypoxia-induced drug-resistance. However, the mechanism still remains to be clarified. Herein, we investigated the role of Egr-1 in hypoxia-induced autophagy and its resulted hypoxia-driven chemo-resistance in Hepatocellular Carcinoma (HCC) cells. Our data demonstrated that Egr-1 was overexpressed in HCC tissues and cells and conferred them drug resistance under hypoxia. Mechanistically, Egr-1 transcriptionally regulated hypoxia-induced autophagy by binding to LC3 promoter in HCC cells, which resulted in resistance of HCC cells to chemotherapeutic agents; while dominant negative Egr-1 could inhibit autophagy level, and thus enhanced the sensitivity of HCC cells to chemotherapeutic agents, indicating that hypoxia-induced Egr-1 expression enhanced drug resistance of HCC cells likely through autophagy. Accordingly, it is suggested that a mechanism of hypoxia/Egr-1/autophagy axis might be involved in drug resistance in HCC.

  14. Hypoxia-induced IL-32β increases glycolysis in breast cancer cells.

    PubMed

    Park, Jeong Su; Lee, Sunyi; Jeong, Ae Lee; Han, Sora; Ka, Hye In; Lim, Jong-Seok; Lee, Myung Sok; Yoon, Do-Young; Lee, Jeong-Hyung; Yang, Young

    2015-01-28

    IL-32β is highly expressed and increases the migration and invasion of gastric, lung, and breast cancer cells. Since IL-32 enhances VEGF production under hypoxic conditions, whether IL-32β is regulated by hypoxia was examined. Hypoxic conditions and a mimetic chemical CoCl2 enhanced IL-32β production. When cells were treated with various inhibitors of ROS generation to prevent hypoxia-induced ROS function, IL-32β production was suppressed by both NADPH oxidase and mitochondrial ROS inhibitors. IL-32β translocated to the mitochondria under hypoxic conditions, where it was associated with mitochondrial biogenesis. Thus, whether hypoxia-induced IL-32β is associated with oxidative phosphorylation (OXPHOS) or glycolysis was examined. Glycolysis under aerobic and anaerobic conditions is impaired in IL-32β-depleted cells, and the hypoxia-induced IL-32β increased glycolysis through activation of lactate dehydrogenase. Src is also known to increase lactate dehydrogenase activity, and the hypoxia-induced IL-32β was found to stimulate Src activation by inhibiting the dephosphorylation of Src. These findings revealed that a hypoxia-ROS-IL-32β-Src-glycolysis pathway is associated with the regulation of cancer cell metabolism.

  15. RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response

    PubMed Central

    Huang, Gaoxiang; Su, Jie; Zhang, Mingzhuo; Jin, Yiduo; Wang, Yan; Zhou, Peng; Lu, Jian

    2017-01-01

    Immune cells, particularly macrophages, play critical roles in the hypoxia-induced inflammatory response. The small GTPase RhoB is usually rapidly induced by a variety of stimuli and has been described as an important regulator of cytoskeletal organization and vesicle and membrane receptor trafficking. However, it is unknown whether RhoB is involved in the hypoxia-induced inflammatory response. Here, we investigated the effect of hypoxia on the expression of RhoB and the mechanism and significance of RhoB expression in macrophages. We found that hypoxia significantly upregulated the expression of RhoB in RAW264.7 cells, mouse peritoneal macrophages, and the spleen of rats. Hypoxia-induced expression of RhoB was significantly blocked by a specific inhibitor of hypoxia-inducible factor-1α (HIF-1α), c-Jun N-terminal kinase (JNK), or extracellular-signal regulated protein kinase (ERK), indicating that hypoxia-activated HIF-1α, JNK, and ERK are involved in the upregulation of RhoB by hypoxia. Knockdown of RhoB expression not only significantly suppressed basal production of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in normoxia but also more markedly decreased the hypoxia-stimulated production of these cytokines. Furthermore, we showed that RhoB increased nuclear factor-kappa B (NF-κB) activity, and the inhibition of NF-κB transcriptional activity significantly decreased the RhoB-increased mRNA levels of IL-1β, IL-6, and TNF-α. Finally, we demonstrated that RhoB enhanced cell adhesion and inhibited cell migration in normoxia and hypoxia. Taken together, these results suggest that RhoB plays an important role in the hypoxia-induced activation of macrophages and the inflammatory response. PMID:26388235

  16. RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response.

    PubMed

    Huang, Gaoxiang; Su, Jie; Zhang, Mingzhuo; Jin, Yiduo; Wang, Yan; Zhou, Peng; Lu, Jian

    2017-03-01

    Immune cells, particularly macrophages, play critical roles in the hypoxia-induced inflammatory response. The small GTPase RhoB is usually rapidly induced by a variety of stimuli and has been described as an important regulator of cytoskeletal organization and vesicle and membrane receptor trafficking. However, it is unknown whether RhoB is involved in the hypoxia-induced inflammatory response. Here, we investigated the effect of hypoxia on the expression of RhoB and the mechanism and significance of RhoB expression in macrophages. We found that hypoxia significantly upregulated the expression of RhoB in RAW264.7 cells, mouse peritoneal macrophages, and the spleen of rats. Hypoxia-induced expression of RhoB was significantly blocked by a specific inhibitor of hypoxia-inducible factor-1α (HIF-1α), c-Jun N-terminal kinase (JNK), or extracellular-signal regulated protein kinase (ERK), indicating that hypoxia-activated HIF-1α, JNK, and ERK are involved in the upregulation of RhoB by hypoxia. Knockdown of RhoB expression not only significantly suppressed basal production of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in normoxia but also more markedly decreased the hypoxia-stimulated production of these cytokines. Furthermore, we showed that RhoB increased nuclear factor-kappa B (NF-κB) activity, and the inhibition of NF-κB transcriptional activity significantly decreased the RhoB-increased mRNA levels of IL-1β, IL-6, and TNF-α. Finally, we demonstrated that RhoB enhanced cell adhesion and inhibited cell migration in normoxia and hypoxia. Taken together, these results suggest that RhoB plays an important role in the hypoxia-induced activation of macrophages and the inflammatory response.Cellular & Molecular Immunology advance online publication, 21 September 2015; doi:10.1038/cmi.2015.78.

  17. Agmatine improves renal function in gentamicin-induced nephrotoxicity in rats.

    PubMed

    El-Kashef, Dalia H; El-Kenawi, Asmaa E; Abdel Rahim, Mona; Suddek, Ghada M; Salem, Hatem A

    2016-03-01

    The present study was designed to explore the possible protective effects of agmatine, a known nitric oxide (NO) synthase inhibitor, against gentamicin-induced nephrotoxicity in rats. For this purpose, we quantitatively evaluated gentamicin-induced renal structural and functional alterations using histopathological and biochemical approaches. Furthermore, the effect of agmatine on gentamicin-induced hypersensitivity of urinary bladder rings to acetylcholine (ACh) was evaluated. Twenty-four male Wistar albino rats were randomly divided into 3 groups, namely control, gentamicin (100 mg/kg, i.p.), and gentamicin plus agmatine (40 mg/kg, orally). At the end of the study, all rats were sacrificed and then blood and urine samples and kidneys were taken. Administration of agmatine significantly decreased kidney/body mass ratio, serum creatinine, lactate dehydrogenase (LDH), renal malondialdehyde (MDA), myeloperoxidase (MPO), NO, and tumor necrosis factor-alpha (TNF-α) while it significantly increased creatinine clearance and renal superoxide dismutase (SOD) activity when compared with the gentamicin-treated group. Additionally, agmatine ameliorated tissue morphology as evidenced by histological evaluation and reduced the responses of isolated bladder rings to ACh. Our study indicates that agmatine administration with gentamicin attenuates oxidative-stress associated renal injury by reducing oxygen free radicals and lipid peroxidation, restoring NO level and inhibiting inflammatory mediators such as TNF-α.

  18. Neonatal epithelial hypoxia inducible factor-1α expression regulates the response of the lung to experimental asthma.

    PubMed

    Greenwood, Krista K; Proper, Steven P; Saini, Yogesh; Bramble, Lori A; Jackson-Humbles, Daven N; Wagner, James G; Harkema, Jack R; LaPres, John J

    2012-03-01

    Allergic airway disease is characterized by a T helper type 2 cell-mediated airway inflammation and airway hyperresponsiveness. Little is known about the role of hypoxia-mediated signaling in the progression of the disease. To address this knowledge gap, a mouse model was created in which doxycycline exposure induces the functional deletion of hypoxia inducible factor-1α from alveolar type II and Clara cells of the lung. When hypoxia inducible factor-1α deletion was induced during the early postnatal development period of the lung, the mice displayed an enhanced response to the ovalbumin model of allergic airway disease. These hypoxia inducible factor-1α-deficient mice exhibit increased cellular infiltrates, eosinophilia in the lavage fluid and parenchyma, and T helper type 2 cytokines, as compared with ovalbumin-treated control mice. Moreover, these hypoxia inducible factor-1α-deficient mice display increased airway resistance when compared with their control counterparts. Interestingly, if the loss of hypoxia inducible factor-1α was induced in early adulthood, the exacerbated phenotype was not observed. Taken together, these results suggest that epithelial hypoxia inducible factor-1α plays an important role in establishing the innate immunity of the lung and epithelial-specific deficiency in the transcription factor, during early postnatal development, increases the severity of inflammation and functional airway resistance, following ovalbumin challenge. Finally, these results might explain some of the chronic respiratory pathology observed in premature infants, especially those that receive supplemental oxygen. This early hyperoxic exposure, from normal ambient and supplemental oxygen, would presumably inhibit normal hypoxia inducible factor-1α signaling, mimicking the functional deletion described.

  19. Agmatine enhances the antidepressant-like effect of lithium in mouse forced swimming test through NMDA pathway.

    PubMed

    Mohseni, Gholmreza; Ostadhadi, Sattar; Imran-Khan, Muhammad; Norouzi-Javidan, Abbas; Zolfaghari, Samira; Haddadi, Nazgol-Sadat; Dehpour, Ahmad-Reza

    2017-04-01

    Depression is one the world leading global burdens leading to various comorbidities. Lithium as a mainstay in the treatment of depression is still considered gold standard treatment. Similar to lithium another agent agmatine has also central protective role against depression. Since, both agmatine and lithium modulate various effects through interaction with NMDA receptor, therefore, in current study we aimed to investigate the synergistic antidepressant-like effect of agmatine with lithium in mouse force swimming test. Also to know whether if such effect is due to interaction with NMDA receptor. In our present study we found that when potent dose of lithium (30mg/kg) was administered, it significantly decreased the immobility time. Also, when subeffective dose of agmatine (0.01mg/kg) was coadministered with subeffective dose of lithium (3mg/kg), it potentiated the antidepressant-like effect of subeffective dose of lithium. For the involvement of NMDA receptor in such effect, we administered NMDA receptor antagonist MK-801 (0.05mg/kg) with a combination of subeffective dose of lithium (3mg/kg) and agmatine (0.001mg/kg). A significant antidepressant-like effect was observed. Furthermore, when subeffective dose (50 and 75mg/kg) of NMDA was given it inhibited the synergistic effect of agmatine (0.01mg/kg) with lithium (3mg/kg). Hence, our finding demonstrate that agmatine have synergistic effect with lithium which is mediated by NMDA receptor pathway.

  20. Salidroside exerts protective effects against chronic hypoxia-induced pulmonary arterial hypertension via AMPKα1-dependent pathways

    PubMed Central

    Chen, Mayun; Cai, Hui; Yu, Chang; Wu, Peiliang; Fu, Yangyang; Xu, Xiaomei; Fan, Rong; Xu, Cunlai; Chen, Yanfan; Wang, Liangxing; Huang, Xiaoying

    2016-01-01

    Salidroside, an active ingredient isolated from Rhodiola rosea, has shown to exert protective effects against chronic hypoxia-induced pulmonary arterial hypertension (PAH). However, the underlying mechanisms were not well known. Based on our recent reports, we predicted the involvement of adenosine monophosphate-activated protein kinase (AMPK) mediated effects in salidroside regulation of PAH. Firstly, to prove the hypothesis, rats were exposed to chronic hypoxia and treated with increasing concentrations of salidroside or a selective AMPK activator-5’-aminoimidazole-4-carboxamide ribonucleoside (AICAR) for 4 weeks. After salidroside or AICAR treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary artery remodeling were attenuated. Then the effects of salidroside or AICAR on hypoxia-induced excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs), which contributed to pulmonary arterial remodeling, were investigated. Our results suggested salidroside, as well as AICAR, reversed hypoxia-induced PASMCs proliferation and apoptosis resistance while AMPK inhibitor Compound C enhanced the effects of hypoxia. To reveal the potential cellular mechanisms, activation of AMPKα1 and expression of the genes related to proliferation and apoptosis were analyzed in PASMCs after salidroside treatment under hypoxia conditions. The results demonstrated salidroside as well as AICAR might inhibit chronic hypoxia-induced PASMCs proliferation via AMPKα1-P53-P27/P21 pathway and reverse apoptosis resistance via AMPKα1-P53-Bax/Bcl-2-caspase 9-caspase 3 pathway. PMID:27069536

  1. Salidroside exerts protective effects against chronic hypoxia-induced pulmonary arterial hypertension via AMPKα1-dependent pathways.

    PubMed

    Chen, Mayun; Cai, Hui; Yu, Chang; Wu, Peiliang; Fu, Yangyang; Xu, Xiaomei; Fan, Rong; Xu, Cunlai; Chen, Yanfan; Wang, Liangxing; Huang, Xiaoying

    2016-01-01

    Salidroside, an active ingredient isolated from Rhodiola rosea, has shown to exert protective effects against chronic hypoxia-induced pulmonary arterial hypertension (PAH). However, the underlying mechanisms were not well known. Based on our recent reports, we predicted the involvement of adenosine monophosphate-activated protein kinase (AMPK) mediated effects in salidroside regulation of PAH. Firstly, to prove the hypothesis, rats were exposed to chronic hypoxia and treated with increasing concentrations of salidroside or a selective AMPK activator-5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) for 4 weeks. After salidroside or AICAR treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary artery remodeling were attenuated. Then the effects of salidroside or AICAR on hypoxia-induced excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs), which contributed to pulmonary arterial remodeling, were investigated. Our results suggested salidroside, as well as AICAR, reversed hypoxia-induced PASMCs proliferation and apoptosis resistance while AMPK inhibitor Compound C enhanced the effects of hypoxia. To reveal the potential cellular mechanisms, activation of AMPKα1 and expression of the genes related to proliferation and apoptosis were analyzed in PASMCs after salidroside treatment under hypoxia conditions. The results demonstrated salidroside as well as AICAR might inhibit chronic hypoxia-induced PASMCs proliferation via AMPKα1-P53-P27/P21 pathway and reverse apoptosis resistance via AMPKα1-P53-Bax/Bcl-2-caspase 9-caspase 3 pathway.

  2. Genome-Wide Transcriptional Analysis Reveals the Protection against Hypoxia-Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways

    PubMed Central

    Gesang, Luobu; Dan, Zeng; Gusang, Lamu

    2016-01-01

    The molecular mechanisms for hypoxic environment causing the injury of intestinal mucosal barrier (IMB) are widely unknown. To address the issue, Han Chinese from 100 m altitude and Tibetans from high altitude (more than 3650 m) were recruited. Histological and transcriptome analyses were performed. The results showed intestinal villi were reduced and appeared irregular, and glandular epithelium was destroyed in the IMB of Tibetans when compared with Han Chinese. Transcriptome analysis revealed 2573 genes with altered expression. The levels of 1137 genes increased and 1436 genes decreased in Tibetans when compared with Han Chinese. Gene ontology (GO) analysis indicated most immunological responses were reduced in the IMB of Tibetans when compared with Han Chinese. Gene microarray showed that there were 25-, 22-, and 18-fold downregulation for growth factor receptor-bound protein 2 (GRB2), epidermal growth factor receptor (EGFR), and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) in the IMB of Tibetans when compared with Han Chinese. The downregulation of EGFR, GRB2, and PTPN11 will reduce the production of reactive oxygen species and protect against oxidative stress-induced injury for intestine. Thus, the transcriptome analysis showed the protecting functions of IMB patients against hypoxia-induced oxidative injury in the intestine of Tibetans via affecting GRB2/EGFR/PTPN11 pathways. PMID:27594973

  3. Silencing of hypoxia inducible factor-1α by RNA interference inhibits growth of SK-NEP-1 Wilms tumour cells in vitro, and suppresses tumourigenesis and angiogenesis in vivo.

    PubMed

    Shi, Bo; Li, Ying; Wang, Xiuli; Yang, Yi; Li, Dan; Liu, Xin; Yang, Xianghong

    2016-06-01

    Wilms tumour is the most common tumour of the pediatric kidney. Elevation of hypoxia-inducible factor 1α (HIF-1α) has been detected in 93% to 100% of human Wilms tumour specimens, suggesting a potential value of HIF-1α as a therapeutic target for Wilms tumour. In the present study, a stable HIF-1α-silenced Wilms tumour cell strain was established by introducing HIF-1α short-hairpin RNA (shRNA) into SK-NEP-1 cells. Silencing of HIF-1α significantly reduced single-cell growth capacity, suppressed proliferation and arrested cell cycle of SK-NEP-1 cells. In addition, reduction of HIF-1α expression induced apoptosis in SK-NEP-1 cells, which was accompanied by increased levels of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP) and Bax as well as downregulation of Bcl-2 in the cells. Furthermore, when inoculated subcutaneously in nude mice, HIF-1α-silenced SK-NEP-1 cells displayed retarded tumour growth and impaired tumour angiogenesis. In summary, the findings of this study suggest that HIF-1α plays a critical role in the development of Wilms tumour, and it may serve as a candidate target of gene therapy for Wilms tumour.

  4. Saururus cernuus Lignans - Potent Small Molecule Inhibitors of Hypoxia-Inducible Factor-1

    PubMed Central

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang, Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors. PMID:15967416

  5. Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1.

    PubMed

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R; Zhang, Lei; Bruick, Richard K; Mohammed, Kaleem A; Agarwal, Ameeta K; Nagle, Dale G; Zhou, Yu-Dong

    2005-08-05

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

  6. Mechanistic studies of the agmatine deiminase from Listeria monocytogenes

    PubMed Central

    Soares, Charles A.; Knuckley, Bryan

    2016-01-01

    Listeria monocytogenes is a Gram-positive food-borne pathogen that is capable of living within extreme environments (i.e. low temperatures and pH). This ability to survive in such conditions may arise, at least in part, from agmatine catabolism via the agmatine deiminase system (AgDS). This catabolic pathway utilizes an agmatine deiminase (AgD) to hydrolyse agmatine into N-carbamoylputrescine (NCP), with concomitant release of ammonia, which increases the pH, thus mitigating the ill effects of the acidic environment. Given the potential significance of this pathway for cell survival, we set out to study the catalytic mechanism of the AgD encoded by L. monocytogenes. In the present paper, we describe the catalytic mechanism employed by this enzyme based on pH profiles, pKa measurements of the active site cysteine and solvent isotope effects (SIE). In addition, we report inhibition of this enzyme by two novel AgD inhibitors, i.e. N-(4-aminobutyl)-2-fluoro-ethanimidamide (ABFA) and N-(4-aminobutyl)-2-chloro-ethanimidamide (ABCA). In contrast with other orthologues, L. monocytogenes AgD does not use the reverse protonation or substrate-assisted mechanism, which requires an active site cysteine with a high pKa and has been commonly seen in other members of the guanidinium-modifying enzyme (GME) superfamily. Instead, the L. monocytogenes AgD has a low pKa cysteine in the active site leading to an alternative mechanism of catalysis. This is the first time that this mechanism has been observed in the GME superfamily and is significant because it explains why previously developed mechanism-based inactivators of AgDs are ineffective against this orthologue. PMID:27034081

  7. Hydrogen sulfide alleviates hypoxia-induced root tip death in Pisum sativum.

    PubMed

    Cheng, Wei; Zhang, Liang; Jiao, Chengjin; Su, Miao; Yang, Tao; Zhou, Lina; Peng, Renyi; Wang, Ranran; Wang, Chongying

    2013-09-01

    Flooding of soils often results in hypoxic conditions surrounding plant roots, which is a harmful abiotic stress to crops. Hydrogen sulfide (H2S) is a highly diffusible, gaseous molecule that modulates cell signaling and is involved in hypoxia signaling in animal cells. However, there have been no previous studies of H2S in plant cells in response to hypoxia. The effects of H2S on hypoxia-induced root tip death were studied in pea (Pisum sativum) via analysis of endogenous H2S and reactive oxygen species (ROS) levels. The activities of key enzymes involved in antioxidative and H2S metabolic pathways were determined using spectrophotometric assays. Ethylene was measured by gas chromatography. We found that exogenous H2S pretreatment dramatically alleviated hypoxia-induced root tip death by protecting root tip cell membranes from ROS damage induced by hypoxia and by stimulating a quiescence strategy through inhibiting ethylene production. Conversely, root tip death induced by hypoxia was strongly enhanced by inhibition of the key enzymes responsible for endogenous H2S biosynthesis. Our results demonstrated that exogenous H2S pretreatment significantly alleviates hypoxia-induced root tip death in pea seedlings and, therefore, enhances the tolerance of the plant to hypoxic stress.

  8. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt

    PubMed Central

    Zeng, Rui; Yao, Ying; Han, Min; Zhao, Xiaoqin; Liu, Xiao-Cheng; Wei, Juncheng; Luo, Yun; Zhang, Juan; Zhou, Jianfeng; Wang, Shixuan; Ma, Ding; Xu, Gang

    2008-01-01

    Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway. PMID:18184861

  9. Structural integration in hypoxia-inducible factors

    SciTech Connect

    Wu, Dalei; Potluri, Nalini; Lu, Jingping; Kim, Youngchang; Rastinejad, Fraydoon

    2015-08-20

    The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

  10. Musashi mediates translational repression of the Drosophila hypoxia inducible factor

    PubMed Central

    Bertolin, Agustina P.; Katz, Maximiliano J.; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M.; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-01-01

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3′ UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available. PMID:27141964

  11. Musashi mediates translational repression of the Drosophila hypoxia inducible factor.

    PubMed

    Bertolin, Agustina P; Katz, Maximiliano J; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-09-19

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.

  12. Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

    SciTech Connect

    Zhang, Cui-Li; Song, Fei; Zhang, Jing; Song, Q.H.

    2010-04-16

    Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs' proliferation and migration. Over-expression of Bcl-2 increased HAECs' tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.

  13. Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages

    PubMed Central

    Chai, Jianshen; Luo, Li; Hou, Fengyan; Fan, Xia; Yu, Jing; Ma, Wei; Tang, Wangqi; Yang, Xue; Zhu, Junyu; Kang, Wenyuan; Yan, Jun; Liang, Huaping

    2016-01-01

    Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. PMID:27685463

  14. Novel Pathway for Hypoxia-Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF-1α, FASN, and mTORC1.

    PubMed

    Lee, Hyun Jik; Ryu, Jung Min; Jung, Young Hyun; Oh, Sang Yub; Lee, Sei-Jung; Han, Ho Jae

    2015-07-01

    The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCB-hMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1α)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCB-hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1α/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. Stem Cells 2015;33:2182-2195.

  15. Hypoxia-Inducible Factor as an Angiogenic Master Switch

    PubMed Central

    Hashimoto, Takuya; Shibasaki, Futoshi

    2015-01-01

    Hypoxia-inducible factors (HIFs) regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. Eukaryotic initiation factor 3 subunit e (eIF3e)/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and von Hippel-Lindau protein. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications

  16. Discovery of Indenopyrazoles as a New Class of Hypoxia Inducible Factor (HIF)-1 Inhibitors

    PubMed Central

    2013-01-01

    The indenopyrazole framework was investigated as a new class of HIF-1α inhibitors. Indenopyrazole 2l was found to most strongly inhibit the hypoxia-induced HIF-1α transcriptional activity (IC50 = 0.014 μM) among all of the known compounds having relatively simple structures, unlike manassantins. Indenopyrazole 2l suppressed HIF-1α transcriptional activity without affecting both HIF-1α protein accumulation and HIF-1α/HIF-1β heterodimerization in nuclei under the hypoxic conditions, suggesting that 2l probably affected the transcriptional pathway induced by the HIF-1α/HIF-1β heterodimer. PMID:24900662

  17. Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    PubMed Central

    Hida, Yasuhiro; Maishi, Nako; Towfik, Alam Mohammad; Inoue, Nobuo; Shindoh, Masanobu; Hida, Kyoko

    2013-01-01

    There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment. PMID:24260373

  18. The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop

    PubMed Central

    Bartoszewska, Sylwia; Kochan, Kinga; Piotrowski, Arkadiusz; Kamysz, Wojciech; Ochocka, Renata J.; Collawn, James F.; Bartoszewski, Rafal

    2015-01-01

    Hypoxia-inducible factors (HIFs) 1 and 2 are dimeric α/β transcription factors that regulate cellular responses to low oxygen. HIF-1 is induced first, whereas HIF-2 is associated with chronic hypoxia. To determine how HIF1A mRNA, the inducible subunit of HIF-1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF-1, increased ∼2.5- and 8-fold at 2–4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR-429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A. Target protector analysis, which interferes with miRNA-mRNA complex formation, confirmed that miR-429 targeted HIF1A message. Desferoxamine treatment, which inhibits the hydroxylases that promote HIF-1α protein degradation, stabilized HIF-1 activity during normoxic conditions and elevated miR-429 levels, demonstrating that HIF-1 promotes miR-429 expression. RNA-sequencing-based transcriptome analysis indicated that inhibition of miRNA-429 in HUVECs up-regulated 209 mRNAs, a number of which regulate angiogenesis. The results demonstrate that HIF-1 is in a negative regulatory loop with miR-429, that miR-429 attenuates HIF-1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF-2 is activated, and this regulatory network helps explain the HIF-1 transition to HIF-2 during chronic hypoxia in endothelial cells.—Bartoszewska, S., Kochan, K., Piotrowski, A., Kamysz, W., Ochocka, R. J., Collawn, J. F., Bartoszewski, R. The hypoxia-inducible miR-429 regulates hypoxia hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop. PMID:25550463

  19. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  20. Agmatine rescues autistic behaviors in the valproic acid-induced animal model of autism.

    PubMed

    Kim, Ji-Woon; Seung, Hana; Kim, Ki Chan; Gonzales, Edson Luck T; Oh, Hyun Ah; Yang, Sung Min; Ko, Mee Jung; Han, Seol-Heui; Banerjee, Sourav; Shin, Chan Young

    2017-02-01

    Autism spectrum disorder (ASD) is an immensely challenging developmental disorder characterized primarily by two core behavioral symptoms of social communication deficits and restricted/repetitive behaviors. Investigating the etiological process and identifying an appropriate therapeutic target remain as formidable challenges to overcome ASD due to numerous risk factors and complex symptoms associated with the disorder. Among the various mechanisms that contribute to ASD, the maintenance of excitation and inhibition balance emerged as a key factor to regulate proper functioning of neuronal circuitry. Interestingly, our previous study involving the valproic acid animal model of autism (VPA animal model) has demonstrated excitatory-inhibitory imbalance (E/I imbalance) due to enhanced differentiation of glutamatergic neurons and reduced GABAergic neurons. Here, we investigated the potential of agmatine, an endogenous NMDA receptor antagonist, as a novel therapeutic candidate in ameliorating ASD symptoms by modulating E/I imbalance using the VPA animal model. We observed that a single treatment of agmatine rescued the impaired social behaviors as well as hyperactive and repetitive behaviors in the VPA animal model. We also observed that agmatine treatment rescued the overly activated ERK1/2 signaling in the prefrontal cortex and hippocampus of VPA animal models, possibly, by modulating over-excitability due to enhanced excitatory neural circuit. Taken together, our results have provided experimental evidence suggesting a possible therapeutic role of agmatine in ameliorating ASD-like symptoms in the VPA animal model of ASD.

  1. Resveratrol prevents hypoxia-induced arginase II expression and proliferation of human pulmonary artery smooth muscle cells via Akt-dependent signaling.

    PubMed

    Chen, Bernadette; Xue, Jianjing; Meng, Xiaomei; Slutzky, Jessica L; Calvert, Andrea E; Chicoine, Louis G

    2014-08-15

    Pulmonary artery smooth muscle cell (PASMC) proliferation plays a fundamental role in the vascular remodeling seen in pulmonary hypertensive diseases associated with hypoxia. Arginase II, an enzyme regulating the first step in polyamine and proline synthesis, has been shown to play a critical role in hypoxia-induced proliferation of human PASMC (hPASMC). In addition, there is evidence that patients with pulmonary hypertension have elevated levels of arginase in the vascular wall. Resveratrol, a natural polyphenol found in red wine and grape skins, has diverse biochemical and physiological actions including antiproliferative properties. Furthermore, resveratrol has been shown to attenuate right ventricular and pulmonary artery remodeling, both pathological components of pulmonary hypertension. The present studies tested the hypothesis that resveratrol would prevent hypoxia-induced pulmonary artery smooth muscle cell proliferation by inhibiting hypoxia-induced arginase II expression. Our data indicate that hypoxia-induced hPASMC proliferation is abrogated following treatment with resveratrol. In addition, the hypoxic induction of arginase II was directly attenuated by resveratrol treatment. Furthermore, we found that the inhibitory effect of resveratrol on arginase II in hPASMC was mediated through the PI3K-Akt signaling pathway. Supporting these in vitro findings, resveratrol normalized right ventricular hypertrophy in an in vivo neonatal rat model of chronic hypoxia-induced pulmonary hypertension. These novel data support the notion that resveratrol may be a potential therapeutic agent in pulmonary hypertension by preventing PASMC arginase II induction and proliferation.

  2. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

    PubMed

    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-01-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  3. Mechanisms Of Hypoxia-Induced Immune Escape In Cancer And Their Regulation By Nitric Oxide.

    PubMed

    Graham, Charles; Barsoum, Ivraym; Kim, Judy; Black, Madison; Siemens, Robert D

    2015-08-01

    The acquired ability of tumour cells to avoid destruction by immune effector mechanisms (immune escape) is important for malignant progression. Also associated with malignant progression is tumour hypoxia, which induces aggressive phenotypes such as invasion, metastasis and drug resistance in cancer cells. Our studies revealed that hypoxia contributes to escape from innate immunity by increasing tumour cell expression of the metalloproteinase ADAM10 in a manner dependent on accumulation of the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α). Increased ADAM10 expression leads to shedding of the NK cell-activating ligand, MICA, from the surface of tumour cells, thereby resulting in resistance to NK cell-mediated lysis. Our more recent studies demonstrated that hypoxia, also via HIF-1α accumulation, increases the expression of the inhibitory co-stimulatory ligand PD-L1 on tumour cells. Elevated PD-L1 expression leads to escape from adaptive immunity via increased apoptosis of CD8(+) cytotoxic T lymphocytes. Accumulating evidence indicates that hypoxia-induced acquisition of malignant phenotypes, including immune escape, is in part due to impaired nitric oxide (NO)-mediated activation of cGMP signalling and that restoration of cGMP signalling prevents such hypoxic responses. We have shown that NO/cGMP signalling inhibits hypoxia-induced malignant phenotypes likely in part by interfering with HIF-1α accumulation via a mechanism involving calpain. These findings indicate that activation of NO/cGMP signalling may have useful applications in cancer therapy.

  4. Hypoxia-induced hypothermia mediated by GABA in the rostral parapyramidal area of the medulla oblongata.

    PubMed

    Osaka, T

    2014-05-16

    Hypoxia evokes a regulated decrease in the body core temperature (Tc) in a variety of animals. The neuronal mechanisms of this response include, at least in part, glutamatergic activation in the lateral preoptic area (LPO) of the hypothalamus. As the sympathetic premotor neurons in the medulla oblongata constitute a cardinal relay station in the descending neuronal pathway from the hypothalamus for thermoregulation, their inhibition can also be critically involved in the mechanisms of the hypoxia-induced hypothermia. Here, I examined the hypothesis that hypoxia-induced hypothermia is mediated by glutamate-responsive neurons in the LPO that activate GABAergic transmission in the rostral raphe pallidus (rRPa) and neighboring parapyramidal region (PPy) of the medulla oblongata in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. Unilateral microinjection of GABA (15nmol) into the rRPa and PPy regions elicited a prompt increase in tail skin temperature (Ts) and decreases in Tc, oxygen consumption rate (VO2), and heart rate. Next, when the GABAA receptor blocker bicuculline methiodide (bicuculline methiodide (BMI), 10pmol) alone was microinjected into the rRPa, it elicited unexpected contradictory responses: simultaneous increases in Ts, VO2 and heart rate and a decrease in Tc. Then, when BMI was microinjected bilaterally into the PPy, no direct effect on Ts was seen; and thermogenic and tachycardic responses were slight. However, pretreatment of the PPy with BMI, but not vehicle saline, greatly attenuated the hypothermic responses evoked by hypoxic (10%O2-90%N2, 5min) ventilation or bilateral microinjections of glutamate (5nmol, each side) into the LPO. The results suggest that hypoxia-induced hypothermia was mediated, at least in part, by the activation of GABAA receptors in the PPy.

  5. Comparison of agmatine with moxonidine and rilmenidine in morphine dependence in vitro: role of imidazoline I(1) receptors.

    PubMed

    Li, Fei; Wu, Ning; Su, Rui-Bin; Liu, Yin; Lu, Xin-Qiang; Li, Jin

    2009-06-10

    Moxonidine and rilmenidine are classical imidazoline I(1) receptor agonists, and used as anti-hypertension drugs in clinical practice. Agmatine is an imidazoline I(1) receptor endogenous ligand as well as its agonist, but more and more evidences suggest it has no influence on blood pressure. In the present study we compared the effects of moxonidine, rilmenidine and agmatine in the development of morphine dependence, and investigated the role of imidazoline I(1) receptor in the effects of these agents. Chinese hamster ovary cells co-expressing mu opioid receptor and imidazoline receptor antisera-selected protein (IRAS), the strong candidate for imidazoline I(1) receptor, were used as the cell line. cAMP overshoot, which represents an opioid dependent state in vitro, was measured to study the effects on morphine dependence. siRNA against IRAS was carried out to investigate the role of imidazoline I(1) receptor. Moxonidine and rilmenidine (0.01-10 microM) were ineffective on cAMP level in the cells when given alone, and failed to inhibit chronic morphine exposure, naloxone-precipitated cAMP overshoot when co-pretreated with morphine. Agmatine (0.01-10 microM) by itself was ineffective but co-pretreated with morphine concentration-dependently inhibited chronic morphine exposure, naloxone-precipitated cAMP overshoot in the cells. Furthermore, we found that the inhibitory effect of agmatine (100 nM and 1 microM) on cAMP overshoot was significantly reduced by siRNA against IRAS. This study indicates that agmatine can inhibit the development of morphine dependence in vitro, whereas moxonidine and rilmenidine have no the effect. Imidazoline I(1) receptor plays an important role in agmatine inhibiting morphine dependence.

  6. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells

    PubMed Central

    Lai, Ming-Chih; Chang, Chiao-May; Sun, H. Sunny

    2016-01-01

    Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia. PMID:27078027

  7. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

    PubMed Central

    Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  8. The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

    PubMed

    Landete, José M; Arena, Mario E; Pardo, Isabel; Manca de Nadra, María C; Ferrer, Sergi

    2010-12-01

    Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

  9. Hypoxia induces heart regeneration in adult mice.

    PubMed

    Nakada, Yuji; Canseco, Diana C; Thet, SuWannee; Abdisalaam, Salim; Asaithamby, Aroumougame; Santos, Celio X; Shah, Ajay M; Zhang, Hua; Faber, James E; Kinter, Michael T; Szweda, Luke I; Xing, Chao; Hu, Zeping; Deberardinis, Ralph J; Schiattarella, Gabriele; Hill, Joseph A; Oz, Orhan; Lu, Zhigang; Zhang, Cheng Cheng; Kimura, Wataru; Sadek, Hesham A

    2017-01-12

    The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.

  10. Chronic Normobaric Hypoxia Induces Pulmonary Hypertension in Rats: Role of NF-κB.

    PubMed

    Fan, Junming; Fan, Xiaofang; Li, Yang; Ding, Lu; Zheng, Qingqing; Guo, Jinbin; Xia, Dongmei; Xue, Feng; Wang, Yongyu; Liu, Shufang; Gong, Yongsheng

    2016-03-01

    To investigate whether nuclear factor-kappa B (NF-κB) activation is involved in chronic normobaric hypoxia-induced pulmonary hypertension (PH), rats were treated with saline or an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 150 mg/kg, sc, twice daily), and exposed to normoxia or chronic normobaric hypoxia with a fraction of inspired oxygen of ∼0.1 for 14 days. Lung tissue levels of NF-κB activity, and interleukin (IL)-1β, IL-6, and cyclooxygenase-2 mRNAs, were determined, and mean pulmonary arterial pressure, right ventricular hypertrophy, and right heart function were evaluated. Compared to the normoxia exposure group, rats exposed to chronic normobaric hypoxia showed an increased NF-κB activity, measured by increased nuclear translocation of p50 and p65 proteins, an increased inflammatory gene expression in the lungs, elevated mean pulmonary arterial blood pressure and mean right ventricular pressure, right ventricular hypertrophy, as assessed by right ventricle-to-left ventricle plus septum weight ratio, and right heart dysfunction. Treatment of hypoxia-exposed rats with PDTC inhibited NF-κB activity, decreased pulmonary arterial blood pressure and right ventricular pressure, and ameliorated right ventricular hypertrophy and right heart dysfunction. Hypoxia exposure increased protein kinase C activity and promoted pulmonary artery smooth muscle cell proliferation in vitro. Our data suggest that NF-κB activation may contribute to chronic normobaric hypoxia-induced PH.

  11. The Effects of Portulaca oleracea on Hypoxia-Induced Pulmonary Edema in Mice

    PubMed Central

    Yue, Tan; Xiaosa, Wen; Ruirui, Qi; Wencai, Shi; Hailiang, Xin

    2015-01-01

    Abstract Tan Yue, Wen Xiaosa, Qi Ruirui, Shi Wencai, Xin Hailiang, and Li Min. The effects of Portulaca oleracea on hypoxia-induced pulmonary edema in mice. High Alt Med Biol 16:43–51, 2015—Portulaca oleracea L. (PO) is known as “a vegetable for long life” due to its antioxidant, anti-inflammatory, and other pharmacological activities. However, the protective activity of the ethanol extract of PO (EEPO) against hypoxia-induced pulmonary edema has not been fully investigated. In this study, we exposed mice to a simulated altitude of 7000 meters for 0, 3, 6, 9, and 12 h to observe changes in the water content and transvascular leakage of the mouse lung. It was found that transvascular leakage increased to the maximum in the mouse lung after 6 h exposure to hypobaric hypoxia. Prophylactic administration of EEPO before hypoxic exposure markedly reduced the transvascular leakage and oxidative stress, and inhibited the upregulation of NF-kB in the mouse lung, as compared with the control group. In addition, EEPO significantly reduced the levels of proinflammatory cytokines and cell adhesion molecules in the lungs of mice, as compared with the hypoxia group. Our results show that EEPO can reduce initial transvascular leakage and pulmonary edema under hypobaric hypoxia conditions. PMID:25761168

  12. Hypoxia Inducible Factor-1α Inactivation Unveils a Link between Tumor Cell Metabolism and Hypoxia-Induced Cell Death

    PubMed Central

    Favaro, Elena; Nardo, Giorgia; Persano, Luca; Masiero, Massimo; Moserle, Lidia; Zamarchi, Rita; Rossi, Elisabetta; Esposito, Giovanni; Plebani, Mario; Sattler, Ulrike; Mann, Thomas; Mueller-Klieser, Wolfgang; Ciminale, Vincenzo; Amadori, Alberto; Indraccolo, Stefano

    2008-01-01

    Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1α (HIF-1α) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1α inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1α in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was also associated with a sharp decrease in intracellular ATP levels and was prevented by glucose supplementation. Stable HIF-1α silencing enhanced hypoxia-induced cell death in vitro due to a lack of cell cycle arrest. Tumors bearing attenuated HIF-1α levels had similar growth rates and vascularization as did controls, but tumors showed higher proliferation levels and increased necrosis. Moreover, tumors formed by HIF-1α deficient cells had higher levels of lactate and lower ATP concentrations than controls as shown by metabolic imaging. The findings that such metabolic properties can affect the survival of cancer cells under hypoxic conditions and that these properties contribute to the determination of the consequences of HIF-1α inactivation could have important implications on the understanding of the effects of anti-angiogenic and HIF-1α-targeting drugs in cancer. PMID:18772337

  13. Protective effect of salidroside against bone loss via hypoxia-inducible factor-1α pathway-induced angiogenesis

    PubMed Central

    Li, Ling; Qu, Ye; Jin, Xin; Guo, Xiao Qin; Wang, Yue; Qi, Lin; Yang, Jing; Zhang, Peng; Li, Ling Zhi

    2016-01-01

    Hypoxia-inducible factor (HIF)-1α plays a critical role in coupling angiogenesis with osteogenesis during bone development and regeneration. Salidroside (SAL) has shown anti-hypoxic effects in vitro and in vivo. However, the possible roles of SAL in the prevention of hypoxia-induced osteoporosis have remained unknown. Two osteoblast cell lines, MG-63 and ROB, were employed to evaluate the effects of SAL on cell viability, apoptosis, differentiation and mineralization in vitro. Rats subjected to ovariectomy-induced bone loss were treated with SAL in vivo. Our results showed that pre-treatment with SAL markedly attenuated the hypoxia-induced reductions in cell viability, apoptosis, differentiation and mineralization. SAL down-regulated HIF-1α expression and inhibited its translocation; however, SAL increased its transcriptional activity and, consequently, up-regulated vascular endothelial growth factor (VEGF). In vivo studies further demonstrated that SAL caused decreases in the mineral, alkaline phosphatase (ALP), and BGP concentrations in the blood of ovariectomized (OVX) rats. Moreover, SAL improved the trabecular bone microarchitecture and increased bone mineral density in the distal femur. Additionally, SAL administration partially ameliorated this hypoxia via the HIF-1α-VEGF signalling pathway. Our results indicate that SAL prevents bone loss by enhancing angiogenesis and osteogenesis and that these effects are associated with the activation of HIF-1α signalling. PMID:27558909

  14. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    PubMed Central

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  15. Hypoxia regulates CD44 expression via hypoxia-inducible factor-1α in human gastric cancer cells

    PubMed Central

    Liang, Gai; Li, Shuang; Du, Wei; Ke, Qinghua; Cai, Jun; Yang, Jiyuan

    2017-01-01

    Hypoxia induces proliferation and invasion in cancer cells via hypoxia-inducible factor (HIF)-1α. The cell adhesion molecule cluster of differentiation (CD) 44 has been associated with increased cell invasion and metastasis. Whether hypoxia regulates the expression of CD44 in gastric cancer cells remains to be established. In the current study, the effects of hypoxia on HIF-1α and CD44 expression levels in human gastric cell lines SGC-7901 and BGC-823 were evaluated. The cells were cultured in 1% O2 for 1 week and then treated with 20 nM rapamycin for 72 h. Cell viability was evaluated using the Cell Counting kit-8 assay, and cell invasion was detected by the Transwell invasion assay. The protein and messenger (m) RNA expression levels of HIF-1α and CD44 were detected using western blotting and reverse transcription-quantitative polymerase chain reaction, respectively. The results revealed that cell viability and invasion increased under hypoxic conditions, but decreased following rapamycin treatment in SGC-7901 and BGC-823 cells. Hypoxia also increased the protein and mRNA expression levels of HIF-1α and CD44 in these two cell lines. However, this hypoxia-induced increase in HIF-1α and CD44 protein and mRNA expression levels was inhibited by rapamycin. These findings suggest that hypoxia induced the proliferation and invasion of SGC-7901 and BGC-823 cells. Furthermore, CD44 expression levels were potentially associated with HIF-1α expression levels. Therefore, in gastric cancer cells, hypoxia may regulate CD44 expression via HIF-1α in order to promote cell proliferation and invasion.

  16. Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

    PubMed

    Zimnicka, Adriana M; Tang, Haiyang; Guo, Qiang; Kuhr, Frank K; Oh, Myung-Jin; Wan, Jun; Chen, Jiwang; Smith, Kimberly A; Fraidenburg, Dustin R; Choudhury, Moumita S R; Levitan, Irena; Machado, Roberto F; Kaplan, Jack H; Yuan, Jason X-J

    2014-01-01

    Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

  17. Targeted genes and interacting proteins of hypoxia inducible factor-1

    PubMed Central

    Liu, Wei; Shen, Shao-Ming; Zhao, Xu-Yun; Chen, Guo-Qiang

    2012-01-01

    Heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) functions as a master regulator of oxygen homeostasis in almost all nucleated mammalian cells. The fundamental process adapted to cellular oxygen alteration largely depends on the refined regulation on its alpha subunit, HIF-1α. Recent studies have unraveled expanding and critical roles of HIF-1α, involving in a multitude of developmental, physiological, and pathophysiological processes. This review will focus on the current knowledge of HIF-1α-targeting genes and its interacting proteins, as well as the concomitant functional relationships between them. PMID:22773957

  18. Wheatgrass Extract Ameliorates Hypoxia-induced Mucin Gene Expression in A549 cells

    PubMed Central

    Sim, Ju hwan; Choi, Moon-Hee; Shin, Hyun-Jae; Lee, Ji-Eun

    2017-01-01

    Background: Wheatgrass is known to have antioxidant, antiaging, and anti-inflammatory effect. However, its protective effect against hypoxia is not yet evaluated. Objective: In this study, we evaluated the protective and anti-inflammatory effect of wheatgrass against the hypoxia in airway epithelial cells. Materials and Methods: A549 human lung adenocarcinoma cells were incubated in a hypoxic condition (CO2 5%/O2 1%) for 24 hr in the presence of different concentration of wheatgrass 50, 75, 100, and 150 μg/mL, and the magnitude of each immunologic response produced by the A549 cells was compared. The mRNA expression level of mucin gene (MUC), 5A, 5B, 8, GM-CSF, TNF-α, and VEGF were evaluated by using real-time polymerase chain reaction. The MUC proteins level before and after knocking out the hypoxia-inducible factor (hif)-1α via short interfering (si) RNA transfection were assessed by immunoblot analysis. Accordingly, the involved cell signaling pathway was evaluated by immunoblot analysis. Results: The inflammatory cytokines (GM-CSF, TNF- α) and the expressions of MUC 5A, 5B, and 8 were augmented by hypoxia. The augmented MUC expression was decreased by the wheatgrass extract administration. Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass. Knockdown of hif-1α by siRNA reduced the mucin gene expression and which was more enhanced by wheatgrass extract. Conclusion: Theses results suggest that wheatgrass may be useful in the treatment of sinonasal disease by inhibiting mucus hypersecretion in airway epithelium. SUMMARY Wheatgrass extract decreases the hypoxia-induced MUC 5A, 5B and 8 expression.Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass.Wheatgrass inhibits p44/42 phosphorylation in hypoxia-exposed airway epithelial cells. Abbreviations used: A549: human lung adenocarcinoma cells, GM-CSF: granulocyte-macrophage colony stimulating factor, HIF: hypoxia inducible factor, IL: interleukin, MUC: mucin, MTT: 3

  19. Integrin-linked kinase: a hypoxia-induced anti-apoptotic factor exploited by cancer cells.

    PubMed

    Abboud, Elizabeth R; Coffelt, Seth B; Figueroa, Yanira G; Zwezdaryk, Kevin J; Nelson, Anne B; Sullivan, Deborah E; Morris, Cindy B; Tang, Yan; Beckman, Barbara S; Scandurro, Aline B

    2007-01-01

    Based on cDNA microarray results, integrin-linked kinase (ILK) emerged as an interesting candidate in hypoxia-mediated survival mechanisms employed by cancer cells. This notion was confirmed here by the following observations: the 5' promoter region of the ilk gene contains hypoxia responsive elements (HRE) that bind hypoxia-inducible factor (HIF) transcription factor complexes and drive HRE-luciferase gene expression in reporter assays; ILK protein and kinase activity are induced following hypoxia; downstream targets of ILK signaling are induced following hypoxia treatment; inhibition of ILK leads to increased apoptosis; and HIF and ILK are co-localized within human cancer tissues. The identification of ILK as a player in hypoxia survival signaling employed by cancer cells further validates ILK as a unique target for cancer therapy.

  20. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

    PubMed Central

    Iliopoulos, O; Levy, A P; Jiang, C; Kaelin, W G; Goldberg, M A

    1996-01-01

    Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855223

  1. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    SciTech Connect

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  2. Hypoxia induced cognitive impairment modulating activity of Cyperus rotundus.

    PubMed

    Kandikattu, Hemanth Kumar; Deep, Satya Narayan; Razack, Sakina; Amruta, Narayanappa; Prasad, Dipti; Khanum, Farhath

    2017-03-27

    Hypobaric hypoxia leads to decrease in cellular oxygen content which subsequently damages the hippocampus with an increase in brain oxidative stress and impairs the memory of the individual. In the present study, we have evaluated the cognitive impairment modulating activity of total oligomeric flavonoids fraction of Cyperus rotundus (TOF) in Sprague Dawley rats. The rats were trained for memory activity for a period of 7days followed by 7days exposure to 25,000ft. altitude and the spatial reference memory was evaluated. Behavioral analysis of the rats by Morris water maze experiment showed that TOF supplementation enhanced the spatial reference memory activity of the rats exposed to hypobaric hypoxia. The decrease in antioxidant status of the animals exposed to hypoxia was restored with TOF supplementation. The increase in ROS, lipid peroxidation products and protein carbonyls of the hippocampus was significantly decreased in animals with TOF administration. The histological assessment of the pyramidal cells of the hippocampus of hypoxia-exposed animals showed nuclear damage and TOF supplementation prevented nuclear damage. TOF administration suppressed hypoxia-induced increase in serotonin, dopamine, and norepinephrine. GABA and Ach levels were decreased by hypoxia which was prevented by TOF supplementation. The increase in GFAP, HIF-1α and VEGF expression in CA3 region of the hippocampus in hypoxia-exposed rats was decreased in TOF administered rats. Taken together, TOF extract ameliorates hypobaric hypoxia induced memory impairment and neurodegeneration in hippocampus through its anti-stress effects.

  3. Dexamethasone impairs hypoxia-inducible factor-1 function

    SciTech Connect

    Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

    2008-07-25

    Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of {alpha}- and {beta}-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1{alpha} levels in the cytosol of HepG2 cells, while nuclear HIF-1{alpha} levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

  4. Hypoxia-Induced Oxidative Stress Modulation with Physical Activity

    PubMed Central

    Debevec, Tadej; Millet, Grégoire P.; Pialoux, Vincent

    2017-01-01

    Increased oxidative stress, defined as an imbalance between prooxidants and antioxidants, resulting in molecular damage and disruption of redox signaling, is associated with numerous pathophysiological processes and known to exacerbate chronic diseases. Prolonged systemic hypoxia, induced either by exposure to terrestrial altitude or a reduction in ambient O2 availability is known to elicit oxidative stress and thereby alter redox balance in healthy humans. The redox balance modulation is also highly dependent on the level of physical activity. For example, both high-intensity exercise and inactivity, representing the two ends of the physical activity spectrum, are known to promote oxidative stress. Numerous to-date studies indicate that hypoxia and exercise can exert additive influence upon redox balance alterations. However, recent evidence suggests that moderate physical activity can attenuate altitude/hypoxia-induced oxidative stress during long-term hypoxic exposure. The purpose of this review is to summarize recent findings on hypoxia-related oxidative stress modulation by different activity levels during prolonged hypoxic exposures and examine the potential mechanisms underlying the observed redox balance changes. The paper also explores the applicability of moderate activity as a strategy for attenuating hypoxia-related oxidative stress. Moreover, the potential of such moderate intensity activities used to counteract inactivity-related oxidative stress, often encountered in pathological, elderly and obese populations is also discussed. Finally, future research directions for investigating interactive effects of altitude/hypoxia and exercise on oxidative stress are proposed. PMID:28243207

  5. HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts

    PubMed Central

    Ferdin, J; Nishida, N; Wu, X; Nicoloso, M S; Shah, M Y; Devlin, C; Ling, H; Shimizu, M; Kumar, K; Cortez, M A; Ferracin, M; Bi, Y; Yang, D; Czerniak, B; Zhang, W; Schmittgen, T D; Voorhoeve, M P; Reginato, M J; Negrini, M; Davuluri, R V; Kunej, T; Ivan, M; Calin, G A

    2013-01-01

    Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category. PMID:24037088

  6. Endothelium-derived hyperpolarizing factor contributes to hypoxia-induced skeletal muscle vasodilation in humans.

    PubMed

    Spilk, Samson; Herr, Michael D; Sinoway, Lawrence I; Leuenberger, Urs A

    2013-12-01

    Systemic hypoxia causes skeletal muscle vasodilation, thereby preserving O2 delivery to active tissues. Nitric oxide (NO), adenosine, and prostaglandins contribute to this vasodilation, but other factors may also play a role. We tested the hypothesis that regional inhibition of endothelium-derived hyperpolarizing factor with the cytochrome P-450 2C9 antagonist fluconazole, alone or combined with the NO synthase antagonist N(G)-monomethyl-L-arginine (L-NMMA), attenuates hypoxia-induced vasodilation. We compared forearm blood flow (FBF) and skin blood flow before and during brachial artery infusion of fluconazole (0.3 mg/min; trial 1) or fluconazole + L-NMMA (50 mg over 10 min; trial 2) and during systemic hypoxia (10 min, arterial Po2 ~37 mmHg) in infused (experimental) and control forearms of 12 healthy humans. During normoxia, fluconazole and fluconazole + L-NMMA reduced (P < 0.05) forearm vascular conductance (FVC) by ~10% and ~18%, respectively. During hypoxia and fluconazole (trial 1), FVC increased by 1.76 ± 0.37 and 0.95 ± 0.35 units in control and experimental forearms, respectively (P < 0.05). During hypoxia and fluconazole + L-NMMA (trial 2), FVC increased by 2.32 ± 0.51 and 0.72 ± 0.22 units in control and experimental forearms, respectively (P < 0.05). Similarly, during hypoxia with L-NMMA alone (trial 3; n = 8) FVC increased by 1.51 ± 0.46 and 0.45 ± 0.32 units in control and experimental forearms, respectively (P < 0.05). These effects were not due to altered skin blood flow. We conclude that endothelium-derived hyperpolarizing factor contributes to basal vascular tone and to hypoxia-induced skeletal muscle vasodilation and could be particularly relevant when other vasodilator systems are impaired.

  7. Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes*

    PubMed Central

    Laukka, Tuomas; Mariani, Christopher J.; Ihantola, Tuukka; Cao, John Z.; Hokkanen, Juho; Kaelin, William G.; Godley, Lucy A.; Koivunen, Peppi

    2016-01-01

    The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1–3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe2+ and 2-oxoglutarate-binding residues increased the Km values for these factors 30–80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400–500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization. PMID:26703470

  8. Hypoxia-inducible factor-1alpha suppresses the expression of macrophage scavenger receptor 1.

    PubMed

    Shirato, Ken; Kizaki, Takako; Sakurai, Takuya; Ogasawara, Jun-Etsu; Ishibashi, Yoshinaga; Iijima, Takehiko; Okada, Chikako; Noguchi, Izumi; Imaizumi, Kazuhiko; Taniguchi, Naoyuki; Ohno, Hideki

    2009-11-01

    Macrophages are distributed in all peripheral tissues and play a critical role in the first line of the innate immune defenses against bacterial infection by phagocytosis of bacterial pathogens through the macrophage scavenger receptor 1 (MSR1). Within tissues, the partial pressure of oxygen (pO2) decreases depending on the distance of cells from the closest O2-supplying blood vessel. However, it is not clear how the expression of MSR1 in macrophages is regulated by low pO2. On the other hand, hypoxia-inducible factor (HIF)-1alpha is well known to control hypoxic responses through regulation of hypoxia-inducible genes. Therefore, we investigated the effects of hypoxia and HIF-1alpha on MSR1 expression and function in the macrophage cell line RAW264. Exposure to 1% O2 or treatment with the hypoxia-mimetic agent cobalt chloride (CoCl2) significantly suppressed the expression of MSR1 mRNA, accompanied by a markedly increase in levels of nuclear HIF-1alpha protein. The overexpression of HIF-1alpha in RAW264 cells suppressed the expression of MSR1 mRNA and protein, transcriptional activity of the MSR1 gene, and phagocytic capacity against the Gram-positive bacteria Listeria monocytogenes. The suppression of MSR1 mRNA by hypoxia or CoCl2 was inhibited by YC-1, an inhibitor of HIF-1alpha, or by the depletion of HIF-1alpha expression by small interference RNA. These results indicate that hypoxia transcriptionally suppresses MSR1 expression through HIF-1alpha.

  9. Wnt5a attenuates hypoxia-induced pulmonary arteriolar remodeling and right ventricular hypertrophy in mice

    PubMed Central

    Jin, Yuling; Wang, Wang; Chai, Sanbao; Liu, Jie

    2015-01-01

    Hypoxic pulmonary hypertension (HPH), which is characterized by pulmonary arteriolar remodeling and right ventricular hypertrophy, is still a life-threatening disease with the current treatment strategies. The underlying molecular mechanisms of HPH remain unclear. Our previously published study showed that Wnt5a, one of the ligands in the Wnt family, was critically involved in the inhibition of hypoxia-induced pulmonary arterial smooth muscle cell proliferation by downregulation of β-catenin/cyclin D1 in vitro. In this study, we investigated the possible functions and mechanisms of Wnt5a in HPH in vivo. Recombinant mouse Wnt5a (rmWnt5a) or phosphate buffered saline (PBS) was administered to male C57/BL6 mice weekly from the first day to the end of the two or four weeks after exposed to hypoxia (10% O2). Hypoxia-induced pulmonary hypertension was associated with a marked increase in β-catenin/cyclin D1 expression in lungs. Right ventricular systolic pressure and right ventricular hypertrophy index were reduced in animals treated with rmWnt5a compared with PBS. Histology showed less pulmonary vascular remodeling and right ventricular hypertrophy in the group treated with rmWnt5a than with PBS. Treatment with rmWnt5a resulted in a concomitant reduction in β-catenin/cyclin D1 levels in lungs. These data demonstrate that Wnt5a exerts its beneficial effects on HPH by regulating pulmonary vascular remodeling and right ventricular hypertrophy in a manner that is associated with reduction in β-catenin/cyclin D1 signaling. A therapy targeting the β-catenin/cyclin D1 signaling pathway might be a potential strategy for HPH treatment. PMID:25956683

  10. Rutaecarpine attenuates hypoxia-induced right ventricular remodeling in rats.

    PubMed

    Li, Wen-Qun; Li, Xiao-Hui; Du, Jie; Zhang, Wang; Li, Dai; Xiong, Xiao-Ming; Li, Yuan-Jian

    2016-07-01

    Rutaecarpine has been shown to exhibit wide pharmacological effects in the cardiovascular system via stimulation of calcitonin gene-related peptide (CGRP) release. In the present study, the effect of rutaecarpine on hypoxia-induced right ventricular (RV) remodeling and the underlying mechanisms were evaluated. RV remodeling was induced by hypoxia (10 % O2, 3 weeks) in rats. Rats were treated with rutaecarpine (20 or 40 mg/kg) by intragastric administration. Proliferation of cardiac fibroblasts was induced by TGF-β1 (5 ng/mL) and determined by MTS and EdU incorporation method. Cardiac fibroblasts were treated with exogenous CGRP (10 or 100 nM). The concentrations of CGRP and TGF-β1 in plasma were measured by ELISA. The expression of eIF3a, p27, α-SMA, collagen-I/III, ANP, and BNP were measured by real-time PCR or western blot. Hypoxia induced an increase of right ventricle systolic pressure (RVSP), ration of RV/LV+S, and RV/tibial length in rats, while cardiac hypertrophy, apoptosis, and fibrosis were detected. The expression of ANP, BNP, α-SMA, collagen-I, collagen-III, eIF3a, and TGF-β1 was up-regulated, and the expression of p27 was down-regulated in the right ventricle of hypoxia-treated rats. The plasma concentration of CGRP was decreased and TGF-β1 was increased in hypoxia-treated rats. All of these effects induced by hypoxia were attenuated by rutaecarpine in a dose-dependent manner. In cultured cardiac fibroblasts, TGF-β1 significantly promoted the proliferation and up-regulated the expression of α-SMA and collagen-I/III, while the expression of eIF3a was up-regulated and the expression of p27 was down-regulated. The effects of TGF-β1 were attenuated by CGRP. CGRP8-37, a selective CGRP receptor antagonist, abolished the effects of CGRP. Rutaecarpine attenuates hypoxia-induced RV remodeling via stimulation of CGRP release, and the effects of rutaecarpine involve the eIF3a/p27 pathway.

  11. Cobalt chloride attenuates hypobaric hypoxia induced vascular leakage in rat brain: molecular mechanisms of action of cobalt chloride.

    PubMed

    Kalpana, S; Dhananjay, S; Anju, B; Lilly, G; Sai Ram, M

    2008-09-15

    This study reports the efficacy of cobalt preconditioning in preventing hypobaric hypoxia induced vascular leakage (an indicator of cerebral edema) using male Sprague-Dawley rats as model system. Exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, reactive oxygen species (ROS), nitric oxide (NO), and vascular endothelial growth factor (VEGF) levels. There was a marked increase in Nuclear Factor kappaB (NFkappaB) DNA binding activity and levels of pro-inflammatory cytokines such as Monocyte chemoattractant protein (MCP-1), Interferon-gamma (IFN-gamma), Interleukin-1 (IL-1), and Tumor Necrosis Factor-alpha (TNF-alpha) and cell adhesion molecules such as Vascular Cell Adhesion Molecule-1 (VCAM-1), and P-selectin. Chemical preconditioning by cobalt for 7 days (12.5 mg Co/kg b.w., oral) significantly attenuated cerebral vascular leakage and the expression of inflammatory mediators induced by hypoxia. Administration of NFkappaB inhibitor, curcumin (50 mg/kg b.w.; i.p.) appreciably inhibited hypoxia induced vascular leakage indicating the involvement of NFkappaB in causing vascular leakage. Interestingly, cobalt when administered at 12.5 mg Co/kg b.w. (i.p.), 1 h before hypoxia could not prevent the vascular leakage indicating that cobalt per se did not have an effect on NFkappaB. The lower levels of NFkappaB observed in the brains of cobalt administered animals might be due to higher levels of antioxidant and anti-inflammatory proteins (hemeoxygenase-1 and metallothionein). To conclude cobalt preconditioning inhibited hypobaric hypoxia induced cerebral vascular leakage by lowering NFkappaB DNA binding activity and its regulated pro-inflammatory mediators. This is contemplated to be mediated by cobalt induced reduction in ROS/NO and increase in HO-1 and MT.

  12. Effect of mesenchymal stem cells on hypoxia-induced desensitization of β2-adrenergic receptors in rat osteosarcoma cells

    PubMed Central

    KIDO, AKIRA; YOSHITANI, KAZUHIRO; SHIMIZU, TAKAMASA; AKAHANE, MANABU; FUJII, HIROMASA; TSUKAMOTO, SHINJI; KONDO, YUMIKO; HONOKI, KANYA; IMANO, MOTOHIRO; TANAKA, YASUHITO

    2012-01-01

    The β2-adrenergic receptor (β2AR) mediates the effects of chronic stress in several neoplasms, however, β2AR signaling is impaired by hypoxia in various tissues. While hypoxia is a common feature significant in the progression of solid tumors, little is known about the effect of hypoxia on β2AR signaling in the tumor microenvironment. Previously, it has been reported that the systemic administration of mesenchymal stem cells (MSCs) increased the engraftment and metastatic colonization of rat osteosarcoma (OS) cells. In the current study, the effect of MSCs on the hypoxia-induced desensitization of the β2AR in OS cells was investigated. Epinephrine, norepinephrine and isoproterenol increased the cellular proliferation of the rat OS cell line COS1NR and rat MSCs in a dose-dependent and β2AR antagonist-sensitive manner. While isoproterenol had significant proliferative effects on MSCs under normoxic and hypoxic conditions, COS1NR cells did not respond under hypoxic conditions. A sensitivity assay for the β2AR revealed that hypoxia impaired the sensitivity of COS1NR cells, whereas hypoxia did not affect MSCs. An immunoassay revealed no significant change in the expression of hypoxia-inducible factor-1α (HIF1α) in COS1NR cells, whilst an immunoassay demonstrated a 15% increase in MSCs following isoproterenol stimulation. In COS1NR cells co-cultured with MSCs under hypoxic conditions, isoproterenol caused a significant increase in proliferation and this effect was inhibited by an anti-interleukin (IL)-6 antibody. A tumor formation assay in syngeneic rats revealed that the systemic administration of MSCs enhances the growth of OS and the effect of MSCs was inhibited by IL-6 neutralization. In conclusion, MSCs are resistant to the hypoxia-induced desensitization to β2AR. Hypoxia caused a siginificant desensitization of the β2AR in COS1NR cells alone, whereas MSCs may support tumor progression through cellular interactions. PMID:23205094

  13. Cobalt chloride attenuates hypobaric hypoxia induced vascular leakage in rat brain: Molecular mechanisms of action of cobalt chloride

    SciTech Connect

    Kalpana, S.; Dhananjay, S.; Anju, B. Lilly, G.; Sai Ram, M.

    2008-09-15

    This study reports the efficacy of cobalt preconditioning in preventing hypobaric hypoxia induced vascular leakage (an indicator of cerebral edema) using male Sprague-Dawley rats as model system. Exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, reactive oxygen species (ROS), nitric oxide (NO), and vascular endothelial growth factor (VEGF) levels. There was a marked increase in Nuclear Factor {kappa}B (NF{kappa}B) DNA binding activity and levels of pro-inflammatory cytokines such as Monocyte chemoattractant protein (MCP-1), Interferon-{gamma} (IFN-{gamma}), Interleukin-1 (IL-1), and Tumor Necrosis Factor-{alpha} (TNF-{alpha}) and cell adhesion molecules such as Vascular Cell Adhesion Molecule-1 (VCAM-1), and P-selectin. Chemical preconditioning by cobalt for 7 days (12.5 mg Co/kg b.w., oral) significantly attenuated cerebral vascular leakage and the expression of inflammatory mediators induced by hypoxia. Administration of NF{kappa}B inhibitor, curcumin (50 mg/kg b.w.; i.p.) appreciably inhibited hypoxia induced vascular leakage indicating the involvement of NF{kappa}B in causing vascular leakage. Interestingly, cobalt when administered at 12.5 mg Co/kg b.w. (i.p.), 1 h before hypoxia could not prevent the vascular leakage indicating that cobalt per se did not have an effect on NF{kappa}B. The lower levels of NF{kappa}B observed in the brains of cobalt administered animals might be due to higher levels of antioxidant and anti-inflammatory proteins (hemeoxygenase-1 and metallothionein). To conclude cobalt preconditioning inhibited hypobaric hypoxia induced cerebral vascular leakage by lowering NF{kappa}B DNA binding activity and its regulated pro-inflammatory mediators. This is contemplated to be mediated by cobalt induced reduction in ROS/NO and increase in HO-1 and MT.

  14. Hypoxia-inducible factors as key regulators of tumor inflammation.

    PubMed

    Mamlouk, Soulafa; Wielockx, Ben

    2013-06-15

    Low levels of oxygen or hypoxia is often an obstacle in health, particularly in pathological disorders like cancer. The main family of transcription factors responsible for cell survival and adaptation under strenuous conditions of hypoxia are the "hypoxia-inducible factors" (HIFs). Together with prolyl hydroxylase domain enzymes (PHDs), HIFs regulates tumor angiogenesis, proliferation, invasion, metastasis, in addition to resistance to radiation and chemotherapy. Additionally, the entire HIF transcription cascade is involved in the "seventh" hallmark of cancer; inflammation. Studies have shown that hypoxia can influence tumor associated immune cells toward assisting in tumor proliferation, differentiation, vessel growth, distant metastasis and suppression of the immune response via cytokine expression alterations. These changes are not necessarily analogous to HIF's role in non-cancer immune responses, where hypoxia often encourages a strong inflammatory response.

  15. Hypoxia-inducible factors as molecular targets for liver diseases.

    PubMed

    Ju, Cynthia; Colgan, Sean P; Eltzschig, Holger K

    2016-06-01

    Liver disease is a growing global health problem, as deaths from end-stage liver cirrhosis and cancer are rising across the world. At present, pharmacologic approaches to effectively treat or prevent liver disease are extremely limited. Hypoxia-inducible factor (HIF) is a transcription factor that regulates diverse signaling pathways enabling adaptive cellular responses to perturbations of the tissue microenvironment. HIF activation through hypoxia-dependent and hypoxia-independent signals have been reported in liver disease of diverse etiologies, from ischemia-reperfusion-induced acute liver injury to chronic liver diseases caused by viral infection, excessive alcohol consumption, or metabolic disorders. This review summarizes the evidence for HIF stabilization in liver disease, discusses the mechanistic involvement of HIFs in disease development, and explores the potential of pharmacological HIF modifiers in the treatment of liver disease.

  16. Hypoxia-inducible factor 1 and breast cancer metastasis*

    PubMed Central

    Liu, Zhao-ji; Semenza, Gregg L.; Zhang, Hua-feng

    2015-01-01

    Accumulating evidence has shown that the hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating breast cancer progression and metastasis. The effects of hypoxia-inducible factor 1 (HIF-1), a master regulator of the hypoxic response, have been extensively studied during these processes. In this review, we focus on the roles of HIF-1 in regulating breast cancer cell metastasis, specifically its effects on multiple key steps of metastasis, such as epithelial-mesenchymal transition (EMT), invasion, extravasation, and metastatic niche formation. We also discuss the roles of HIF-1-regulated non-coding RNAs in breast cancer metastasis, and therapeutic opportunities for breast cancer through targeting the HIF-1 pathway. PMID:25559953

  17. Myeloid hypoxia-inducible factors in inflammatory diseases.

    PubMed

    Aragonés, Julian; Elorza, Ainara; Acosta-Iborra, Barbara; Landázuri, Manuel O

    2011-01-01

    Hypoxia inducible factors (HIF1 and HIF2) have emerged as central regulators of the activity of myeloid cells at inflammatory sites where O(2) is frequently limited. Novel insights in the field have revealed that the expression of HIFs by myeloid cells is not exclusively induced by hypoxia but also in response to central inflammatory mediators independently of O(2) shortage. This has substantially elevated the biological significance of HIFs in the context of inflammatory diseases. As a consequence, the loss of HIF1 or HIF2 in myeloid cells specifically compro-mises some of the processes driven by myeloid cells, such as bactericidal activity and myeloid invasion, as well as inflammation-associated detrimental consequences.

  18. Hypoxia-induced expression of RTEF-1 (related transcriptional enhancer factor-1) in endothelial cells is independent of HIF-1 (hypoxia-inducible factor-1)

    SciTech Connect

    Zhang, Cuili; Song, Q.H.; Li, Jian; Tian, Ye

    2009-04-10

    Related transcriptional enhancer factor-1 (RTEF-1) plays an important role in transcriptional regulation of angiogenic genes in hypoxic endothelial cells. The mechanisms involved in the induction of RTEF-1 expression in hypoxia are poorly understood. In bovine aortic endothelial cells (BAEC) subjected to hypoxia, Western blot and quantitative PCR analysis revealed that RTEF-1 protein and mRNA levels were significantly increased by hypoxia. To address the potential role of the hypoxia-inducible factor-1 (HIF-1) in RTEF-1 induction, a hepatoma cell line deficient in HIF-1 (c4) and a control HIF-1 positive cell line (vT{l_brace}2{r_brace}) were exposed to hypoxia. We report that RTEF-1 protein expression assessed by either Western blotting or immunofluorescence was increased in both cell lines. This demonstrates that HIF-1 is not required for RTEF-1 upregulation by hypoxia. Conversely, RTEF-1 appeared to regulate the expression of HIF-1: HIF-1{alpha} promoter activity was increased (3.6-fold) by RTEF-1 overexpression in BAEC. Furthermore, RTEF-1 enhanced BAEC proliferation and tubule formation; these were inhibited by RTEF-1 knockdown with siRNA. We propose that RTEF-1, acting via HIF-1, is a key regulator of angiogenesis in response to hypoxia.

  19. Diacetoxyscirpenol as a new anticancer agent to target hypoxia-inducible factor 1

    PubMed Central

    Choi, Yong-Joon; Shin, Hyun-Woo; Chun, Yang-Sook; Leutou, Alain Simplice; Son, Byeng Wha; Park, Jong-Wan

    2016-01-01

    Hypoxia activates hypoxia-inducible factor 1, which promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. Thus, HIF-1 is one of the most compelling targets for treating cancers. The aim of this study was to find a small molecule that inhibits HIF-1 under hypoxia in cancer cells. 7,280 compounds in a chemical library were tested in a cancer cell line expressing luciferase HIF-dependently. Through three rounds of screening, we finally picked up a compound that originates from a marine bacterium parasitizing red alga. The antibiotic potently inhibited HIF-1 expression and its transcriptional activity in cancer cells exposed to hypoxia. Through two-step fractionation, diacetoxyscirpenol was purified and identified as a HIF-inhibiting ingredient. Mechanistically, diacetoxyscirpenol inhibits the synthesis of HIF-1α protein and also interferes with the dimerization of HIF-1α and ARNT. It attenuates HIF-mediated gene expression in cancer cells exposed to hypoxia, and by doing so reduces tumorigenic and angiogenic potentials of cancer cells. More importantly, diacetoxyscirpenol retarded tumor growth in mice, and reduced HIF-1α expression and vascular formation in the tumors. Overall, diacetoxyscirpenol is considered a potential drug deregulating the HIF-1 signaling pathway, and it could be beneficially employed for treating malignant tumors with hypoxic microenvironment. PMID:27613833

  20. Involvement of nitric oxide in anticompulsive-like effect of agmatine on marble-burying behaviour in mice.

    PubMed

    Gawali, Nitin B; Chowdhury, Amrita A; Kothavade, Pankaj S; Bulani, Vipin D; Nagmoti, Dnyaneshwar M; Juvekar, Archana R

    2016-01-05

    In view of the reports that nitric oxide modulates the neurotransmitters implicated in obsessive-compulsive disorder (OCD), patients with OCD exhibit higher plasma nitrate levels, and drugs useful in OCD influence nitric oxide. Agmatine is a polyamine and widely distributed in mammalian brain which interacts with nitrergic systems. Hence, the present study was carried out to understand the involvement of nitrergic systems in the anticompulsive-like effect of agmatine. We used marble-burying behaviour (MBB) of mice as the animal model of OCD, and nitric oxide levels in hippocampus (HC) and cortex homogenate were measured. Results revealed that, agmatine (20 and 40mg/kg, i.p) significantly inhibited the MBB. Intraperitoneal administration of nitric oxide enhancers viz. nitric oxide precursor - l-arginine (l-ARG) (400mg/kg and 800mg/kg) increased MBB as well as brain nitrites levels, whereas treatment with N(G)-nitro-l-arginine methyl ester (l-NAME) neuronal nitric oxide synthase inhibitor (30mg/kg and 50mg/kg, i.p.) and 7-nitroindazole (7-NI) (20mg/kg and 40mg/kg) attenuated MBB and nitrites levels in brain. Further, in combination studies, the anticompulsive-like effect of agmatine (20mg/kg, ip) was exacerbated by prior administration of l-ARG (400mg/kg) and conversely l-NAME (15mg/kg) or 7-NI (10.0mg/kg) attenuated OCD-like behaviour with HC and cortex changes in the levels of NO. None of the above treatment had any significant influence on locomotor activity. In conclusion, Agmatine is effective in ameliorating the compulsive-like behaviour in mice which appears to be related to nitric oxide in brain.

  1. Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine-displacing substance and agmatine.

    PubMed

    Reis, D J; Li, G; Regunathan, S

    1995-07-12

    1. There are several endogenous ligands that bind to I-receptors of both the I1 and I2 subclass. These include: (a) classic CDS, a partially purified entity isolated by the criteria that it displaces binding ligands to alpha 2- and I-receptors; (b) immunoreactive (ir)-CDS, a moiety that binds to antibodies raised against clonidine, para-amino-clonidine, or idazoxan; and (c) agmatine. 2. Classic-CDS, not yet defined structurally, binds to I1, I2, and alpha 2-adrenergic receptors, is neither a peptide nor a catecholamine, and has purportedly a molecular weight of 588 Da. By ligand binding assays, it was found in brain, serum, CSF, and placenta and in a neural-glial cell line. Partially purified classic CDS is bioactive. Like clonidine, it contracts aorta and vas deferens and inhibits platelet aggregation, effects largely attributable to agonism at alpha 2-adrenergic receptors. Unlike clonidine, it contracts rat gastric fundus and releases catecholamines from chromaffin cells, effects attributable to actions at I-receptors. Injected into the RVL, classic CDS alters arterial pressure, but the direction of change of pressure has differed between groups of investigators. However, in the absence of structure, it is possible that ligand binding and bioactivity may be attributable to different molecules. 3. Ir-CDS, also of unknown structure, is a material(s) that binds to antibodies raised against clonidine, PAC, or idazoxan. Ir-CDS, measured by radioimmunoassay, is unevenly distributed in brain with highest concentrations in the hypothalamus, midbrain, and dorsal medulla. It is contained in the gastric fundus, adrenal gland, heart, kidney, and serum in amounts substantially higher than found in brain. Ir-CDS may be elevated in the serum of some patients with hypertension and in the CSF of patients with structural brain disease. The concentration of ir-CDS and bioactivity on gastric fundus directly correlates, suggesting that it may share similarities with classic

  2. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration

    PubMed Central

    Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  3. A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network.

    PubMed

    Nguyen, Lan K; Cavadas, Miguel A S; Scholz, Carsten C; Fitzpatrick, Susan F; Bruning, Ulrike; Cummins, Eoin P; Tambuwala, Murtaza M; Manresa, Mario C; Kholodenko, Boris N; Taylor, Cormac T; Cheong, Alex

    2013-03-15

    Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1α pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1α transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1α transcriptional activity, but paradoxically decreases HIF-1α stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1α to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1α transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

  4. Expression of DDX3 Is Directly Modulated by Hypoxia Inducible Factor-1 Alpha in Breast Epithelial Cells

    PubMed Central

    Botlagunta, Mahendran; Krishnamachary, Balaji; Vesuna, Farhad; Winnard, Paul T.; Bol, Guus M.; Patel, Arvind H.; Raman, Venu

    2011-01-01

    DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region. PMID:21448281

  5. ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function

    PubMed Central

    Cui, Dan; Arima, Mitsuru; Takubo, Keiyo; Kimura, Tokuhiro; Horiuchi, Keisuke; Minagawa, Takuya; Matsuda, Satoshi; Ikeda, Eiji

    2015-01-01

    Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases. PMID:26242473

  6. Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells.

    PubMed

    Bach, Duc-Hiep; Kim, Seong-Hwan; Hong, Ji-Young; Park, Hyen Joo; Oh, Dong-Chan; Lee, Sang Kook

    2015-11-19

    Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

  7. HIF-1α-induced microRNA-210 reduces hypoxia-induced osteoblast MG-63 cell apoptosis.

    PubMed

    Sun, Guanwen; Peng, Hao

    2015-01-01

    To better understand the ischemic-hypoxia-induced fracture healing impairment, we determined in this study the microRNA-210 expression in broken bone specimens and in osteoblasts under hypoxia and then determined the influence of microRNA-210 overexpression on the osteoblast cell proliferation and apoptosis. Results demonstrated that microRNA-210 expression was upregulated with an association with HIF-1α overexpression in clinical human catagmatic tissues and was upregulated HIF-1α-dependently in response to hypoxia in osteoblast MG-63 cells. CCK-8 assay indicated that microRNA-210 upregulation by microRNA-210 mimics reduced the chemotherapeutic 5-FU-induced osteoblast cell death, and colony formation assay demonstrated that microRNA-210 mimics promoted osteoblast cells growth. Moreover, the microRNA-210 mimics transfection inhibited the hypoxia-induced MG-63 cell apoptosis via inhibiting the activation of caspase 3 and caspase 9. Therefore, our research indicated a protective role of microRNA-210 in response to hypoxia. And microRNA-210 might serve as a protective role in bone fracture healing.

  8. ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

    PubMed

    Cui, Dan; Arima, Mitsuru; Takubo, Keiyo; Kimura, Tokuhiro; Horiuchi, Keisuke; Minagawa, Takuya; Matsuda, Satoshi; Ikeda, Eiji

    2015-08-05

    Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

  9. KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

    PubMed Central

    Hedegaard, E R; Nielsen, B D; Kun, A; Hughes, A D; Krøigaard, C; Mogensen, S; Matchkov, V V; Fröbert, O; Simonsen, U

    2014-01-01

    BACKGROUND AND PURPOSE Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca2+ concentration ([Ca2+]i) by opening of K channels and release of H2S. EXPERIMENTAL APPROACH Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca2+]i, and the expression of voltage-gated K channels KV7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (KCa1.1) was examined. Voltage clamp assessed the role of KV7 channels in hypoxia. KEY RESULTS Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca2+]i in PGF2α (10 μM)-contracted arteries whereas no fall in [Ca2+]i was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and KCa1.1 inhibited hypoxia-induced dilatation in PGF2α-contracted arteries; this inhibition was more marked in the presence of the Kv7 channel blockers, XE991 and linopirdine, while a KV7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H2S- and adenosine-induced vasodilatation. PCR revealed the expression of KV7.1, KV7.4, KV7.5 and KCa1.1 channels, and KCa1.1, KV7.4 and KV7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions. CONCLUSION The KV7.4 and KV7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of KV7 channels in this vasodilatation. Activation of these KV7 channels may be induced by H2S and adenosine. PMID:24111896

  10. Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes Containing Elongins B/C and Cul2

    PubMed Central

    Lonergan, Kim M.; Iliopoulos, Othon; Ohh, Michael; Kamura, Takumi; Conaway, Ronald C.; Conaway, Joan Weliky; Kaelin, William G.

    1998-01-01

    The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis. PMID:9447969

  11. Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

    PubMed Central

    Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

    2012-01-01

    Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

  12. Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

    PubMed Central

    Werth, Nadine; Beerlage, Christiane; Rosenberger, Christian; Yazdi, Amir S.; Edelmann, Markus; Amr, Amro; Bernhardt, Wanja; von Eiff, Christof; Becker, Karsten; Schäfer, Andrea; Peschel, Andreas; Kempf, Volkhard A. J.

    2010-01-01

    Background Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. Methodology/Principal Findings Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i) in all ex vivo in biopsies of patients suffering from skin infections, (ii) in vitro using cell culture infection models and (iii) in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs) of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. Conclusions/Significance Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections. PMID:20644645

  13. Endothelin-converting enzyme is a plausible target gene for hypoxia-inducible factor.

    PubMed

    Khamaisi, Mogher; Toukan, Hala; Axelrod, Jonathan H; Rosenberger, Christian; Skarzinski, Galia; Shina, Ahuva; Meidan, Rina; Koesters, Robert; Rosen, Seymour; Walkinshaw, Gail; Mimura, Imari; Nangaku, Masaomi; Heyman, Samuel N

    2015-04-01

    Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.

  14. Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation

    PubMed Central

    Liu, Xing; Chen, Zhu; Xu, Chenxi; Leng, Xiaoqian; Cao, Hong; Ouyang, Gang; Xiao, Wuhan

    2015-01-01

    Hypoxia-inducible factor (HIF)-1α and HIF-2α are the main regulators of cellular responses to hypoxia. Post-translational modifications of HIF-1α and 2α are necessary to modulate their functions. The methylation of non-histone proteins by Set7, an SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. In this study, we show that Set7 methylates HIF-1α at lysine 32 and HIF-2α at lysine K29; this methylation inhibits the expression of HIF-1α/2α targets by impairing the occupancy of HIF-α on hypoxia response element of HIF target gene promoter. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes. Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 or inhibition of Set7 by the inhibitor subjected to hypoxia display an increased glucose uptake and intracellular adenosine triphosphate levels. These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis. PMID:25897119

  15. Hypoxia induces discoidin domain receptor-2 expression via the p38 pathway in vascular smooth muscle cells to increase their migration.

    PubMed

    Chen, Shih-Chung; Wang, Bao-Wei; Wang, Danny Ling; Shyu, Kou-Gi

    2008-10-03

    Discoidin domain receptor-2 (DDR2) is a receptor tyrosine kinase that binds to the extracellular matrix. We investigated the role of hypoxia in DDR2 expression in vascular smooth muscle cells (VSMCs) and the underlying mechanism. Subjecting VSMCs to hypoxia (2.5% O(2)) induced DDR2 expression; treatments with a specific inhibitor (SB203580) of p38 mitogen-activated protein kinase (MAPK) or p38-specific small interference RNA (siRNA) abolished this hypoxia-induced DDR2 expression. Gel shifting assays showed that hypoxia increased the Myc-Max-DNA binding activity in the promoter region of DDR2; inhibition of p38 MAPK activation by SB203580 and p38-specific siRNA blocked hypoxia-induced DDR2 promoter activity. Hypoxia also induced matrix metalloproteinase-2 (MMP-2) activity in VSMCs and increased their migration. These VSMC responses to hypoxia were inhibited by DDR2- and p38-specific siRNAs. Our results suggested that hypoxia induces DDR2 expression in VSMCs at the transcriptional level, which is mediated by the p38 MAPK pathway and contributes to VSMC migration.

  16. Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

    PubMed

    Eguchi, Ryoji; Naitou, Hirotaka; Kunimasa, Kazuhiro; Ayuzawa, Rie; Fujimori, Yoshihiro; Ohashi, Norio; Kaji, Kazuhiko; Ohta, Toshiro

    2008-07-01

    We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

  17. Hypoxia Inducible Factor-1 as a Target for Neurodegenerative Diseases

    PubMed Central

    Zhang, Ziyan; Yan, Jingqi; Chang, Yanzhong; Yan, Shirley ShiDu; Shi, Honglian

    2011-01-01

    Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor responsible for cellular and tissue adaption to low oxygen tension. HIF-1, a heterodimer consisting of a constitutively expressed β subunit and an oxygen-regulated α subunit, regulates a series of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival. The activity of HIF-1 is controlled by post-translational modifications on different amino acid residues of its subunits, mainly the alpha subunit. Besides in ischemic stroke (see review [1]), emerging evidence has revealed that HIF-1 activity and expression of its down-stream genes, such as vascular endothelial growth factor and erythropoietin, are altered in a range of neurodegenerative diseases. At the same time, experimental and clinical evidence has demonstrated that regulating HIF-1 might ameliorate the cellular and tissue damage in the neurodegenerative diseases. These new findings suggest HIF-1 as a potential medicinal target for the neurodegenerative diseases. This review focuses on HIF-1α protein modifications and HIF-1’s potential neuroprotective roles in Alzheimer’s (AD), Parkinson’s (PD), Huntington’s diseases (HD), and amyotrophic lateral sclerosis (ALS). PMID:21861815

  18. Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERβ or GPR30 signalling.

    PubMed

    Rzemieniec, J; Litwa, E; Wnuk, A; Lason, W; Gołas, A; Krzeptowski, W; Kajta, M

    2015-02-01

    Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in

  19. A Dialogue between the Hypoxia-Inducible Factor and the Tumor Microenvironment

    PubMed Central

    Dayan, Frédéric; Mazure, Nathalie M.; Brahimi-Horn, M. Christiane

    2008-01-01

    The hypoxia-inducible factor is the key protein responsible for the cellular adaptation to low oxygen tension. This transcription factor becomes activated as a result of a drop in the partial pressure of oxygen, to hypoxic levels below 5% oxygen, and targets a panel of genes involved in maintenance of oxygen homeostasis. Hypoxia is a common characteristic of the microenvironment of solid tumors and, through activation of the hypoxia-inducible factor, is at the center of the growth dynamics of tumor cells. Not only does the microenvironment impact on the hypoxia-inducible factor but this factor impacts on microenvironmental features, such as pH, nutrient availability, metabolism and the extracellular matrix. In this review we discuss the influence the tumor environment has on the hypoxia-inducible factor and outline the role of this factor as a modulator of the microenvironment and as a powerful actor in tumor remodeling. From a fundamental research point of view the hypoxia-inducible factor is at the center of a signaling pathway that must be deciphered to fully understand the dynamics of the tumor microenvironment. From a translational and pharmacological research point of view the hypoxia-inducible factor and its induced downstream gene products may provide information on patient prognosis and offer promising targets that open perspectives for novel “anti-microenvironment” directed therapies. PMID:19308685

  20. Effect of multidrug resistance 1/P-glycoprotein on the hypoxia-induced multidrug resistance of human laryngeal cancer cells.

    PubMed

    Li, Dawei; Zhou, Liang; Huang, Jiameng; Xiao, Xiyan

    2016-08-01

    In a previous study, it was demonstrated that hypoxia upregulated the multidrug resistance (MDR) of laryngeal cancer cells to chemotherapeutic drugs, with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp) expression also being upregulated. The present study aimed to investigate the role and mechanism of MDR1/P-gp on hypoxia-induced MDR in human laryngeal carcinoma cells. The sensitivity of laryngeal cancer cells to multiple drugs and cisplatin-induced apoptosis was determined by CCK-8 assay and Annexin-V/propidium iodide staining analysis, respectively. The accumulation of rhodamine 123 (Rh123) in the cells served as an estimate of drug accumulation and was evaluated by flow cytometry (FCM). MDR1/P-gp expression was inhibited using interference RNA, and the expression of the MDR1 gene was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. As a result, the sensitivity to multiple chemotherapeutic agents and the apoptosis rate of the hypoxic laryngeal carcinoma cells increased following a decrease in MDR1/P-gp expression (P<0.05). Additionally, FCM analysis of fluorescence intensity indicated that the downregulated expression of MDR1/P-gp markedly increased intracellular Rh123 accumulation (P<0.05). Such results suggest that MDR1/P-gp serves an important role in regulating hypoxia-induced MDR in human laryngeal carcinoma cells through a decrease in intracellular drug accumulation.

  1. Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes.

    PubMed

    Cirillo, Federica; Resmini, Giulia; Ghiroldi, Andrea; Piccoli, Marco; Bergante, Sonia; Tettamanti, Guido; Anastasia, Luigi

    2017-02-10

    Regeneration of skeletal muscle is a complex process that requires the activation of quiescent adult stem cells, called satellite cells, which are resident in hypoxic niches in the tissue. Hypoxia has been recognized as a key factor to maintain stem cells in an undifferentiated state. Herein we report that hypoxia plays a fundamental role also in activating myogenesis. In particular, we found that the activation of the hypoxia-inducible factor (HIF)-1α under hypoxia, in murine skeletal myoblasts, leads to activation of MyoD through the noncanonical Wnt/β-catenin pathway. Moreover, chemical inhibition of HIF-1α activity significantly reduces differentiation, thus confirming its crucial role in the process. Furthermore, hypoxia-preconditioned myoblasts, once induced to differentiate under normoxic conditions, tend to form hypertrophic myotubes. These results support the notion that hypoxia plays a pivotal role in activating the regeneration process by directly inducing myogenesis through HIF-1α. Although preliminary, these findings may suggest new perspective for novel therapeutic targets in the treatment of several muscle diseases.-Cirillo, F., Resmini, G., Ghiroldi, A., Piccoli, M., Bergante, S., Tettamanti, G., Anastasia, L. Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes.

  2. Serotonin-mediated modulation of hypoxia-induced intracellular calcium responses in glomus cells isolated from rat carotid body.

    PubMed

    Yokoyama, Takuya; Nakamuta, Nobuaki; Kusakabe, Tatsumi; Yamamoto, Yoshio

    2015-06-15

    In the present study, we examined serotonin (5-HT)-induced intracellular Ca(2+) ([Ca(2+)]i) responses to hypoxia in glomus cells isolated from carotid body (CB) of the rat. 5-HT did not induce any [Ca(2+)]i responses in clustered glomus cells during normoxia (21% O2), whereas, the perfusion of hypoxic solution (1% O2) induced repetitive increases in [Ca(2+)]i in the same specimens. The frequency and magnitude of hypoxia-induced [Ca(2+)]i changes observed in the glomus cells were enhanced in the presence of 5-HT, and this response was inhibited by the 5-HT2 receptor antagonist, ketanserin. Furthermore, RT-PCR analysis detected the expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT3A, and 5-HT3B receptor mRNAs in extracts of the CB. These results suggest that 5-HT increases hypoxia-induced [Ca(2+)]i responses in glomus cells. 5-HT may elevate hypoxic responses in glomus cells in order to increase chemosensory activity of the CB.

  3. Hypoxia-induced CCL28 promotes recruitment of regulatory T cells and tumor growth in liver cancer

    PubMed Central

    Wang, Li; Zhu, Zhifeng; Lu, Rong; Yao, Zhi

    2016-01-01

    Tumor cells craft microenvironment to overcome growth disadvantages and adjust to escape the immunosurveillance during tumorigenesis and metastasis. The evolving adaption to the changing microenvironment is exemplified by the development of strategies to deal with hypoxia resulted from fast proliferation of the tumor cells. In this study, we found that hypoxia hepatocellular carcinoma (HCC) cells recruited Regulatory T cells (Tregs) and expressed more Chemokine (C-C motif) ligand 28 (CCL28). Deletion of CCL28 inhibited Treg recruitment. Furthermore, overexpression of CCL28 promoted tumor growth and Treg infiltration in vivo. Enhanced angiogenesis and VEGF expression was also observed. Moreover, inhibition of HIF1α reversed hypoxia-induced CCL28 upregulation. Taken together, our results demonstrate that HCC recruits Tregs to promote angiogenesis under hypoxic condition by upregulating CCL28 expression. These findings establish a link between Tregs and hypoxia in HCC growth and may provide a new potential therapeutic target for treating HCC. PMID:27716621

  4. Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1)

    PubMed Central

    Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, ancd effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors. PMID:16515532

  5. Hypoxia inducible factor-1α regulates a pro-invasive phenotype in acute monocytic leukemia

    PubMed Central

    Migliavacca, Jessica; Percio, Stefano; Valsecchi, Roberta; Ferrero, Elisabetta; Spinelli, Antonello; Ponzoni, Maurilio; Tresoldi, Cristina; Pattini, Linda

    2016-01-01

    Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1α can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type. In this study, we find that HIF-1α promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1α-target genes to different AML sub-types, we identified a HIF-1α signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF-1α impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1α-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination. PMID:27447550

  6. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    SciTech Connect

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  7. Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

    PubMed Central

    Zhang, Wei; Wang, Wang; Liu, Jie; Li, Jinna; Wang, Juan; Zhang, Yunxia; Zhang, Zhifei; Liu, Yafei; Jin, Yankun; Li, Jifeng; Cao, Jie; Wang, Chen; Ning, Wen; Wang, Jun

    2017-01-01

    Pulmonary hypertension (PH) remains a life-limiting disease characterized by pulmonary vascular remodelling due to aberrant proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), thus leading to raised pulmonary arterial pressure and right ventricular hypertrophy. Secreted glycoprotein follistatin-like 1 (FSTL1) has been reported to ameliorate tissue remodelling in cardiovascular injuries. However, the role of FSTL1 in deranged pulmonary arteries remains elusive. We found that there were higher serum levels of FSTL1 in patients with PH related to chronic obstructive pulmonary diseases (COPD) and in mice model of hypoxia-induced PH (HPH). Haploinsufficiency of Fstl1 in mice contributed to an exacerbated HPH, as demonstrated by increased right ventricular systolic pressure, pulmonary arterial muscularization and right ventricular hypertrophy index. Conversely, FSTL1 administration attenuated HPH. In cultured human PASMCs, hypoxia-promoted cellular viability, DNA synthesis and migration were suppressed by exogenous FSTL1 but enhanced by small interfering RNA targeting FSTL1. Additionally, FSTL1 inhibited the proliferation and migration of PASMCs via extracellular regulated kinase (ERK) signal pathway. All these findings indicate that FSTL1 imposed a protective modulation on pulmonary vascular remodelling, thereby suggesting its role in the regulation of HPH. PMID:28361925

  8. Hypoxia increases sirtuin 1 expression in a hypoxia-inducible factor-dependent manner.

    PubMed

    Chen, Rui; Dioum, Elhadji M; Hogg, Richard T; Gerard, Robert D; Garcia, Joseph A

    2011-04-22

    Hypoxia-inducible factors (HIFs) are stress-responsive transcriptional regulators of cellular and physiological processes involved in oxygen metabolism. Although much is understood about the molecular machinery that confers HIF responsiveness to oxygen, far less is known about HIF isoform-specific mechanisms of regulation, despite the fact that HIF-1 and HIF-2 exhibit distinct biological roles. We recently determined that the stress-responsive genetic regulator sirtuin 1 (Sirt1) selectively augments HIF-2 signaling during hypoxia. However, the mechanism by which Sirt1 maintains activity during hypoxia is unknown. In this report, we demonstrate that Sirt1 gene expression increases in a HIF-dependent manner during hypoxia in Hep3B and in HT1080 cells. Impairment of HIF signaling affects Sirt1 deacetylase activity as decreased HIF-1 signaling results in the appearance of acetylated HIF-2α, which is detected without pharmacological inhibition of Sirt1. We also find that Sirt1 augments HIF-2 mediated, but not HIF-1 mediated, transcriptional activation of the isolated Sirt1 promoter. These data in summary reveal a bidirectional link of HIF and Sirt1 signaling during hypoxia.

  9. Prodeath or Prosurvival: Two Facets of Hypoxia Inducible Factor-1 in Perinatal Brain Injury

    PubMed Central

    Chen, Wanqiu; Ostrowski, Robert P.; Obenaus, Andre; Zhang, John H.

    2009-01-01

    Hypoxia, which occurs in the brain when oxygen availability drops below the normal level, is a major cause of perinatal hypoxic-ischemic injury (HII). The transcriptional factor hypoxia inducible factor-1 (HIF-1) is a key regulator in the pathophysiological response to the stress of hypoxia. Genes regulated by HIF-1 are involved in energy metabolism, erythropoiesis, angiogenesis, vasodilatation, cell survival and apoptosis. Compared with the adult brain, the neonatal brain is different in physiological structure, function, cellular composition and signaling pathways related gene activation and response after hypoxia. The purpose of this review is to determine if developmental susceptibility of the brain after hypoxic/ischemic injury is related to HIF-1a, which also plays a pivotal role in the normal brain development. HIF-1a regulates both prosurvival and prodeath responses in the neonatal brain and various mechanisms underlie the apparent contradictory effects, including duration of ischemic injury and severity, cell-types, and/or dependent on the nature of the stimulus after HII. Studies report an excessive induction of HIF-1 in the immature brain, which suggests that a cell death promoting role of HIF may prevail. Inhibition of HIF-1a and targeted activation of its prosurvival genes appear as a favorable therapeutic strategy. However, a better understanding of multifaceted HIF-1 function during brain development is required to explore potential targets for further therapeutic interventions in the neonate. PMID:19041643

  10. Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice.

    PubMed

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M; Huttner, Wieland; Weidemann, Alexander; Wielockx, Ben

    2013-09-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

  11. Functional regulation of hypoxia inducible factor-1α by SET9 lysine methyltransferase

    PubMed Central

    Liu, Qiong; Geng, Hao; Xue, Changhui; Beer, Tomasz M.; Qian, David Z.

    2015-01-01

    HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells. PMID:25637186

  12. Hypoxia-inducible factor-1alpha suppresses squamous carcinogenic progression and epithelial-mesenchymal transition.

    PubMed

    Scortegagna, Marzia; Martin, Rebecca J; Kladney, Raleigh D; Neumann, Robert G; Arbeit, Jeffrey M

    2009-03-15

    Hypoxia-inducible factor-1 (HIF-1) is a known cancer progression factor, promoting growth, spread, and metastasis. However, in selected contexts, HIF-1 is a tumor suppressor coordinating hypoxic cell cycle suppression and apoptosis. Prior studies focused on HIF-1 function in established malignancy; however, little is known about its role during the entire process of carcinogenesis from neoplasia induction to malignancy. Here, we tested HIF-1 gain of function during multistage murine skin chemical carcinogenesis in K14-HIF-1alpha(Pro402A564G) (K14-HIF-1alphaDPM) transgenic mice. Transgenic papillomas appeared earlier and were more numerous (6 +/- 3 transgenic versus 2 +/- 1.5 nontransgenic papillomas per mouse), yet they were more differentiated, their proliferation was lower, and their malignant conversion was profoundly inhibited (7% in transgenic versus 40% in nontransgenic mice). Moreover, transgenic cancers maintained squamous differentiation whereas epithelial-mesenchymal transformation was frequent in nontransgenic malignancies. Transgenic basal keratinocytes up-regulated the HIF-1 target N-myc downstream regulated gene-1, a known tumor suppressor gene in human malignancy, and its expression was maintained in transgenic papillomas and cancer. We also discovered a novel HIF-1 target gene, selenium binding protein-1 (Selenbp1), a gene of unknown function whose expression is lost in human cancer. Thus, HIF-1 can function as a tumor suppressor through transactivation of genes that are themselves targets for negative selection in human cancers.

  13. Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma.

    PubMed

    Teng, Hongming; Yang, Yazong; Wei, Hengyun; Liu, Zundong; Liu, Zhichao; Ma, Yanhong; Gao, Zixiang; Hou, Lin; Zou, Xiangyang

    2015-06-03

    Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.

  14. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  15. Myeloid derived hypoxia inducible factor 1-alpha is required for protection against pulmonary Aspergillus fumigatus infection.

    PubMed

    Shepardson, Kelly M; Jhingran, Anupam; Caffrey, Alayna; Obar, Joshua J; Suratt, Benjamin T; Berwin, Brent L; Hohl, Tobias M; Cramer, Robert A

    2014-09-01

    Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections.

  16. Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

    PubMed Central

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M.; Huttner, Wieland; Weidemann, Alexander

    2013-01-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds. PMID:23798557

  17. Interplay between sirtuins, MYC and hypoxia-inducible factor in cancer-associated metabolic reprogramming

    PubMed Central

    Zwaans, Bernadette M. M.; Lombard, David B.

    2014-01-01

    In the early twentieth century, Otto Heinrich Warburg described an elevated rate of glycolysis occurring in cancer cells, even in the presence of atmospheric oxygen (the Warburg effect). Despite the inefficiency of ATP generation through glycolysis, the breakdown of glucose into lactate provides cancer cells with a number of advantages, including the ability to withstand fluctuations in oxygen levels, and the production of intermediates that serve as building blocks to support rapid proliferation. Recent evidence from many cancer types supports the notion that pervasive metabolic reprogramming in cancer and stromal cells is a crucial feature of neoplastic transformation. Two key transcription factors that play major roles in this metabolic reprogramming are hypoxia inducible factor-1 (HIF1) and MYC. Sirtuin-family deacetylases regulate diverse biological processes, including many aspects of tumor biology. Recently, the sirtuin SIRT6 has been shown to inhibit the transcriptional output of both HIF1 and MYC, and to function as a tumor suppressor. In this Review, we highlight the importance of HIF1 and MYC in regulating tumor metabolism and their regulation by sirtuins, with a main focus on SIRT6. PMID:25085992

  18. Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

    PubMed

    Myllymäki, Mikko N M; Määttä, Jenni; Dimova, Elitsa Y; Izzi, Valerio; Väisänen, Timo; Myllyharju, Johanna; Koivunen, Peppi; Serpi, Raisa

    2017-01-15

    Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2(gt/gt)) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

  19. Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

    PubMed

    Kato, Hiroko; Izumi, Kenji; Uenoyama, Atsushi; Shiomi, Aki; Kuo, Shiuhyang; Feinberg, Stephen E

    2014-01-01

    The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

  20. A positive feedback loop between ROS and Mxi1-0 promotes hypoxia-induced VEGF expression in human hepatocellular carcinoma cells.

    PubMed

    Hu, Zhenzhen; Dong, Na; Lu, Dian; Jiang, Xiuqin; Xu, Jinjin; Wu, Zhiwei; Zheng, Datong; Wechsler, Daniel S

    2017-02-01

    VEGF expression induced by hypoxia plays a critical role in promoting tumor angiogenesis. However, the molecular mechanism that modulates VEGF expression under hypoxia is still poorly understood. In this study, we found that VEGF induction in hypoxic HepG2 cells is ROS-dependent. ROS mediates hypoxia-induced VEGF by upregulation of Mxi1-0. Furthermore, PI3K/AKT/HIF-1α signaling pathway is involved in ROS-mediated Mxi1-0 and VEGF expression in hypoxic HepG2 cells. Finally, Mxi1-0 could in turn regulate ROS generation in hypoxic HepG2 cells, creating a positive feedback loop. Taken together, this study demonstrate a positive regulatory feedback loop in which ROS mediates hypoxia-induced Mxi1-0 via activation of PI3K/AKT/HIF-1α pathway, events that in turn elevate ROS generation and promote hypoxia-induced VEGF expression. These findings could provide a rationale for designing new therapies based on inhibition of hepatocellular carcinoma (HCC) angiogenesis.

  1. Suppression of the proliferation of hypoxia-Induced retinal pigment epithelial cell by rapamycin through the /mTOR/HIF-1α/VEGF/ signaling.

    PubMed

    Liu, Ning-Ning; Zhao, Ning; Cai, Na

    2015-06-01

    Rapamycin, a highly specific inhibitor of mammalian target of rapamycin (mTOR), exhibits significant antitumor/antiangiogenic activity in human cancer cells. Its effect on the retinal pigment epithelial (RPE) cells was rarely investigated. This study assessed the proliferation of hypoxia-induced RPE and the inhibitory effects of rapamycin using 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and examined the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells with or without rapamycin under normoxic and hypoxic conditions using real-time PCR and Western blot. We found that hypoxia increased the levels of mTOR, HIF-1α, and VEGF. The suppression of HIF-1α and VEGF by rapamycin was associated with dephosphorylation of mTOR and the downstream effector ribosomal protein S6 kinase (P70S6K) and 4E-binding protein-1 (4E-BP1) of mTORC1. Rapamycin only inhibited the protein levels and did not change the mRNA expression of HIF-1α. No cytotoxicity to the RPE cells by rapamycin was caused under either normoxia or hypoxia. Our data suggest that rapamycin suppresses hypoxia-induced RPE cell proliferation through a mechanism related to the targeting of mTOR/HIF-1α/VEGF signaling. Rapamycin may potentially provide a safe and effective novel treatment for choroidal vascular disease.

  2. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

    PubMed Central

    Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L.

    2016-01-01

    Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation. PMID:27590511

  3. Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas.

    PubMed

    Lu, Huimin; Li, Yan; Shu, Minfeng; Tang, Jianjun; Huang, Yijun; Zhou, Yuxi; Liang, Yingjie; Yan, Guangmei

    2009-12-01

    Aberrant differentiation is a characteristic feature of neoplastic transformation, while hypoxia in solid tumors is believed to be linked to aggressive behavior and poor prognosis. However, the possible relationship between hypoxia and differentiation in malignancies remains poorly defined. Here we show that rat C6 and primary human malignant glioma cells can be induced to differentiate into astrocytes by the well-known adenylate cyclase activator forskolin. However, hypoxia-inducible factor-1alpha expression stimulated by the hypoxia mimetics cobalt chloride or deferoxamine blocks this differentiation and this effectiveness is reversible upon withdrawal of the hypoxia mimetics. Importantly, knockdown of hypoxia inducible factor-1alpha by RNA interference restores the differentiation capabilities of the cells, even in the presence of cobalt chloride, whereas stabilization of hypoxia-inducible factor-1alpha through retarded ubiquitination by von Hippel-Lindau tumor suppressor gene silence abrogates the induced differentiation. Moreover, targeting of HIF-1 using chetomin, a disrupter of HIF-1 binding to its transcriptional co-activator CREB-binding protein (CBP)/p300, abolishes the differentiation-inhibitory effect of hypoxia-inducible factor-1alpha. Administration of chetomin in combination with forskolin significantly suppresses malignant glioma growth in an in vivo xenograft model. Analysis of 95 human glioma tissues revealed an increase of hypoxia-inducible factor-1alpha protein expression with progressing tumor grade. Taken together, these findings suggest a key signal transduction pathway involving hypoxia-inducible factor-1alpha that contributes to a differentiation defect in malignant gliomas and sheds new light on the differentiation therapy of solid tumors by targeting hypoxia-inducible factor-1alpha.

  4. Hypoxia-Inducible Factor-1α Causes Renal Cyst Expansion through Calcium-Activated Chloride Secretion

    PubMed Central

    Schley, Gunnar; Faria, Diana; Kroening, Sven; Willam, Carsten; Schreiber, Rainer; Klanke, Bernd; Burzlaff, Nicolai; Jantsch, Jonathan; Kunzelmann, Karl; Eckardt, Kai-Uwe

    2014-01-01

    Polycystic kidney diseases are characterized by numerous bilateral renal cysts that continuously enlarge and, through compression of intact nephrons, lead to a decline in kidney function over time. We previously showed that cyst enlargement is accompanied by regional hypoxia, which results in the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) in the cyst epithelium. Here we demonstrate a correlation between cyst size and the expression of the HIF-1α–target gene, glucose transporter 1, and report that HIF-1α promotes renal cyst growth in two in vitro cyst models—principal-like MDCK cells (plMDCKs) within a collagen matrix and cultured embryonic mouse kidneys stimulated with forskolin. In both models, augmenting HIF-1α levels with the prolyl hydroxylase inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate enhanced cyst growth. In addition, inhibition of HIF-1α degradation through tubule-specific knockdown of the von Hippel-Lindau tumor suppressor increased cyst size in the embryonic kidney cyst model. In contrast, inhibition of HIF-1α by chetomin and knockdown of HIF-1α both decreased cyst growth in these models. Consistent with previous reports, plMDCK cyst enlargement was driven largely by transepithelial chloride secretion, which consists, in part, of a calcium-activated chloride conductance. plMDCKs deficient for HIF-1α almost completely lacked calcium-activated chloride secretion. We conclude that regional hypoxia in renal cysts contributes to cyst growth, primarily due to HIF-1α–dependent calcium-activated chloride secretion. These findings identify the HIF system as a novel target for inhibition of cyst growth. PMID:24203996

  5. Sodium hydrosulfide prevents hypoxia-induced pulmonary arterial hypertension in broilers.

    PubMed

    Yang, Y; Zhang, B K; Liu, D; Nie, W; Yuan, J M; Wang, Z; Guo, Y M

    2012-01-01

    1. The aim of the study was to determine if H(2)S is involved in the development of hypoxia-induced pulmonary hypertension in broilers, a condition frequently observed in a variety of cardiac and pulmonary diseases. 2. Two-week-old broilers were reared under normoxic conditions or exposed to normobaric hypoxia (6 h/day) with tissue levels of H(2)S adjusted by administering sodium hydrosulfide (NaHS, 10 µmol/kg body weight/day). Mean pulmonary arterial pressure, right ventricular mass, plasma and tissue H(2)S levels, the expression of cystathionine-β-synthase (CSE) and vascular remodeling were determined at 35 d of age. 3. Exposure to hypoxia-induced pulmonary arterial hypertension was characterized by elevated pulmonary pressure, right ventricular hypertrophy and vascular remodeling. This was accompanied by decreased expression of CSE and decreased concentrations of plasma and tissue H(2)S. 4. Hypoxia-induced pulmonary hypertension was significantly reduced by administration of NaHS but this protective effect was largely abolished by D, L-propargylglycerine, an inhibitor of CSE. 5. The results indicate that H(2)S is involved in the development of hypoxia-induced pulmonary hypertension. Supplementing NaHS or H(2)S could be a strategy for reducing hypoxia-induced hypertension in broilers.

  6. Hypoxia-induced force increase (HIFI) is a novel mechanism underlying the strengthening of labor contractions, produced by hypoxic stresses.

    PubMed

    Alotaibi, Mohammed; Arrowsmith, Sarah; Wray, Susan

    2015-08-04

    For successful birth, contractions need to become progressively stronger. The underlying mechanisms are unknown, however. We have found that a novel mechanism, hypoxia-induced force increase (HIFI), is switched on selectively, at term, and is essential to strengthening contractions. HIFI is initiated as contractions cyclically reduce blood flow and produce repeated hypoxic stresses, with associated metabolic and transcriptomic changes. The increases in contractility are a long-lasting, oxytocin-independent, intrinsic mechanism present only in the full-term pregnant uterus. HIFI is inhibited by adenosine receptor antagonism and blockade of cyclooxygenase-2 signaling, and partially reproduced by brief episodes of acidic (but not alkalotic) pH. HIFI explains how labor can progress despite paradoxical metabolic challenge, and provides a new mechanistic target for the 1 in 10 women suffering dysfunctional labor because of poor contractions.

  7. Effect of chemical stabilizers of hypoxia-inducible factors on early lung development.

    PubMed

    Groenman, Freek A; Rutter, Martin; Wang, Jinxia; Caniggia, Isabella; Tibboel, Dick; Post, Martin

    2007-09-01

    Low oxygen stimulates pulmonary vascular development and airway branching and involves hypoxia-inducible factor (HIF). HIF is stable and initiates expression of angiogenic factors under hypoxia, whereas normoxia triggers hydroxylation of the HIF-1alpha subunit by prolyl hydroxylases (PHDs) and subsequent degradation. Herein, we investigated whether chemical stabilization of HIF-1alpha under normoxic (20% O(2)) conditions would stimulate vascular growth and branching morphogenesis in early lung explants. Tie2-LacZ (endothelial LacZ marker) mice were used for visualization of the vasculature. Embryonic day 11.5 (E11.5) lung buds were dissected and cultured in 20% O(2) in the absence or presence of cobalt chloride (CoCl(2), a hypoxia mimetic), dimethyloxalylglycine (DMOG; a nonspecific inhibitor of PHDs), or desferrioxamine (DFO; an iron chelator). Vascularization was assessed by X-gal staining, and terminal buds were counted. The fine vascular network surrounding the developing lung buds seen in control explants disappeared in CoCl(2)- and DFO-treated explants. Also, epithelial branching was reduced in the explants treated with CoCl(2) and DFO. In contrast, DMOG inhibited branching but stimulated vascularization. Both DFO and DMOG increased nuclear HIF-1alpha protein levels, whereas CoCl(2) had no effect. Since HIF-1alpha induces VEGF expression, the effect of SU-5416, a potent VEGF receptor (VEGFR) blocker, on early lung development was also investigated. Inhibition of VEGFR2 signaling in explants maintained under hypoxic (2% O(2)) conditions completely abolished vascularization and slightly decreased epithelial branching. Taken together, the data suggest that DMOG stabilization of HIF-1alpha during early development leads to a hypervascular lung and that airway branching proceeds without the vasculature, albeit at a slower rate.

  8. Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes.

    PubMed

    Jeon, Yu Jin; Kim, Hyoung Seok; Song, Kyung-Sik; Han, Ho Jae; Park, Soo Hyun; Chang, Woochul; Lee, Min Young

    2015-04-01

    Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2-induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.

  9. MicroRNA-322 protects hypoxia-induced apoptosis in cardiomyocytes via BDNF gene

    PubMed Central

    Yang, Liguo; Song, Shigang; Lv, Hang

    2016-01-01

    Background: Cardiomyocytes apoptosis under hypoxia condition contributes significantly to various cardiovascular diseases. In this study, we investigated the role of microRNA-322 (miR-322) in regulating hypoxia-induced apoptosis in neonatal murine cardiomyocytes in vitro. Method: Cardiomyocytes of C57BL/6J mice were treated with hypoxia condition in vitro. Cardiomyocyte apoptosis was measured by TUNEL assay. Gene expression pattern of miR-322 was measured by qRT-PCR. Stable downregulation of miR-322 in cardiomyocytes were achieved by lentiviral transduction, and the effect of miR-322 downregulation on hypoxia-induced cardiomyocyte apoptosis was investigated. Possible regulation of miR-322 on its downstream target gene, brain derived neurotrophic factor (BDNF) was investigated in cardiomyocytes. BDNF was then genetically silenced by siRNA to evaluate its role in miR-137 mediated cardiomyocyte apoptosis protection under hypoxia condition. Results: Under hypoxia condition, significant apoptosis was induced and miR-322 was significantly upregulated in cardiomyocytes in vitro. Through lentiviral transduction, miR-322 was efficiently knocked down in cardiomyocytes. Downregulation of miR-322 protected hypoxia-induced cardiomyocyte apoptosis. Luciferase assay showed BDNF was the target gene of miR-322. QRT-PCR showed BDNF expression was associated with miR-322 regulation on hypoxia-induced cardiomyocyte apoptosis. Silencing BDNF in cardiomyocyte through siRNA transfection reversed the protective effect of miR-322 downregulation on hypoxia-induced apoptosis. Conclusion: Our study revealed that miR-322, in association with BDNF, played important role in regulating hypoxia-induced apoptosis in cardiomyocyte. PMID:27398164

  10. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

    SciTech Connect

    Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.; Hales, Charles A. . E-mail: chales@partners.org

    2006-07-14

    The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.

  11. Ketosis may promote brain macroautophagy by activating Sirt1 and hypoxia-inducible factor-1.

    PubMed

    McCarty, Mark F; DiNicolantonio, James J; O'Keefe, James H

    2015-11-01

    Ketogenic diets are markedly neuroprotective, but the basis of this effect is still poorly understood. Recent studies demonstrate that ketone bodies increase neuronal levels of hypoxia-inducible factor-1α (HIF-1α), possibly owing to succinate-mediated inhibition of prolyl hydroxylase activity. Moreover, there is reason to suspect that ketones can activate Sirt1 in neurons, in part by increasing cytoplasmic and nuclear levels of Sirt1's obligate cofactor NAD(+). Another recent study has observed reduced activity of mTORC1 in the hippocampus of rats fed a ketogenic diet - an effect plausibly attributable to Sirt1 activation. Increased activities of HIF-1 and Sirt1, and a decrease in mTORC1 activity, could be expected to collaborate in the induction of neuronal macroautophagy. Considerable evidence points to moderate up-regulation of neuronal autophagy as a rational strategy for prevention of neurodegenerative disorders; elimination of damaged mitochondria that overproduce superoxide, as well as clearance of protein aggregates that mediate neurodegeneration, presumably contribute to this protection. Hence, autophagy may mediate some of the neuroprotective benefits of ketogenic diets. Brain-permeable agents which activate AMP-activated kinase, such as metformin and berberine, as well as the Sirt1 activator nicotinamide riboside, can also boost neuronal autophagy, and may have potential for amplifying the impact of ketogenesis on this process. Since it might not be practical for most people to adhere to ketogenic diets continuously, alternative strategies are needed to harness the brain-protective potential of ketone bodies. These may include ingestion of medium-chain triglycerides or coconut oil, intermittent ketogenic dieting, and possibly the use of supplements that promote hepatic ketogenesis - notably carnitine and hydroxycitrate - in conjunction with dietary regimens characterized by long daily episodes of fasting or carbohydrate avoidance.

  12. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

    PubMed Central

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-01-01

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed. PMID:19660100

  13. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression.

    PubMed

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-08-06

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

  14. Copper is required for cobalt-induced transcriptional activity of hypoxia-inducible factor-1.

    PubMed

    Qiu, Liying; Ding, Xueqin; Zhang, Zhen; Kang, Y James

    2012-08-01

    Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1α (HIF-1α) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl(2)) at concentrations above 25 μM for 2 h resulted in an accumulation of HIF-1α, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 μM did not significantly affect the accumulation of HIF-1α but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 μM copper sulfate (CuSO(4)). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1α accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1α in the cells.

  15. NOTCH SIGNALLING MODULATES HYPOXIA-INDUCED NEUROENDOCRINE DIFFERENTIATION OF HUMAN PROSTATE CANCER CELLS

    PubMed Central

    Danza, Giovanna; Di Serio, Claudia; Rosati, Fabiana; Lonetto, Giuseppe; Sturli, Niccolò; Kacer, Doreen; Pennella, Antonio; Ventimiglia, Giuseppina; Barucci, Riccardo; Piscazzi, Annamaria; Prudovsky, Igor; Landriscina, Matteo; Marchionni, Niccolò; Tarantini, Francesca

    2012-01-01

    Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation has been associated with tumor progression, poor prognosis and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavourable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells, in vitro. Results exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent down regulation of Notch-mediated signalling, as demonstrated by reduced levels of the Notch target genes, Hes1 and Hey1. Neuroendocrine differentiation was promoted by attenuation of Hes1 transcription, as cells expressing a dominant negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia down regulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen independent cell lines, PC3 and Du145, it did not change the extent of NE differentiation in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions hypoxia induces neuroendocrine differentiation of LNCaP cells in vitro, which appears to be driven by the inhibition of Notch signalling with subsequent down-regulation of Hes1 transcription. PMID:22172337

  16. Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension

    SciTech Connect

    Satwiko, Muhammad Gahan; Ikeda, Koji; Nakayama, Kazuhiko; Yagi, Keiko; Hocher, Berthold; Hirata, Ken-ichi; Emoto, Noriaki

    2015-09-25

    Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH. - Highlights: • Role of endothelin-1 in pulmonary arterial hypertension (PAH) was investigated. • The endothelin-1 transgenic (ETTG) and wild type (WT) mice were analyzed. • ETTG mice spontaneously developed PAH under normoxia conditions. • SU5416 further aggravated PAH in ETTG mice. • Enhanced endothelin system activity could be a causative factor in

  17. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    PubMed

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  18. Hypoxia-inducible factor-1alpha signaling in aquaporin upregulation after traumatic brain injury.

    PubMed

    Ding, Jamie Y; Kreipke, Christian W; Speirs, Susan L; Schafer, Patrick; Schafer, Steven; Rafols, José A

    2009-03-27

    Previous studies have demonstrated that traumatic brain injury (TBI) causes brain edema via aquaporins (AQPs), the water-transporting proteins. In the present study, we determined the role of hypoxia inducible factor-1alpha (HIF-1alpha), which is a transcription factor in response to physiological hypoxia, in regulating expression of AQP4 and AQP9. Adult male Sprague-Dawley rats (400-425g) received a closed head injury using the Marmarou weight drop model with a 450g weight and survived for 1, 4, 24 and 48h. Some animals were administered 30min after injury with 2-methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol which is known to post-transcriptionally down-regulate HIF-1alpha expression, and sacrificed 4h after injury. Real-time PCR and Western blot were used, respectively, to detect gene and protein expressions of manganese superoxide dismutase (MnSOD, showing hypoxic stress), HIF-1alpha, AQP4, and AQP9. ANOVA analysis demonstrated a significant (p<0.05) increase in gene expression of MnSOD, HIF-1alpha, AQP4, and AQP9, starting at 1h after injury through 48h. Western blot analysis further indicated a significant (p<0.05) increase in protein expression of these molecules at the same time points. Pharmacological inhibition of HIF-1alpha by 2ME2 reduced the up-regulated levels of AQP4 and AQP9 after TBI. The present study suggests that hypoxic conditions determined by MnSOD expression after closed head injury contribute to HIF-1alpha expression. HIF-1alpha, in turn, up-regulates expression of AQP4 and AQP9. These results characterize the pathophysiological mechanisms, and suggest possible therapeutic targets for TBI patients.

  19. Hypoxia-Inducible Factor-1α Signaling in Aquaporin Upregulation After Traumatic Brain Injury

    PubMed Central

    Ding, Jamie Y.; Kreipke, Christian W.; Speirs, Susan L.; Schafer, Patrick; Schafer, Steven; Rafols, José A.

    2009-01-01

    Previous studies have demonstrated that traumatic brain injury (TBI) causes brain edema via aquaporins (AQPs), the water transporting proteins. In the present study, we determined the role of hypoxia inducible factor-1α (HIF-1α), which is a transcription factor in response to physiological hypoxia, in regulating expression of AQP4 and AQP9. Adult male Sprague-Dawley rats (400–425g) received a closed head injury using the Marmarou weight drop model with a 450 g weight and survived for 1, 4, 24 and 48 hours. Some animals were administered 30 minutes after injury with 2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol which is known to post-transcriptionally down-regulate HIF-1α expression, and sacrificed 4 hours after injury. Real-time PCR and Western blot were used, respectively, to detect gene and protein expressions of manganese superoxide dismutase (MnSOD, showing hypoxic stress), HIF-1α, AQP4, and AQP9. ANOVA analysis demonstrated a significant (p<0.05) increase in gene expression of MnSOD, HIF-1α, AQP4, and AQP9, starting at 1 hour after injury through 48 hours. Western blot analysis further indicated a significant (p<0.05) increase in protein expression of these molecules at the same time points. Pharmacological inhibition of HIF-1α by 2ME2 reduced the up-regulated levels of AQP4 and AQP9 after TBI. The present study suggests that hypoxic conditions determined by MnSOD expression after closed head injury contribute to HIF-1α expression. HIF-1α, in turn, up-regulates expression of AQP4 and AQP9. These results characterize the pathophysiological mechanisms, and suggest possible therapeutic targets for TBI patients. PMID:19429018

  20. Hypoxia inducible factor 1α promotes survival of mesenchymal stem cells under hypoxia

    PubMed Central

    Lv, Bingke; Li, Feng; Fang, Jie; Xu, Limin; Sun, Chengmei; Han, Jianbang; Hua, Tian; Zhang, Zhongfei; Feng, Zhiming; Jiang, Xiaodan

    2017-01-01

    Mesenchymal stem cells (MSCs) are ideal materials for cell therapy. Research has indicated that hypoxia benefits MSC survival, but little is known about the underlying mechanism. This study aims to uncover potential mechanisms involving hypoxia inducible factor 1α (HIF1A) to explain the promoted MSC survival under hypoxia. MSCs were obtained from Sprague-Dawley rats and cultured under normoxia or hypoxia condition. The overexpression vector or small interfering RNA of Hif1a gene was transfected to MSCs, after which cell viability, apoptosis and expression of HIF1A were analyzed by MTT assay, flow cytometry, qRT-PCR and Western blot. Factors in p53 pathway were detected to reveal the related mechanisms. Results showed that hypoxia elevated MSCs viability and up-regulated HIF1A (P < 0.05) as previously reported. HIF1A overexpression promoted viability (P < 0.01) and suppressed apoptosis (P < 0.001) under normoxia. Correspondingly, HIF1A knockdown inhibited viability (P < 0.05) and promoted apoptosis (P < 0.01) of MSCs under hypoxia. Expression analysis suggested that p53, phosphate-p53 and p21 were repressed by HIF1A overexpression and promoted by HIF1A knockdown, and B-cell CLL/lymphoma 2 (BCL2) expression had the opposite pattern (P < 0.05). These results suggest that HIF1A may improve viability and suppress apoptosis of MSCs, implying the protective effect of HIF1A on MSC survival under hypoxia. The underlying mechanisms may involve the HIF1A-suppressed p53 pathway. This study helps to explain the mechanism of MSC survival under hypoxia, and facilitates the application of MSCs in cell therapy. PMID:28386377

  1. Tumor cycling hypoxia induces chemoresistance in glioblastoma multiforme by upregulating the expression and function of ABCB1

    PubMed Central

    Chou, Chii-Wen; Wang, Chi-Chung; Wu, Chung-Pu; Lin, Yu-Jung; Lee, Yu-Chun; Cheng, Ya-Wen; Hsieh, Chia-Hung

    2012-01-01

    Tumor cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. However, how tumor cycling hypoxia impacts chemotherapy is unclear. In the present study, we explored the impact and the mechanism of cycling hypoxia on tumor microenvironment-mediated chemoresistance. Hoechst 33342 staining and hypoxia-inducible factor–1 (HIF-1) activation labeling together with immunofluorescence imaging and fluorescence-activated cell sorting were used to isolate hypoxic tumor subpopulations from human glioblastoma xenografts. ABCB1 expression, P-glycoprotein function, and chemosensitivity in tumor cells derived from human glioblastoma xenografts or in vitro cycling hypoxic stress-treated glioblastoma cells were determined using Western blot analysis, drug accumulation and efflux assays, and MTT assay, respectively. ABCB1 expression and P-glycoprotein function were upregulated under cycling hypoxia in glioblastoma cells concomitant with decreased responses to doxorubicin and BCNU. However, ABCB1 knockdown inhibited these effects. Moreover, immunofluorescence imaging and flow cytometric analysis for ABCB1, HIF-1 activation, and Hoechst 3342 in glioblastoma revealed highly localized ABCB1 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion in the solid tumor microenvironment. The cycling hypoxic tumor cells derived from glioblastoma xenografts exhibited higher ABCB1 expression, P-glycoprotein function, and chemoresistance, compared with chronic hypoxic and normoxic cells. Tumor-bearing mice that received YC-1, an HIF-1α inhibitor, exhibited suppressed tumor microenvironment-induced ABCB1 induction and enhanced survival rate in BCNU chemotherapy. Cycling hypoxia plays a vital role in tumor microenvironment-mediated chemoresistance through the HIF-1–dependent induction of ABCB1. HIF-1 blockade before and concurrent with chemotherapy could suppress cycling hypoxia-induced chemoresistance. PMID:22946104

  2. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

  3. Hypoxia-induced downregulation of miR-30c promotes epithelial-mesenchymal transition in human renal cell carcinoma.

    PubMed

    Huang, Jiwei; Yao, Xiaoying; Zhang, Jin; Dong, Baijun; Chen, Qi; Xue, Wei; Liu, Dongming; Huang, Yiran

    2013-12-01

    MicroRNAs (miRNAs), which negatively regulate protein expression by binding protein-coding mRNAs, have been integrated into cancer development and progression as either oncogenes or tumor suppressor genes. miR-30c was reported to be downregulated in several types of cancer. However, its role in human renal cell carcinoma (RCC) remains largely unknown. Here, we show that miR-30c is significantly downregulated in human RCC tissues and cell lines. We found that miR-30c downregulation could be induced by hypoxia in RCC cells in a hypoxia-inducible factors (HIFs) dependent manner. Repression of miR-30c through its inhibitor resulted in reduction of E-cadherin production and promotion of epithelial-mesenchymal transition (EMT), while overexpression of miR-30c inhibited EMT in RCC cells. We identified Slug as a direct target of miR-30c in RCC cells. Slug was upregulated in RCC tissues and its expression could be induced by hypoxia, which is consistent with downregulation of miR-30c by hypoxia. Forced overexpression of Slug in 786-O cells reduced E-cadherin production, and promoted EMT as well as cell migration. Moreover, Slug overexpression abrogated the inhibitory role of miR-30c in regulating EMT and cell migration, indicating miR-30c regulates EMT through Slug in RCC cells. Our findings propose a model that hypoxia induces EMT in RCC cells through downregulation of miR-30c, which leads to subsequent increase of Slug expression and repression of E-cadherin production, and suggest a potential application of miR-30c in RCC treatment.

  4. Focused Review: Agmatine in Fermented Foods

    PubMed Central

    Galgano, Fernanda; Caruso, Marisa; Condelli, Nicola; Favati, Fabio

    2012-01-01

    Polyamines (PAs) are ubiquitous substances considered to be bioregulators of numerous cell functions; they take part in cell growth, division, and differentiation. These biogenic amines are also involved in tissue repair and in intracellular signaling; in fact, because of their polycationic character, they interact to a large extent with membrane phospholipids and may play an important role in the regulation of membrane-linked enzymes. The intracellular polyamine content derives from the simultaneous regulation of the synthesis, catabolism, uptake, and elimination of the polyamines; furthermore, PAs are present in all cell types at different concentrations, but the highest levels are found in rapid-turnover tissues. In addition to spermidine, spermine, and putrescine, also agmatine (AGM), deriving from arginine and identified in mammals in the 1990s, is a polyamine and several studies have reported its potentially positive role in the production of secretagogues, and in neuronal, vascular, metabolic, and therapeutic functions. Because of the low arginine decarboxylase (ADC) activity in mammalians, the amounts of AGM found in their tissues can be only minimally ascribed to an endogenous de novo synthesis by ADC, while a substantial quantity of AGM may be of dietary origin. Several food products contain only small amounts of polyamines, while higher concentrations can be found in fermented foods. PAs could also be considered as indicators of freshness in fish and meat products; as these moieties are produced during food storage, it would seem to confirm the main role of microorganisms in their synthesis. In particular, high levels of AGM are present in alcoholic beverages, such as wine, beer, sake, which would seem to confirm the role of yeasts in AGM production. Although many biological functions have been attributed to polyamines, high levels of these compounds in foodstuffs can have toxicological effects; however, no safe level for the intake of polyamines in a

  5. Attenuated effects of Neu2000 on hypoxia-induced synaptic activities in a rat hippocampus.

    PubMed

    Noh, Jihyun; Koh, Young-Hyun; Chung, Jun-Mo

    2014-02-01

    Neu2000 (NEU; 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid), a recently developed derivative of acetylsalicylic acid and sulfasalazine, potently protects against neuronal cell death following ischemic brain injury by antagonizing NMDA receptor-mediated neuronal toxicity and oxidative stress. However, it has yet to be determined whether NEU can attenuate hypoxia-induced impairment of neuronal electrical activity. In this study, we carried out extracellular recordings of hippocampal slices in order to investigate the effects of NEU on the electrical activity of neurons exposed to a hypoxic insult (oxygen and glucose deprivation). NEU prominently suppressed hypoxia-induced impairment of neuronal activity in a concentration-dependent manner. NEU, at a low dose (1 μM), competently depressed the hypoxia-induced convulsive activity in a manner similar to trolox. Furthermore, high concentrations of NEU (50 μM) markedly abolished all hypoxia-mediated impairment of neuronal activity and accelerated the slow recovery of neuronal activity more efficiently than ifenprodil and APV. These results suggest that NEU attenuates hypoxia-induced impairment of neuronal activity more potently than the antioxidant, trolox, and the NMDA receptor antagonists, ifenprodil and APV. We propose that NEU is a striking pharmacological candidate for neuroprotection against hypoxia because of its defensive action on hypoxia-mediated impairment of electrical neurotransmission as well as its neuroprotective action against neuronal cell death induced by exposure to pathological hypoxic conditions.

  6. Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

    PubMed

    Kumar, Sunil; Saragadam, Tejaswani; Punekar, Narayan S

    2015-08-15

    Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

  7. Changes of hypoxia-inducible factor-1 signaling and the effect of cilostazol in chronic cerebral ischemia

    PubMed Central

    Chen, Han; Wei, Aixuan; He, Jinting; Yu, Ming; Mang, Jing; Xu, Zhongxin

    2013-01-01

    Hypoxia-inducible factor-1 and its specific target gene heme oxygenase-1, are involved in acute cerebral ischemia. However, very few studies have examined in detail the changes in the hypoxia-inducible factor-1/heme oxygenase-1 signaling pathway in chronic cerebral ischemia. In this study, a rat model of chronic cerebral ischemia was established by permanent bilateral common carotid artery occlusion, and these rats were treated with intragastric cilostazol (30 mg/kg) for 9 weeks. Morris water maze results showed that cognitive impairment gradually worsened as the cerebral ischemia proceeded. Immunohistochemistry, semi-quantitative PCR and western blot analysis showed that hypoxia-inducible factor-1α and heme oxygenase-1 expression levels increased after chronic cerebral ischemia, with hypoxia-inducible factor-1α expression peaking at 3 weeks and heme oxygenase-1 expression peaking at 6 weeks. These results suggest that the elevated levels of hypoxia-inducible factor-1α may upregulate heme oxygenase-1 expression following chronic cerebral ischemia and that the hypoxia-inducible factor-1/heme oxygenase-1 signaling pathway is involved in the development of cognitive impairment induced by chronic cerebral ischemia. Cilostazol treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, decreased hypoxia-inducible factor-1α and heme oxygenase-1 expression levels, and reduced apoptosis in the frontal cortex. These findings demonstrate that cilostazol can protect against cognitive impairment induced by chronic cerebral ischemic injury through an anti-apoptotic mechanism. PMID:25206477

  8. Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1 alpha.

    PubMed

    Qian, David Z; Kachhap, Sushant K; Collis, Spencer J; Verheul, Henk M W; Carducci, Michael A; Atadja, Peter; Pili, Roberto

    2006-09-01

    Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

  9. In cultured astrocytes, p53 and MDM2 do not alter hypoxia-inducible factor-1alpha function regardless of the presence of DNA damage.

    PubMed

    Rempe, David A; Lelli, Katherine M; Vangeison, Grace; Johnson, Randall S; Federoff, Howard J

    2007-06-01

    A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Several studies describe a binding of p53 to HIF-1alpha in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1alpha. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1alpha plasmid constructs and/or p53 and HIF-1alpha reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1alpha-mediated transactivation of endogenous HIF-1alpha targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1alpha targets that were most dependent on HIF-1alpha function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1alpha-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1alpha target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1alpha target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1alpha targets, regardless of p53 activation, in primary astrocytes.

  10. Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice.

    PubMed

    So, Kanako; Tei, Yuna; Zhao, Meng; Miyake, Takahito; Hiyama, Haruka; Shirakawa, Hisashi; Imai, Satoshi; Mori, Yasuo; Nakagawa, Takayuki; Matsubara, Kazuo; Kaneko, Shuji

    2016-03-17

    Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15-60 min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10 min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H2O2-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80 mmHg) for 30 min. This hypoxia-induced TRPA1 sensitisation to H2O2 was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H2O2-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1.

  11. Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.

    PubMed

    Kuiper, Caroline; Dachs, Gabi U; Currie, Margaret J; Vissers, Margreet C M

    2014-04-01

    Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

  12. NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells

    PubMed Central

    Nakaoka, Hiroki J.; Hara, Toshiro; Yoshino, Seiko; Kanamori, Akane; Matsui, Yusuke; Shimamura, Teppei; Sato, Hiroshi; Murakami, Yoshinori; Seiki, Motoharu; Sakamoto, Takeharu

    2016-01-01

    Unlike most cells, cancer cells activate hypoxia inducible factor-1 (HIF-1) to use glycolysis even at normal oxygen levels, or normoxia. Therefore, HIF-1 is an attractive target in cancer therapy. However, the regulation of HIF-1 during normoxia is not well characterised, although Mint3 was recently found to activate HIF-1 in cancer cells and macrophages by suppressing the HIF-1 inhibitor, factor inhibiting HIF-1 (FIH-1). In this study, we analysed Mint3-binding proteins to investigate the mechanism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified N-terminal EF-hand calcium binding protein 3 (NECAB3) as a novel factor regulating HIF-1 activity via Mint3. NECAB3 bound to the phosphotyrosine-binding domain of Mint3, formed a ternary complex with Mint3 and FIH-1, and co-localised with Mint3 at the Golgi apparatus. Depletion of NECAB3 decreased the expression of HIF-1 target genes and reduced glycolysis in normoxic cancer cells. NECAB3 mutants that binds Mint3 but lacks an intact monooxygenase domain also inhibited HIF-1 activation. Inhibition of NECAB3 in cancer cells by either expressing shRNAs or generating a dominant negative mutant reduced tumourigenicity. Taken together, the data indicate that NECAB3 is a promising new target for cancer therapy. PMID:26948053

  13. Mechanistic studies of Agmatine Deiminase from multiple bacterial species†

    PubMed Central

    Jones, Justin E.; Dreyton, Christina J.; Flick, Heather; Causey, Corey P.; Thompson, Paul R.

    2010-01-01

    One subfamily within the guanidino-group modifying enzymes (GMEs) is the agmatine Deiminases (AgD). These enzymes catalyze the conversion of agmatine (decarboxylated arginine) to N-carbamoyl putrescine and ammonia. In plants, viruses, and bacteria, these enzymes are thought to be involved in energy production, biosynthesis of polyamines, and biofilm formation. In particular, we are interested in the role that this enzyme plays in pathogenic bacteria. Previously we reported the initial kinetic characterization of the agmatine deiminase from Helicobacter pylori and described the synthesis and characterization the two most potent inactivators AgD inactivators. Herein we have expanded our initial efforts to characterize the catalytic mechanisms of AgD from H. pylori as well Streptococcus mutans, and Porphromonas gingivalis. Through the use of pH rate profiles, pKa measurements of the active site cysteine, solvent isotope effects, and solvent viscosity effects, we have determined that the AgDs, like PADs 1 and 4, utilize a reverse protonation mechanism. PMID:20939536

  14. Circulating factors are involved in hypoxia-induced hepcidin suppression.

    PubMed

    Ravasi, Giulia; Pelucchi, Sara; Greni, Federico; Mariani, Raffaella; Giuliano, Andrea; Parati, Gianfranco; Silvestri, Laura; Piperno, Alberto

    2014-12-01

    Hepcidin transcription is strongly down-regulated under hypoxic conditions, however whether hypoxia inhibits hepcidin directly or indirectly is still unknown. We investigated the time course of hypoxia-mediated hepcidin down-regulation in vivo in healthy volunteers exposed to hypobaric hypoxia at high altitude and, based on the hypothesis that circulating factors are implicated in hepcidin inhibition, we analyzed the effect of sera of these volunteers exposed to normoxia and hypoxia on hepcidin expression in Huh-7 cell lines. Hypoxia led to a significant hepcidin down-regulation in vivo that was almost complete within 72h of exposure and followed erythropoietin induction. This delay in hepcidin down-regulation suggests the existence of soluble factor/s regulating hepcidin production. We then stimulated HuH-7 cells with normoxic and hypoxic sera to analyze the effects of sera on hepcidin regulation. Hypoxic sera had a significant inhibitory effect on hepcidin promoter activity assessed by a luciferase assay, although the amount of such decrease was not as relevant as that observed in vivo. Cellular mRNA analysis showed that a number of volunteers' sera inhibited hepcidin expression, concurrently with ID1 inhibition, suggesting that inhibitory factor(s) may act through the SMAD-pathway.

  15. The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis.

    PubMed

    Arndt, Mary Ann; Battaglia, Valentina; Parisi, Eva; Lortie, Mark J; Isome, Masato; Baskerville, Christopher; Pizzo, Donald P; Ientile, Riccardo; Colombatto, Sebastiano; Toninello, Antonio; Satriano, Joseph

    2009-06-01

    Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca(2+)-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca(2+)-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease.

  16. Hypoxia inducible BHLHB2 is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma

    SciTech Connect

    Wang, Weibin; Reiser-Erkan, Carolin; Michalski, Christoph W.; Raggi, Matthias C.; Quan, Liao; Yupei, Zhao; Friess, Helmut; Erkan, Mert; Kleeff, Joerg

    2010-10-22

    cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p = 0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio = 2.348, 95% CI = 1.250-4.411, p = 0.008). Conclusions: Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine.

  17. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  18. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  19. The hypoxia-inducible-factor hydroxylases bring fresh air into hypoxia signalling

    PubMed Central

    Berra, Edurne; Ginouvès, Amandine; Pouysségur, Jacques

    2006-01-01

    Metazoans rapidly respond to changes in oxygen availability by regulating gene expression. The transcription factor hypoxia-inducible-factor (HIF), which controls the expression of several genes, ‘senses' the oxygen concentration indirectly through the hydroxylation of two proline residues that earmarks the HIF-α subunits for proteasomal degradation. We review the expression, regulation and function of the HIF prolyl hydroxylases or prolyl hydroxylases domain proteins, which are genuine oxygen sensors. PMID:16391536

  20. Sildenafil and an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

    PubMed

    Binns-Loveman, Karen M; Kaplowitz, Mark R; Fike, Candice D

    2005-07-01

    Devising therapies that might prevent the onset or progression of pulmonary hypertension in newborns has received little attention. Our major objective was to determine whether sildenafil, a selective phosphodiesterase inhibitor, prevents the development of an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs. Another objective was to determine whether sildenafil causes pulmonary vasodilation without systemic vasodilation in piglets with chronic pulmonary hypertension. Piglets were raised in room air (control, n = 5) or 10-11% O(2) (hypoxic, n = 17) for 3 days. Some piglets (n = 4) received oral sildenafil, 12 mg/kg/day, throughout exposure to hypoxia. All piglets were anesthetized and catheterized, and pulmonary arterial pressure (Ppa), pulmonary wedge pressure (Pw), aortic pressure (Ao), and cardiac output (CO) were measured. Then for some piglets raised in hypoxia for 3 days, a single oral sildenafil dose (3 mg/kg, n = 6) or placebo (n = 5) was given, and hemodynamic measurements were repeated. For piglets raised in hypoxia for 3 days, mean Ppa and calculated PVR were elevated above respective values in control piglets. Mean Ppa and PVR did not differ between piglets that received sildenafil throughout exposure to hypoxia and those that did not. For piglets with chronic hypoxia-induced pulmonary hypertension that received a single oral dose of sildenafil, mean Ppa and PVR decreased, while mean Pw, CO, mean Ao, and systemic vascular resistance remained the same. All hemodynamic measurements were unchanged after placebo. Oral sildenafil did not influence the early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets. However, a single oral dose of sildenafil caused pulmonary vasodilation, without systemic vasodilation, in piglets with chronic hypoxia-induced pulmonary hypertension, which may have therapeutic implications.

  1. Hunterian Lecture. Regulation of hypoxia-inducible factor 1 (HIF-1) by nitric oxide in oral squamous cell carcinoma.

    PubMed Central

    Brennan, P. A.; Quintero, M.

    2005-01-01

    INTRODUCTION: Hypoxia is a common feature of many cancers, contributing to tumour progression as well as potentially compromising radiotherapy and chemotherapy. Hypoxia-inducible factor 1 (HIF-1) is an essential component in changing the transcriptional response of tumours under hypoxia and it targets the transcription of many genes involved in cancer biology. Over-expression of HIF-1 has been associated with increased patient mortality in several cancer types. Regulation of HIF-1 by the signalling molecule nitric oxide (NO) is becoming increasingly recognised. METHOD: Three oral cancer cell lines were used to investigate the effects of NO synthase enzymes (NOS) on HIF-1alpha expression under both normal oxygen and hypoxic conditions. The effect of NOS inhibition was evaluated with the drug L-NMMA. Protein expression was determined with Western blotting. RESULTS & CONCLUSIONS: HIF-1alpha expression occurred following exposure of cells to 3% oxygen for 8 h in all three cell lines, and its expression was found to be dependent on NOS activity, being reduced or inhibited by L-NMMA. Although the mechanism remains to be established, NO appears to play a role in the expression of HIF-1alpha in oral cancer. The possible clinical implications of targeting HIF-1 in cancer are discussed. PMID:15901372

  2. n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems.

    PubMed

    Kimura, Motohide; Takabuchi, Satoshi; Tanaka, Tomoharu; Murata, Miyahiko; Nishi, Kenichiro; Oda, Seiko; Oda, Tomoyuki; Kanai, Michiyuki; Fukuda, Kazuhiko; Kizaka-Kondoh, Shinae; Adachi, Takehiko; Takabayashi, Arimichi; Semenza, Gregg L; Hirota, Kiichi

    2008-04-01

    HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

  3. Hypoxia negatively regulates antimetastatic PEDF in melanoma cells by a hypoxia inducible factor-independent, autophagy dependent mechanism.

    PubMed

    Fernández-Barral, Asunción; Orgaz, José Luis; Gomez, Valentí; del Peso, Luis; Calzada, María José; Jiménez, Benilde

    2012-01-01

    Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.

  4. Isolation of hypoxia-inducible factor 1 (HIF-1) inhibitors from frankincense using a molecularly imprinted polymer.

    PubMed

    Lakka, Achillia; Mylonis, Ilias; Bonanou, Sophia; Simos, George; Tsakalof, Andreas

    2011-10-01

    Hypoxia-Inducible Factor 1 (HIF-1), a transcriptional activator, is highly involved in the pathology of cancer. Inhibition of HIF-1 retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to prepare a Molecularly Imprinted Polymer (MIP), which was subsequently used for the selective isolation of new inhibitors from frankincense, a gum resin used as anticancer remedy in traditional medicine. The frankincense components isolated by Solid Phase Extraction on MIP (MIP-SPE), efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. The selective retention of acetyl 11-ketoboswellic acid (AKBA, one of the main bioactive components of frankincense) by MIP led to the revealing of its inhibitory activity on the HIF-1 signaling pathway. AKBA was selectively retained by SPE on the quercetin imprinted polymer, with an imprinting effect of 8.1 ± 4.6. Overall, this study demonstrates the potential of MIP application in the screening, recognition and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until.

  5. Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

    PubMed Central

    Olmos, Gabriel; DeGregorio-Rocasolano, Nuria; Regalado, M Paz; Gasull, Teresa; Boronat, M Assumpció; Trullas, Ramón; Villarroel, Alvaro; Lerma, Juan; García-Sevilla, Jesús A

    1999-01-01

    This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells.Exposure (30 min) of energy deprived cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine).Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L-glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole] (101)>RX821002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors.Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding.In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV.It is concluded that imidazol(ine) drugs and agmatine are

  6. Glucose-6-Phosphate Isomerase (G6PI) Mediates Hypoxia-Induced Angiogenesis in Rheumatoid Arthritis

    PubMed Central

    Lu, Ying; Yu, Shan-Shan; Zong, Ming; Fan, Sha-Sha; Lu, Tian-Bao; Gong, Ru-Han; Sun, Li-Shan; Fan, Lie-Ying

    2017-01-01

    The higher level of Glucose-6-phosphate isomerase (G6PI) has been found in both synovial tissue and synovial fluid of rheumatoid arthritis (RA) patients, while the function of G6PI in RA remains unclear. Herein we found the enrichment of G6PI in microvascular endothelial cells of synovial tissue in RA patients, where a 3% O2 hypoxia environment has been identified. In order to determine the correlation between the high G6PI level and the low oxygen concentration in RA, a hypoxia condition (~3% O2) in vitro was applied to mimic the RA environment in vivo. Hypoxia promoted cellular proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and induced cell migration and angiogenic tube formation of human dermal microvascular endothelial cells (HDMECs), which were accompanied with the increased expression of G6PI and HIF-1α. Through application of G6PI loss-of-function assays, we confirmed the requirement of G6PI expression for those hypoxia-induced phenotype in RA. In addition, we demonstrated for the first time that G6PI plays key roles in regulating VEGF secretion from RASFs to regulate the hypoxia-induced angiogenesis in RA. Taken together, we demonstrated a novel pathway regulating hypoxia-induced angiogenesis in RA mediated by G6PI. PMID:28067317

  7. HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

    SciTech Connect

    Liu, Junye; Zhang, Jing; Wang, Xiaowu; Li, Yan; Chen, Yongbin; Li, Kangchu; Zhang, Jian; Yao, Libo; Guo, Guozhen

    2010-07-15

    Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

  8. Twist1 Enhances Hypoxia Induced Radioresistance in Cervical Cancer Cells by Promoting Nuclear EGFR Localization

    PubMed Central

    Xiong, Hua; Nie, Xin; Zou, Yanmei; Gong, Chen; Li, Yang; Wu, Hua; Qiu, Hong; Yang, Lin; Zhuang, Liang; Zhang, Peng; Zhang, Jing; Wang, Yihua; Xiong, Huihua

    2017-01-01

    Twist1 is a crucial transcription factor that regulates epithelial mesenchymal transition and involves in metastasis. Recent evidence suggests that Twist1 plays important role in hypoxia-induced radioresistance, but the underlying mechanism remains elusive. Here we investigated the change of Twist1 expression in human cervical squamous cancer cell line SiHa after hypoxia treatment. We also explored the role of Twist1 in radioresistance by manipulating the expression level of Twist1. We observed that hypoxia treatment elevated the expression of Twist1 in SiHa cells. Knockdown of Twist1 with siRNA increased the radiosensitivity of SiHa cells under hypoxia condition, accompanied by reduced levels of nuclear Epidermal Growth Factor Receptor (EGFR) and DNA-dependent protein kinase (DNA-PK). Conversely, overexpression of Twist1 led to increased radioresistance of SiHa cells, which in turn increased nuclear EGFR localization and expression levels of nuclear DNA-PK. Moreover, concomitant high expression of hypoxia-inducible factor-1α (HIF-1α) and Twist1 in primary tumors of cervical cancer patients correlated with the worse prognosis after irradiation treatment. Taken together, these data provide new insights into molecular mechanism underlying hypoxia-induced radioresistance in cervical cancer cells, and suggest that Twist1 is a promising molecular target to improve the efficacy of cancer radiotherapy. PMID:28261334

  9. Hypobaric-hypoxia-induced pulmonary damage in rats ameliorated by antioxidant erdosteine.

    PubMed

    Uzun, Ozge; Balbay, Oner; Comunoğlu, Nil Ustündağ; Yavuz, Ozlem; Nihat Annakkaya, Ali; Güler, Selver; Silan, Coşkun; Erbaş, Mete; Arbak, Peri

    2006-01-01

    Free radical-mediated injury to lung and pulmonary vasculature is an important mechanism in hypoxia-induced lung damage. In this study, we aimed to investigate the potential protective effects of erdosteine as an antioxidant agent on hypobaric hypoxia-induced pulmonary hypertension. Adult male rats were assigned randomly to three groups. The first group of rats was exposed to hypobaric-hypoxia and the second group was treated with erdosteine (20mg/kg, daily) for 2 weeks, during which time they were in a hypoxic chamber. These groups were compared with normoxic controls. All rats were sacrificed after 2 weeks. The hypoxia-induced increase in right ventricle to left ventricle plus septum weight ratio (from 0.20+/-0.01 to 0.26+/-0.01) was reduced significantly in the erdosteine-treated group (0.23+/-0.01). Malondialdehyde levels were elevated (from 0.33+/-0.11 to 0.59+/-0.02) and total antioxidant status was not changed significantly (from 1.77+/-0.42 to 2.61+/-0.23) by hypoxia. In contrast to the hypoxia-exposed group, malondialdehyde levels were significantly decreased in the erdosteine-treated group (0.37+/-0.02). Total antioxidant status (4.03+/-0.22) was significantly higher in erdosteine-treated rats when compared to non-treated rats. Histopathological examination demonstrated that erdosteine prevented inflammation and protected lung parenchyma and pulmonary endothelium of hypoxia-exposed rats.

  10. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  11. A novel hypoxia-induced miR-147a regulates cell proliferation through a positive feedback loop of stabilizing HIF-1α

    PubMed Central

    Wang, Fan; Zhang, Haoxiang; Xu, Naihan; Huang, Nunu; Tian, Caiming; Ye, Anlin; Hu, Guangnan; He, Jie; Zhang, Yaou

    2016-01-01

    ABSTRACT Hypoxia is a general event in solid tumor growth. Therefore, induced cellular responses by hypoxia are important for tumorigenesis and tumor growth. MicroRNAs (miRNAs) have recently emerged as important regulators of hypoxia induced cellular responses. Here we report that miR-147a is a novel and crucial hypoxia induced miRNA. HIF-1α up-regulates the expression of miR-147a, and miR-147a in turn stabilizes and accumulates HIF-1α protein via directly targeting HIF-3α, a dominant negative regulator of HIF-1α. Subsequent studies in xenograft mouse model reveal that miR-147a is capable of inhibiting tumor growth. Collectively, these data demonstrate a positive feedback loop between HIF-1α, miR-147a and HIF-3α, which provide a new insight into the mechanism of miR-147a induced cell proliferation arrest under hypoxia. PMID:27260617

  12. Upstream stimulatory factor 2 and hypoxia-inducible factor 2α (HIF2α) cooperatively activate HIF2 target genes during hypoxia.

    PubMed

    Pawlus, Matthew R; Wang, Liyi; Ware, Katie; Hu, Cheng-Jun

    2012-11-01

    While the functions of hypoxia-inducible factor 1α (HIF1α)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2α/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2α protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2α, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2α knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.

  13. A3 Adenosine Receptors Modulate Hypoxia-Inducible Factor-1α Expression in Human A375 Melanoma Cells

    PubMed Central

    Merighi, Stefania; Benini, Annalisa; Mirandola, Prisco; Gessi, Stefania; Varani, Katia; Leung, Edward; MacLennan, Stephen; Baraldi, Pier Giovanni; Borea, Pier Andrea

    2005-01-01

    Abstract Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% O2), adenosine upregulates HIF-1α protein expression in a dose-dependent and time-dependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2) protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1α and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells. PMID:16242072

  14. Hypoxia induces H19 expression through direct and indirect Hif-1α activity, promoting oncogenic effects in glioblastoma

    PubMed Central

    Wu, Weining; Hu, Qi; Nie, Er; Yu, Tianfu; Wu, Youzhi; Zhi, Tongle; Jiang, Kuan; Shen, Feng; Wang, Yingyi; Zhang, Junxia; You, Yongping

    2017-01-01

    H19 expression is elevated in many human tumors including glioblastomas, suggesting an oncogenic role for the long noncoding RNA; yet the upregulation of H19 in glioblastomas remains unclear. Here we report that hypoxia significantly stimulated H19 expression in glioblastoma cell lines, which was related to hypoxia-inducible factors 1α (Hif-1α). Hif-1α promoted H19 expression in U87 and U251 cells. Meanwhile PTEN is an advantageous factor to affect H19 expression, through attenuating Hif-1α stability. Hif-1α also positively correlates with H19 in human glioblastoma samples depending on PTEN status. ChIP and luciferase reporter assays showed that Hif-1α induced H19 transcription through directly binding to the H19 promoter. Furthermore, Hif-1α upregulated specific protein 1 (SP1) expression in glioblastomas cells in vitro and in vivo, and SP1 also strongly interacted with the H19 promoter to promote H19 expression under hypoxia. We also showed that H19 acts as a molecular sponge that binds miR-181d, relieving inhibition of β-catenin expression. Therefore, H19 participates in hypoxia-driven migration and invasion in glioblastoma cells. In summary, our results uncover the mechanisms that stimulate H19 expression under hypoxia to promote malignant effects in glioblastomas and suggest H19 might be a promising therapeutic target. PMID:28327666

  15. 15-LOX-1 suppression of hypoxia-induced metastatic phenotype and HIF-1α expression in human colon cancer cells.

    PubMed

    Wu, Yuanqing; Mao, Fei; Zuo, Xiangsheng; Moussalli, Micheline J; Elias, Elias; Xu, Weiguo; Shureiqi, Imad

    2014-06-01

    The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis.

  16. Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

    PubMed

    Okumura, Fumihiko; Joo-Okumura, Akiko; Nakatsukasa, Kunio; Kamura, Takumi

    2017-01-01

    Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis.

  17. 15-LOX-1 suppression of hypoxia-induced metastatic phenotype and HIF-1α expression in human colon cancer cells

    PubMed Central

    Wu, Yuanqing; Mao, Fei; Zuo, Xiangsheng; Moussalli, Micheline J; Elias, Elias; Xu, Weiguo; Shureiqi, Imad

    2014-01-01

    The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis. PMID:24634093

  18. Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

    SciTech Connect

    Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa; Lee, Jaewon; Kang, Keon Wook . E-mail: kwkang@chosun.ac.kr

    2007-06-01

    The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.

  19. Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α.

    PubMed

    Zhao, Wei; Chang, Cunjie; Cui, Yangyan; Zhao, Xiaozhi; Yang, Jun; Shen, Lan; Zhou, Ji; Hou, Zhibo; Zhang, Zhen; Ye, Changxiao; Hasenmayer, Donald; Perkins, Robert; Huang, Xiaojing; Yao, Xin; Yu, Like; Huang, Ruimin; Zhang, Dianzheng; Guo, Hongqian; Yan, Jun

    2014-04-18

    Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

  20. Induction of WNT11 by hypoxia and hypoxia-inducible factor-1α regulates cell proliferation, migration and invasion

    PubMed Central

    Mori, Hiroyuki; Yao, Yao; Learman, Brian S.; Kurozumi, Kazuhiko; Ishida, Joji; Ramakrishnan, Sadeesh K.; Overmyer, Katherine A.; Xue, Xiang; Cawthorn, William P.; Reid, Michael A.; Taylor, Matthew; Ning, Xiaomin; Shah, Yatrik M.; MacDougald, Ormond A.

    2016-01-01

    Changes in cellular oxygen tension play important roles in physiological processes including development and pathological processes such as tumor promotion. The cellular adaptations to sustained hypoxia are mediated by hypoxia-inducible factors (HIFs) to regulate downstream target gene expression. With hypoxia, the stabilized HIF-α and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF-β) heterodimer bind to hypoxia response elements (HREs) and regulate expression of target genes. Here, we report that WNT11 is induced by hypoxia in many cell types, and that transcription of WNT11 is regulated primarily by HIF-1α. We observed induced WNT11 expression in the hypoxic area of allograft tumors. In addition, in mice bearing orthotopic malignant gliomas, inhibition with bevacizumab of vascular endothelial growth factor, which is an important stimulus for angiogenesis, increased nuclear HIF-1α and HIF-2α, and expression of WNT11. Gain- and loss-of-function approaches revealed that WNT11 stimulates proliferation, migration and invasion of cancer-derived cells, and increases activity of matrix metalloproteinase (MMP)-2 and 9. Since tumor hypoxia has been proposed to increase tumor aggressiveness, these data suggest WNT11 as a possible target for cancer therapies, especially for tumors treated with antiangiogenic therapy. PMID:26861754

  1. Potential regulation of glioma through the induction of apoptosis signaling via Egl-9 family hypoxia-inducible factor 3

    PubMed Central

    Mao, Ke; You, Chao; Lei, Ding; Zhang, Heng

    2017-01-01

    Glioma is an aggressive form of brain cancer that occurs following the abnormal proliferation of glial cells. Although glioma cannot spread to other organs, the morbidity and mortality rates of the disease are high, even following surgery, radiotherapy and chemotherapy. The function of Egl-9 family hypoxia-inducible factor 3 (Egln3) in cancer is controversial, and it is debated as to whether Egln3 positively or negatively regulates tumors. In the present study, a mouse model of low-grade glioma was successfully established. Through the use of immunohistochemical and western blot analyses, it was demonstrated that Egln3 expression in glioma tissue performed an important role in regulation by amplifying the signals for apoptosis, as determined by an increase in DNA fragments. Furthermore, Egln3 expression was inhibited by the administration of dimethyloxalylglycine, and the downregulated expression of Egln3 had marked effects on the regulation of glioma through apoptosis. The present study therefore provides evidence of an association between Egln3 expression and apoptosis in low-grade glioma. PMID:28356975

  2. Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3

    PubMed Central

    Selvendiran, Karuppaiyah; Bratasz, Anna; Kuppusamy, M. Lakshmi; Tazi, Mia F.; Rivera, Brian K.; Kuppusamy, Periannan

    2010-01-01

    Signal transducer and activator of transcription 3 (STAT3) is activated in a variety of human cancers, including ovarian cancer. The molecular mechanism by which the STAT3 is activated in cancer cells is poorly understood. We observed that human ovarian xenograft tumors (A2780) in mice were severely hypoxic (pO2 ∼ 2 mmHg). We further observed that hypoxic exposure significantly increased the phosphorylation of STAT3 (pSTAT3) at the Tyr705 residue in A2780 cell line. The pSTAT3 (Tyr705) level was highly dependent on cellular oxygenation levels, with a significant increase at <2% O2, and without any change in the pSTAT3 (Ser727) or total STAT3 levels. The pSTAT3 (Tyr705) elevation following hypoxic exposure could be reversed within 12 hr after returning the cells to normoxia. The increased level of pSTAT3 was partly mediated by increased levels of reactive oxygen species generation in the hypoxic cancer cells. Conventional chemotherapeutic drugs cisplatin and taxol were far less effective in eliminating the hypoxic ovarian cancer cells suggesting a role for pSTAT3 in cellular resistance to chemotherapy. Inhibition of STAT3 by AG490 followed by treatment with cisplatin or taxol resulted in a significant increase in apoptosis suggesting that hypoxia-induced STAT3 activation is responsible for chemoresistance. The results have important clinical implications for the treatment of hypoxic ovarian tumors using STAT3-specific inhibitors. PMID:19623660

  3. Identification of the putrescine biosynthetic genes in Pseudomonas aeruginosa and characterization of agmatine deiminase and N-carbamoylputrescine amidohydrolase of the arginine decarboxylase pathway.

    PubMed

    Nakada, Yuji; Itoh, Yoshifumi

    2003-03-01

    Putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC; the speC product) or indirectly from arginine via arginine decarboxylase (ADC; the speA product). The authors identified the speA and speC genes in Pseudomonas aeruginosa PAO1. The activities of the two decarboxylases were similar and each enzyme alone appeared to direct sufficient formation of the polyamine for normal growth. A mutant defective in both speA and speC was a putrescine auxotroph. In this strain, agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were initially identified as the catabolic enzymes of agmatine, biosynthetically convert agmatine to putrescine in the ADC pathway: a double mutant of aguAB and speC was a putrescine auxotroph. AguA was purified as a homodimer of 43 kDa subunits and AguB as a homohexamer of 33 kDa subunits. AguA specifically deiminated agmatine with K(m) and K(cat) values of 0.6 mM and 4.2 s(-1), respectively. AguB was specific to N-carbamoylputrescine and the K(m) and K(cat) values of the enzyme for the substrate were 0.5 mM and 3.3 s(-1), respectively. Whereas AguA has no structural relationship to any known C-N hydrolases, AguB is a protein of the nitrilase family that performs thiol-assisted catalysis. Inhibition by SH reagents and the conserved cysteine residue in AguA and its homologues suggested that this enzyme is also involved in thiol-mediated catalysis.

  4. Preventive mechanisms of agmatine against ischemic acute kidney injury in rats.

    PubMed

    Sugiura, Takahiro; Kobuchi, Shuhei; Tsutsui, Hidenobu; Takaoka, Masanori; Fujii, Toshihide; Hayashi, Kentaro; Matsumura, Yasuo

    2009-01-28

    The excitation of renal sympathetic nervous system plays an important role in the development of ischemic acute kidney injury in rats. Recently, we found that agmatine, an adrenaline alpha(2)/imidazoline I(1)-receptor agonist, has preventive effects on ischemic acute kidney injury by suppressing the enhanced renal sympathetic nerve activity during renal ischemia and by decreasing the renal venous norepinephrine overflow after reperfusion. In the present study, we investigated preventive mechanisms of agmatine against ischemic acute kidney injury in rats. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the contralateral nephrectomy. Pretreatment with efaroxan (30 mumol/kg, i.v.), an alpha(2)/I(1)-receptor antagonist, abolished the suppressive effects of agmatine on the enhanced renal sympathetic nerve activity during renal ischemia and on the elevated norepinephrine overflow after reperfusion, and eliminated the preventing effects of agmatine on the ischemia/reperfusion-induced renal dysfunction and histological damage. On the other hand, pretreatment with yohimbine (6 mumol/kg, i.v.), an alpha(2)-receptor antagonist, eliminated the preventing effects of agmatine on the ischemia/reperfusion-induced renal injury and norepinephrine overflow, without affecting the lowering effect of agmatine on renal sympathetic nerve activity. These results indicate that agmatine prevents the ischemic renal injury by sympathoinhibitory effect probably via I(1) receptors in central nervous system and by suppressing the norepinephrine overflow through alpha(2) or I(1) receptors on sympathetic nerve endings.

  5. Evidences for the agmatine involvement in antidepressant like effect of bupropion in mouse forced swim test.

    PubMed

    Kotagale, Nandkishor R; Tripathi, Sunil J; Aglawe, Manish M; Chopde, Chandrabhan T; Umekar, Milind J; Taksande, Brijesh G

    2013-06-01

    Although bupropion has been widely used in the treatment of depression, the precise mechanism of its therapeutic actions is not fully understood. The present study investigated the role of agmatine in an antidepressant like effect of bupropion in mouse forced swim test. The antidepressant like effect of bupropion was potentiated by pretreatment with agmatine (10-20mg/kg, ip) and by the drugs known to increase endogenous agmatine levels in brain viz., l-arginine (40 μg/mouse, icv), an agmatine biosynthetic precursor, ornithine decarboxylase inhibitor, dl-α-difluoromethyl ornithine hydrochloride, DFMO (12.5 μg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 μg/mouse, icv) and agmatinase inhibitor, arcaine (50 μg/mouse, icv) as well as imidazoline I1 receptor agonists, moxonidine (0.25mg/kg, ip) and clonidine (0.015 mg/kg, ip) and imidazoline I2 receptor agonist, 2-(2-benzofuranyl)-2-imidazoline hydrochloride, 2-BFI (5mg/kg, ip). Conversely, prior administration of I1 receptor antagonist, efaroxan (1mg/kg, ip) and I2 receptor antagonist, idazoxan (0.25mg/kg, ip) blocked the antidepressant like effect of bupropion and its synergistic combination with agmatine. These results demonstrate involvement of agmatine in the antidepressant like effect of bupropion and suggest agmatine and imidazoline receptors as a potential therapeutic target for the treatment of depressive disorders.

  6. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine

    PubMed Central

    del Rio, Beatriz; Redruello, Begoña; Martin, M. Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P.; Ladero, Victor; Alvarez, Miguel A.

    2015-01-01

    The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1], [2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR) via glucose, but not by other sugars such as lactose or galactose [1], [3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1], [3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17) as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under Accession no. GSE74808. PMID:26981381

  7. Sex-related effects of agmatine on caffeine-induced locomotor activity in Swiss Webster mice.

    PubMed

    Uzbay, Tayfun; Kose, Akin; Kayir, Hakan; Ulusoy, Gokhan; Celik, Turgay

    2010-03-25

    In mammalian brain, agmatine is an endogenous amine that is synthesized through the decarboxylation of l-arginine by arginine decarboxylase. It has been proposed as a new neurotransmitter and/or neuromodulator. It was shown that agmatine had some beneficial effects in animal models of opioid and alcohol addiction. Locomotor stimulant properties of drugs such as ethanol, caffeine, nicotine and amphetamine have been linked to their addictive properties. The present study investigates the effects of agmatine on caffeine-induced locomotor activity both in male and female mice. Adult Swiss Webster mice were used in the study. Locomotor activity was measured for 30min immediately following caffeine (2.5, 5, 10 and 20mg/kg, i.p.) or saline treatments. Agmatine (5, 10 and 20mg/kg, i.p.) were injected 20min before caffeine (2.5 and 5mg/kg, i.p.) administration. In both sexes, agmatine (5-20mg/kg) were also tested for ability to depress or stimulate locomotor activity in the absence of caffeine. Caffeine (5mg/kg) induced a significant increase in locomotor activity of both male and female mice. There was no significant difference in the locomotor-activating effects of caffeine between male and female mice. Agmatine blocked the caffeine (5mg/kg)-induced locomotor stimulation dose dependently in male but not female mice. Agmatine had not any effect on the lower dose (2.5mg/kg) of caffeine in both sexes. These results suggest that agmatine has sex-related inhibitory effects on caffeine-induced locomotor activity in Swiss Webster mice, and male mice are more sensitive than the females to the effect of agmatine.

  8. Structure and stability of complexes of agmatine with some functional receptor residues of proteins

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2017-04-01

    The paper reports the results of a theoretical study of the conformational behavior and basicity of biogenic amine agmatine. The complexes modelling of agmatine - protein interaction are also under scrutiny of our investigation using the Becke3LYP and B97D levels of the density functional theory. The relative stabilities (Gibbs energies) of individual complexes are by both DFT methods described equally. Hydration has a dramatic effect on the hydrogen bonded complexes studied. The pairing acidic carboxylate group with different agmatine species resulted in charged hydrogen bond complexes containing negatively charged acetate species acting as proton acceptors.

  9. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

    PubMed

    Wang, Pengzhen; Zhang, Fengjie; He, Qiling; Wang, Jianqi; Shiu, Hoi Ting; Shu, Yinglan; Tsang, Wing Pui; Liang, Shuang; Zhao, Kai; Wan, Chao

    2016-01-01

    Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes

  10. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

    PubMed Central

    He, Qiling; Wang, Jianqi; Shiu, Hoi Ting; Shu, Yinglan; Tsang, Wing Pui; Liang, Shuang; Zhao, Kai; Wan, Chao

    2016-01-01

    Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10−6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes

  11. C-reactive protein augments hypoxia-induced apoptosis through mitochondrion-dependent pathway in cardiac myocytes.

    PubMed

    Yang, Jin; Wang, Junhong; Zhu, Shushu; Chen, Xiangjian; Wu, Hengfang; Yang, Di; Zhang, Jinan

    2008-03-01

    C-reactive protein (CRP) is an important predictive factor for cardiac disorders including acute myocardial infarction. Therapeutic inhibition of CRP has been shown to be a promising new approach to cardioprotection in acute myocardial infarction in rat models, but the direct effects of CRP on cardiac myocytes are poorly defined. In this study, we investigated the effects of CRP on cardiac myocytes and its molecular mechanism involved. Neonatal rat cardiac myocytes were exposed to hypoxia for 8 h. Hypoxia induced myocyte apoptosis under serum-deprived conditions, which was accompanied by cytochrome c release from mitochondria into cytosol, as well as activation of Caspase-9, Caspase-3. Hypoxia also increased Bax and decreased Bcl-2 mRNA and protein expression, thereby significantly increasing Bax/Bcl-2 ratio. Cotreatment of CRP (100 mug/ml) under hypoxia significantly increased the percentage of apoptotic myocytes, translocation of cytochrome c, Bax/Bcl-2 ratio, and the activity of Caspase-9 and Caspase-3. However, no effects were observed on myocyte apoptosis when cotreatment of CRP under normoxia. Furthermore, Bcl-2 overexpression significantly improved cellular viability through inhibition of hypoxia or cotreatment with CRP induced Bax/Bcl-2 ratio changes and cytochrome c release from mitochondria to cytosol, and significantly blocked the activity of Caspase-9 and Caspase-3. The present study demonstrates that CRP could enhance apoptosis in hypoxia-stimulated myocytes through the mitochondrion-dependent pathway but CRP alone has no effects on neonatal rat cardiac myocytes under normoxia. Bcl-2 overexpression might prevent CRP-induced apoptosis by inhibiting cytochrome c release from the mitochondria and block activation of Caspase-9 and Caspase-3.

  12. Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice

    PubMed Central

    So, Kanako; Tei, Yuna; Zhao, Meng; Miyake, Takahito; Hiyama, Haruka; Shirakawa, Hisashi; Imai, Satoshi; Mori, Yasuo; Nakagawa, Takayuki; Matsubara, Kazuo; Kaneko, Shuji

    2016-01-01

    Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15–60 min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10 min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H2O2-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80 mmHg) for 30 min. This hypoxia-induced TRPA1 sensitisation to H2O2 was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H2O2-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1. PMID:26983498

  13. Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1α

    PubMed Central

    Chintala, Sreenivasulu; Tóth, Károly; Cao, Shousong; Durrani, Farukh A.; Vaughan, Mary M.; Jensen, Randy L.; Rustum, Youcef M.

    2010-01-01

    Purpose Hypoxic tumor cells overexpressing hypoxia-inducible factor 1alpha (HIF-1α) are generally resistant to chemo/radiotherapy. We have reported that Se-methylselenocysteine (MSC) therapeutically enhances the efficacy and selectivity of irinotecan against human tumor xenografts. The aim of this study was to delineate the mechanism responsible for the observed efficacy targeting on HIF-1α and its transcriptionally regulated genes VEGF and CAIX. Methods We investigated the mechanism of HIF-1α inhibition by MSC and its critical role in the therapeutic outcome by generating HIF-1α stable knockdown (KD) human head and neck squamous cell carcinoma, FaDu by transfecting HIF-1α short hairpin RNA. Results While cytotoxic efficacy in combination with methylselenic acid (MSA) with SN-38 (active metabolites of MSC and irinotecan) could not be confirmed in vitro against normoxic tumor cells, the hypoxic tumor cells were more sensitive to the combination. Reduction in HIF-1α either by MSA or shRNA knockdown resulted in significant increase in cytotoxicity of SN38 in vitro against hypoxic, but not the normoxic tumor cells. Similarly, in vivo, either MSC in combination with irinotecan treatment of parental xenografts or HIF-1α KD tumors treated with irinotecan alone resulted in comparable therapeutic response and increase in the long-term survival of mice bearing FaDu xenografts. Conclusions Our results show that HIF-1α is a critical target for MSC and its inhibition was associated with enhanced antitumor activity of irinotecan. Inhibition of HIF-1α appeared to be mediated through stabilization of PHD2, 3 and downregulation of ROS by MSC. Thus, our findings support the development of MSC as a HIF-1α inhibitor in combination chemotherapy. PMID:20066420

  14. Interactions between Nitric Oxide and Hypoxia-Inducible Factor Signaling Pathways in Inflammatory Disease

    PubMed Central

    Olson, Nels; van der Vliet, Albert

    2011-01-01

    Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O2) consumption and redistribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders. PMID

  15. Differential sensitivity of oxidative and glycolytic muscles to hypoxia-induced muscle atrophy.

    PubMed

    de Theije, C C; Langen, R C J; Lamers, W H; Gosker, H R; Schols, A M W J; Köhler, S E

    2015-01-15

    Hypoxia as a consequence of acute and chronic respiratory disease has been associated with muscle atrophy. This study investigated the sensitivity of oxidative and glycolytic muscles to hypoxia-induced muscle atrophy. Male mice were exposed to 8% normobaric oxygen for up to 21 days. Oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles were isolated, weighed, and assayed for expression profiles of the ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and glucocorticoid receptor (GR) and hypoxia-inducible factor-1α (HIF1α) signaling. Fiber-type composition and the capillary network were investigated. Hypoxia-induced muscle atrophy was more prominent in the EDL than the soleus muscle. Although increased expression of HIF1α target genes showed that both muscle types sensed hypoxia, their adaptive responses differed. Atrophy consistently involved a hypoxia-specific effect (i.e., not attributable to a hypoxia-mediated reduction of food intake) in the EDL only. Hypoxia-specific activation of the UPS and ALP and increased expression of the glucocorticoid receptor (Gr) and its target genes were also mainly observed in the EDL. In the soleus, stimulation of gene expression of those pathways could be mimicked to a large extent by food restriction alone. Hypoxia increased the number of capillary contacts per fiber cross-sectional area in both muscles. In the EDL, this was due to type II fiber atrophy, whereas in the soleus the absolute number of capillary contacts increased. These responses represent two distinct modes to improve oxygen supply to muscle fibers, but may aggravate muscle atrophy in chronic obstructive pulmonary disease patients who have a predominance of type II fibers.

  16. The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

    PubMed

    Novo, Erica; Povero, Davide; Busletta, Chiara; Paternostro, Claudia; di Bonzo, Lorenzo Valfrè; Cannito, Stefania; Compagnone, Alessandra; Bandino, Andrea; Marra, Fabio; Colombatto, Sebastiano; David, Ezio; Pinzani, Massimo; Parola, Maurizio

    2012-03-01

    Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

  17. Hypoxic preconditioning with cobalt attenuates hypobaric hypoxia-induced oxidative damage in rat lungs.

    PubMed

    Shukla, Dhananjay; Saxena, Saurabh; Jayamurthy, Purushotman; Sairam, Mustoori; Singh, Mrinalini; Jain, Swatantra Kumar; Bansal, Anju; Ilavazaghan, Govindaswamy

    2009-01-01

    Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

  18. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling

    PubMed Central

    Rey, Sergio; Semenza, Gregg L.

    2010-01-01

    The vascular system delivers oxygen and nutrients to every cell in the vertebrate organism. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic/ischaemic vascular responses, driving transcriptional activation of hundreds of genes involved in vascular reactivity, angiogenesis, arteriogenesis, and the mobilization and homing of bone marrow-derived angiogenic cells. This review will focus on the pivotal role of HIF-1 in vascular homeostasis, the involvement of HIF-1 in vascular diseases, and recent advances in targeting HIF-1 for therapy in preclinical models. PMID:20164116

  19. Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells.

    PubMed

    Eguchi, Ryoji; Toné, Shigenobu; Suzuki, Akio; Fujimori, Yoshihiro; Nakano, Takashi; Kaji, Kazuhiko; Ohta, Toshiro

    2009-01-15

    We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.

  20. Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

    SciTech Connect

    Jeon, You-Kyoung; Park, Sae-Gwang; Choi, Il-Whan; Lee, Soo-Woong; Lee, Sang Min

    2015-04-03

    Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

  1. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain

    PubMed Central

    Chan, Mun Chiang; Atasoylu, Onur; Hodson, Emma; Tumber, Anthony; Leung, Ivanhoe K. H.; Chowdhury, Rasheduzzaman; Gómez-Pérez, Verónica; Demetriades, Marina; Rydzik, Anna M.; Holt-Martyn, James; Tian, Ya-Min; Bishop, Tammie; Claridge, Timothy D. W.; Kawamura, Akane; Pugh, Christopher W.; Ratcliffe, Peter J.; Schofield, Christopher J.

    2015-01-01

    As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke. PMID:26147748

  2. Hypoxia-inducible factor-1α contributes to brain edema after stroke by regulating aquaporins and glycerol distribution in brain.

    PubMed

    Higashida, Tetsuhiro; Peng, Changya; Li, Jie; Dornbos, David; Teng, Kailing; Li, Xiaohua; Kinni, Harish; Guthikonda, Murali; Ding, Yuchuan

    2011-02-01

    Brain edema following stroke is a critical clinical problem due to its association with increased morbidity and mortality. Despite its significance, present treatment for brain edema simply provides symptomatic relief due to the fact that molecular mechanisms underlying brain edema remain poorly understood. The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α) and aquaporins (AQP-4 and -9) in regulating cerebral glycerol accumulation and inducing brain edema in a rodent model of stroke. Two-hours of middle cerebral artery occlusion (MCAO) followed by reperfusion was performed in male Sprague-Dawley rats (250-280 g). Anti-AQP-4 antibody, anti-AQP-9 antibody, or 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α) was given at the time of MCAO. The rats were sacrificed at 1 and 24 hours after reperfusion and their brains were examined. Extracellular and intracellular glycerol concentration of brain tissue was calculated with an enzymatic glycerol assay. The protein expressions of HIF-1α, AQP-4 and AQP-9 were determined by Western blotting. Brain edema was measured by brain water content. Compared to control, edema (p < 0.01), increased glycerol (p < 0.05), and enhanced expressions of HIF-1α, AQP-4, and AQP-9 (p < 0.05) were observed after stroke. With inhibition of AQP-4, AQP-9 or HIF-1α, edema and extracellular glycerol were significantly (p < 0.01) decreased while intracellular glycerol was increased (p < 0.01) 1 hour after stroke. Inhibition of HIF-1α with 2ME2 suppressed (p < 0.01) the expression of AQP-4 and AQP-9. These findings suggest that HIF-1α serves as an upstream regulator of cerebral glycerol concentrations and brain edema via a molecular pathway involving AQP-4 and AQP-9. Pharmacological blockade of this pathway in stroke patients may provide novel therapeutic strategies.

  3. Synapse loss regulated by matrix metalloproteinases in traumatic brain injury is associated with hypoxia inducible factor-1alpha expression.

    PubMed

    Ding, Jamie Y; Kreipke, Christian W; Schafer, Patrick; Schafer, Steven; Speirs, Susan L; Rafols, José A

    2009-05-01

    The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor up-regulated during hypoxia, in the regulation of MMP-2 and -9 expression post-TBI. Adult male Sprague-Dawley rats (n=6 per group, 400 g-425 g) were injured using Marmarou's closed-head acceleration impact model and allowed to survive for 1, 4, 24 and 48 h. In another set of experiments, 30 min after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1alpha) and sacrificed at 4 h after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1alpha, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 h through 48 h after TBI. A significant increase in gene and protein expressions of HIF-1alpha, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1alpha significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1alpha reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1alpha in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals.

  4. Synapse Loss Regulated by Matrix Metalloproteinases in Traumatic Brain Injury Is Associated with Hypoxia-Inducible Factor-1α Expression

    PubMed Central

    Ding, Jamie Y.; Kreipke, Christian W.; Schafer, Patrick; Schafer, Steven; Speirs, Susan L.; Rafols, José A.

    2009-01-01

    The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1α (HIF-1α a transcription factor upregulated during hypoxia, in the regulation of MMP-2 and -9 expression post TBI. Adult male Sprague-Dawley rats (n=6 per group, 400g-425g) were injured using Marmarou's closed head acceleration impact model and allowed to survive for 1, 4, 24 and 48 hours. In another set of experiments, 30 minutes after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1α) and sacrificed at 4 hours after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1α, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 hour through 48 hours after TBI. A significant increase in gene and protein expressions of HIF-1α, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1α significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1α reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1α in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals. PMID:19285046

  5. Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

    PubMed

    Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

    2017-03-15

    The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca(2+). Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

  6. Hypoxia-induced vasodilation and effects of regional phentolamine in awake patients with sleep apnea.

    PubMed

    Moradkhan, Raman; Spitnale, Brett; McQuillan, Patrick; Hogeman, Cynthia; Gray, Kristen S; Leuenberger, Urs A

    2010-05-01

    Obstructive sleep apnea (OSA) is associated with increased sympathetic nerve activity, endothelial dysfunction, and premature cardiovascular disease. To determine whether hypoxia is associated with impaired skeletal muscle vasodilation, we compared femoral artery blood flow (ultrasound) and muscle sympathetic nerve activity (peroneal microneurography) during exposure to acute systemic hypoxia (fraction of inspired oxygen 0.1) in awake patients with OSA (n=10) and controls (n=8). To assess the role of elevated sympathetic nerve activity, in a separate group of patients with OSA (n=10) and controls (n=10) we measured brachial artery blood flow during hypoxia before and after regional alpha-adrenergic block with phentolamine. Despite elevated sympathetic activity, in OSA the vascular responses to hypoxia in the leg did not differ significantly from those in controls [P=not significant (NS)]. Following regional phentolamine, in both groups the hypoxia-induced increase in brachial blood flow was markedly enhanced (OSA pre vs. post, 84+/-13 vs. 201+/-34 ml/min, P<0.002; controls pre vs. post 62+/-8 vs. 140+/-26 ml/min, P<0.01). At end hypoxia after phentolamine, the increase of brachial blood flow above baseline was similar (OSA vs. controls +61+/-16 vs. +48+/-6%; P=NS). We conclude that despite high sympathetic vasoconstrictor tone and prominent sympathetic responses to acute hypoxia, hypoxia-induced limb vasodilation is preserved in OSA.

  7. p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

    PubMed Central

    Feng, Xi; Liu, Xing; Zhang, Wei; Xiao, Wuhan

    2011-01-01

    Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stoke. PMID:21792176

  8. Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Mediated Induction of Glycogen Synthase 1

    PubMed Central

    Pescador, Nuria; Garcia-Rocha, Mar; Ortiz-Barahona, Amaya; Vazquez, Silvia; Ordoñez, Angel; Cuevas, Yolanda; Saez-Morales, David; Garcia-Bermejo, Maria Laura; Landazuri, Manuel O.; Guinovart, Joan; del Peso, Luis

    2010-01-01

    When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia. PMID:20300197

  9. The glial cell response is an essential component of hypoxia-induced erythropoiesis in mice

    PubMed Central

    Weidemann, Alexander; Kerdiles, Yann M.; Knaup, Karl X.; Rafie, Christopher A.; Boutin, Adam T.; Stockmann, Christian; Takeda, Norihiko; Scadeng, Miriam; Shih, Andy Y.; Haase, Volker H.; Simon, M. Celeste; Kleinfeld, David; Johnson, Randall S.

    2009-01-01

    A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1α and HIF-2α and their negative regulator von Hippel–Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis. PMID:19809162

  10. Perspective in chronic kidney disease: targeting hypoxia-inducible factor (HIF) as potential therapeutic approach.

    PubMed

    Deshmukh, Aaishwarya B; Patel, Jayvadan K; Prajapati, Ashish R; Shah, Shreya

    2012-01-01

    Tissue hypoxia is a pathologic feature of many human diseases like cancer, myocardial infarction, stroke, and kidney disease. Convincing data from clinical studies in patients with chronic renal failure point to chronic hypoxia of kidneys as the end result of multiple processes and mechanisms. In acute as well as chronic diseases, tissue hypoxia not only implies a risk of energy deprivation but also induces regulatory mechanisms with profound influence on gene expression. Moreover, once established, accumulating evidence points to this chronic hypoxia as the central player along with final common pathway to end-stage renal disease (ESRD). An evolutionarily preserved oxygen-sensing mechanism enables cells to adapt and maintain homeostasis under hypoxic conditions by transcriptional activation of a host of genes mediating metabolic adaptation, angiogenesis, energy conservation, erythropoiesis, in addition to cell survival. The endogenous oxygen-sensing mechanism incorporates hypoxia-inducible factors (HIFs) that hub cellular response to hypoxia and comprises a family of oxygen-sensitive basic helix-loop-helix proteins that control the cellular transcriptional response to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is thus a significant mediator of physiological responses to acute and chronic hypoxia. Since HIF is activated to suboptimal levels in pathogenic renal states, therapeutic activation holds a promising novel and effective approach to the treatment of ESRD. Current insights into the regulation of HIF may augment the understanding of the role of hypoxia in renal failure progression and may unbolt new options to improve hypoxia tolerance and induce nephroprotection.

  11. Hypoxia and hypoxia-inducible factors (HIFs): master regulators of metastasis

    PubMed Central

    Lu, Xin; Kang, Yibin

    2010-01-01

    Hypoxia is a common condition found in a wide range of solid tumors and is often associated with poor prognosis. Hypoxia increases tumor glycolysis, angiogenesis and other survival response as well as invasion and metastasis by activating relevant gene expressions through hypoxia-inducible factors (HIFs). HIF-1α and HIF-2α undergo oxygen-dependent regulation and their overexpression is frequently associated with metastasis and poor clinical outcomes. Recent studies show that each step of the metastasis process, from the initial epithelial-mesenchymal transition to the ultimate organotropic colonization, can potentially be regulated by hypoxia, suggesting a master regulator role of hypoxia and HIFs in metastasis. Furthermore, modulation of cancer stem cell self-renewal by HIFs may also contribute to the hypoxia-regulated metastasis program. Hypoxia-induced metastatic phenotype may be one of the reasons for the modest efficacy of antiangiogenic therapies and may well explain the recent provocative findings that antiangiogenic therapy increased metastasis in preclinical models. Multiple approaches to targeting hypoxia and HIFs, including HIF inhibitors, hypoxia-activated bioreductive prodrugs and gene therapies may become effective treatments to prevent or reduce metastasis. PMID:20962028

  12. Neuroprotective effect of cobalt chloride on hypobaric hypoxia-induced oxidative stress.

    PubMed

    Shrivastava, Kalpana; Shukla, Dhananjay; Bansal, Anju; Sairam, Mustoori; Banerjee, P K; Ilavazhagan, Govindaswamy

    2008-02-01

    Hypobaric hypoxia, characteristic of high altitude is known to increase the formation of reactive oxygen and nitrogen species (RONS), and decrease effectiveness of antioxidant enzymes. RONS are involved and may even play a causative role in high altitude related ailments. Brain is highly susceptible to hypoxic stress and is involved in physiological responses that follow. Exposure of rats to hypobaric hypoxia (7619 m) resulted in increased oxidation of lipids and proteins due to increased RONS and decreased reduced to oxidized glutathione (GSH/GSSG) ratio. Further, there was a significant increase in superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) levels. Increase in heme oxygenase 1 (HO-1) and heat shock protein 70 (HSP70) was also noticed along with metallothionein (MT) II and III. Administration of cobalt appreciably attenuated the RONS generation, oxidation of lipids and proteins and maintained GSH/GSSH ratio similar to that of control cells via induction of HO-1 and MT offering efficient neuroprotection. It can be concluded that cobalt reduces hypoxia oxidative stress by maintaining higher cellular HO-1 and MT levels via hypoxia inducible factor 1alpha (HIF-1alpha) signaling mechanisms. These findings provide a basis for possible use of cobalt for prevention of hypoxia-induced oxidative stress.

  13. Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

    PubMed

    Kast, Brigitte; Schori, Christian; Grimm, Christian

    2016-05-01

    Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors.

  14. Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives

    PubMed Central

    Bishop, Tammie; Ratcliffe, Peter J

    2014-01-01

    By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. PMID:27774477

  15. Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia

    PubMed Central

    Ladroue, Charline; Hoogewijs, David; Gad, Sophie; Carcenac, Romain; Storti, Federica; Barrois, Michel; Gimenez-Roqueplo, Anne-Paule; Leporrier, Michel; Casadevall, Nicole; Hermine, Olivier; Kiladjian, Jean-Jacques; Baruchel, André; Fakhoury, Fadi; Bressac-de Paillerets, Brigitte; Feunteun, Jean; Mazure, Nathalie; Pouysségur, Jacques; Wenger, Roland H.; Richard, Stéphane; Gardie, Betty

    2012-01-01

    Background Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma. Design and Methods Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively. Results This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category. Conclusions As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for

  16. Spatial learning results in elevated agmatine levels in the rat brain.

    PubMed

    Liu, Ping; Collie, Nicola D; Chary, Sree; Jing, Yu; Zhang, Hu

    2008-01-01

    Accumulating evidence suggests that agmatine, a metabolite of L-arginine by arginine decarboxylase, is a novel neurotransmitter, and exogenous agmatine can modulate behavior functions including learning and memory. However, direct evidence of its involvement in learning and memory processes is currently lacking. This study measured agmatine levels in the hippocampus, parahippocampal region, cerebellum, and vestibular nucleus in rats that were trained to find a hidden escape platform in the water-maze task, or forced to swim in the pool with no platform presented, or kept in the holding-box, using liquid chromatography/mass spectrometry. Compared with the swimming only group and holding-box group, agmatine levels were significantly increased in the CA1 and dentate gyrus subregions of the hippocampus, the entorhinal cortex and the vestibular nucleus in the water-maze training group. These results, for the first time, demonstrate spatial learning-induced region-specific elevation in agmatine, and raise a novel issue of the involvement of agmatine in the processes of learning and memory.

  17. Lack of mitochondrial DNA impairs chemical hypoxia-induced autophagy in liver tumor cells through ROS-AMPK-ULK1 signaling dysregulation independently of HIF-1α.

    PubMed

    Marin, Jose J G; Lozano, Elisa; Perez, Maria J

    2016-12-01

    Alterations in mitochondrial DNA (mtDNA) and autophagy activation are common events in tumors. Here we have investigated the effect of mitochondrial genome depletion on chemical hypoxia-induced autophagy in liver tumor cells. Human SK-Hep-1 wild-type and mtDNA-depleted (Rho) cells were exposed to the hypoxia mimetic agents CoCl2 and deferoxamine (DFO). Up-regulation of HIF-1α, but not HIF-2α was observed. The expression of several HIF-1α target genes was also found. In human SK-Hep-1 and mouse Hepa 1-6 liver tumor cells, but not in the counterpart Rho derived lines, chemical hypoxia increased the abundance of autophagosomes and autolysosomes. In wild-type and Rho cells, chemical hypoxia induced down-regulation of HIF-1α-dependent autophagy inhibitors Bcl-2 and mTOR, whereas activation of AMPK/ULK1-mediated pro-autophagy pathway occurred only in wild-type cells. Chemical (compound C) and genetic (shRNA) inhibition of AMPK activation resulted in reduced autophagy. ATP levels were similar in both cell types, whereas constitutive and chemical hypoxia-induced reactive oxygen species (ROS) generation was lower in Rho cells. In wild-type cells, the antioxidant N-acetylcysteine blocked CoCl2- and DFO-induced AMPK and autophagy activation, but not endoplasmic reticulum stress induced by CoCl2. Enhanced Bax-α/Bcl-2 ratio and cell death was induced by hypoxia mimetic agents more markedly in wild-type than in Rho cells. Upon blocking autophagy activation with 3-methyladenine, DFO-induced cell death was partially prevented whereas that induced by CoCl2 was increased, but only in wild-type cells. These results suggest that mitochondrial dysfunction associated with the lack of mtDNA impairs the signaling pathways mediated by ROS, controlling autophagy activation in liver tumor cells, which may contributes to cancer development.

  18. Apigenin ameliorates hypertension-induced cardiac hypertrophy and down-regulates cardiac hypoxia inducible factor-lα in rats.

    PubMed

    Zhu, Zeng-Yan; Gao, Tian; Huang, Yan; Xue, Jie; Xie, Mei-Lin

    2016-04-01

    Apigenin is a natural flavonoid compound that can inhibit hypoxia-inducible factor (HIF)-1α expression in cultured tumor cells under hypoxic conditions. Hypertension-induced cardiac hypertrophy is always accompanied by abnormal myocardial glucolipid metabolism due to an increase of HIF-1α. However, whether or not apigenin may ameliorate the cardiac hypertrophy and abnormal myocardial glucolipid metabolism remains unknown. This study aimed to examine the effects of apigenin. Rats with cardiac hypertrophy induced by renovascular hypertension were treated with apigenin 50-100 mg kg(-1) (the doses can be achieved by pharmacological or dietary supplementation for an adult person) by gavage for 4 weeks. The results showed that after treatment with apigenin, the blood pressure, heart weight, heart weight index, cardiomyocyte cross-sectional area, serum angiotensin II, and serum and myocardial free fatty acids were reduced. It is important to note that apigenin decreased the expression level of myocardial HIF-1α protein. Moreover, apigenin simultaneously increased the expression levels of myocardial peroxisome proliferator-activated receptor (PPAR) α, carnitine palmitoyltransferase (CPT)-1, and pyruvate dehydrogenase kinase (PDK)-4 proteins and decreased the expression levels of myocardial PPARγ, glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter (GLUT)-4 proteins. These findings demonstrated that apigenin could improve hypertensive cardiac hypertrophy and abnormal myocardial glucolipid metabolism in rats, and its mechanisms might be associated with the down-regulation of myocardial HIF-1α expression and, subsequently increasing the expressions of myocardial PPARα and its target genes CPT-1 and PDK-4, and decreasing the expressions of myocardial PPARγ and its target genes GPAT and GLUT-4.

  19. Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

    PubMed Central

    Asghar, Muhammad Yasir; Bergelin, Nina; Jaakkola, Panu; Törnquist, Kid

    2013-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1. PMID:23824493

  20. TCDD Induces the Hypoxia-Inducible Factor (HIF)-1α Regulatory Pathway in Human Trophoblastic JAR Cells

    PubMed Central

    Liao, Tien-Ling; Chen, Su-Chee; Tzeng, Chii-Reuy; Kao, Shu-Huei

    2014-01-01

    The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α) stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS) and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or N-acetylcysteine (a ROS scavenger). The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development. PMID:25272228

  1. Hypoxia-inducible Factor 1α Regulates a SOCS3-STAT3-Adiponectin Signal Transduction Pathway in Adipocytes*

    PubMed Central

    Jiang, Changtao; Kim, Jung-Hwan; Li, Fei; Qu, Aijuan; Gavrilova, Oksana; Shah, Yatrik M.; Gonzalez, Frank J.

    2013-01-01

    Obesity has been identified as a major risk factor for type 2 diabetes, characterized by insulin resistance in insulin target tissues. Hypoxia-inducible factor 1α (HIF1α) regulates pathways in energy metabolism that become dysregulated in obesity. Earlier studies revealed that HIF1α in adipose tissue is markedly elevated in high-fat diet-fed mice that are obese and insulin-resistant. Genetic ablation of HIF1α in adipose tissue decreased insulin resistance and obesity, accompanied by increased serum adiponectin levels. However, the exact mechanism whereby HIF1α regulates adiponectin remains unclear. Here, acriflavine (ACF), an inhibitor of HIF1α, induced the expression of adiponectin and reduced the expression of SOCS3 in cultured 3T3-L1 adipocytes. Mechanistic studies revealed that HIF1α suppressed the expression of adiponectin through a SOCS3-STAT3 pathway. Socs3 was identified as a novel HIF1α target gene based on chromatin immunoprecipitation and luciferase assays. STAT3 directly regulated adiponectin in vitro in cultured 3T3-L1 adipocytes. ACF was found to prevent diet-induced obesity and insulin resistance. In vivo, ACF also regulated the SOCS3-STAT3-adiponectin pathway, and inhibition of HIF1α in adipose tissue was essential for ACF to improve the SOCS3-STAT3-adiponectin pathway to counteract insulin resistance. This study provides evidence for a novel target gene and signal transduction pathway in adipocytes and indicates that inhibitors of HIF1α have potential utility for the treatment of obesity and type 2 diabetes. PMID:23255598

  2. STARS knockout attenuates hypoxia-induced pulmonary arterial hypertension by suppressing pulmonary arterial smooth muscle cell proliferation.

    PubMed

    Shi, Zhaoling; Wu, Huajie; Luo, Jianfeng; Sun, Xin

    2017-03-01

    STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein, which expressed early in cardiac development and involved in pathological remodeling. Abundant evidence indicated that STARS could regulate cell proliferation, but it's exact function remains unclear. In this study, we aimed to investigate the role of STARS in the proliferation of pulmonary arterial smooth muscle cells (PASMC) and the potential effect on the progression of pulmonary arterial hypertension (PAH). In this study, we established a PAH mouse model through chronic hypoxia exposure as reflected by the increased RVSP and RVHI. Western blot and RT-qPCR detected the increased STARS protein and mRNA levels in PAH mice. Next, we cultured the primary PASMC from PAH mice. After STARS overexpression in PASMC, STARS, SRF and Egr-1 were up-regulated significantly. The MTT assay revealed an increase in cell proliferation. Flow cytometry showed a marked inhibition of cell apoptosis. However, STARS silence in PASMC exerted opposite effects with STARS overexpression. SRF siRNA transfection blocked the effects of STARS overexpression in PASMC. In order to further confirm the role of STARS in PAH mice in vivo, we exposed STARS knockout mice to hypoxia and found lower RVSP and RVHI in knockout mice as compared with controls. Our results not only suggest that STARS plays a crucial role in the development of PAH by increasing the proliferation of PASMC through activation of the SRF/Egr-1 pathway, but also provides a new mechanism for hypoxia-induced PAH. In addition, STARS may represent a potential treatment target.

  3. Hypoxia-induced miR-181b enhances angiogenesis of retinoblastoma cells by targeting PDCD10 and GATA6.

    PubMed

    Xu, Xiaofang; Ge, Shengfang; Jia, Renbing; Zhou, Yixiong; Song, Xin; Zhang, He; Fan, Xianqun

    2015-06-01

    Previous findings showed that miR-181b is upregulated under hypoxic conditions in retinoblastoma cells. Since hypoxia is a common feature of retinoblastoma that affects tumor progression as well as tumor therapy, in the present study, we investigated the regulatory mechanism of miR-181b under hypoxic conditions, and examined the role of miR-181b in retinoblastoma responses to hypoxia (chemoresistance and angiogenesis) and possible downstream genes. The level of hypoxia-inducible factor-1α (HIF-1α) and miR-181b was detected to examine the link between them. Tube formation and cell cytotoxicity assays were used to clarify the effects of miR-181b on hypoxic responses of retinoblastoma cells. Bioinformatics analysis was performed to predict potential targets of miR-181b and western blotting was used to verify these targets. The results showed a significantly increased expression of HIF-1α in hypoxia-treated retinoblastoma cells. Downregulation of HIF-1α using a small-interfering RNA (siRNA) knockdown technology did not decrease the expression of miR-181b. Through gain- and loss-of-function studies, miR-181b was demonstrated to significantly stimulate the ability of capillary tube formation of endothelial cells. Programmed cell death-10 (PDCD10) and GATA binding protein 6 (GATA6) were identified as the target genes of miR‑181b. To the best of our knowledge, results of the present study provide the first evidence that miR-181b was upregulated by hypoxia in retinoblastoma in an HIF-1α-independent manner. miR-181b increased tumor angiogenesis of retinoblastoma cells. Additionally, miR-181b exerts its angiogenic function, at least in part, by inhibiting PDCD10 and GATA6. Thus, it is a new potentially useful therapeutic target for retinoblastoma.

  4. Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms*

    PubMed Central

    Mwaikambo, Bupe R.; Yang, Chun; Chemtob, Sylvain; Hardy, Pierre

    2009-01-01

    Neovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1α down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway. PMID:19640849

  5. Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain.

    PubMed

    Mun, Chin Hee; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2010-09-01

    Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

  6. Comparative survey of putrescine production from agmatine deamination in different bacteria.

    PubMed

    Landete, J M; Arena, M E; Pardo, I; Manca de Nadra, M C; Ferrer, S

    2008-10-01

    This article aims to study putrescine production in Lactobacillus hilgardii strain X(1)B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148(T). The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all microorganisms. In other cases, the effect differed between P. aeruginosa PAO1 and the other microorganisms. Histamine and tyramine poorly influenced the utilization of agmatine, although a small increase in putrescine production was observed in P. aeruginosa PAO1. Succinate, spermidine and spermine also led to an increase in putrescine production in P. aeruginosa PAO1, whereas the succinate had no effect in the other microorganisms. Spermine and spermidine always produced a diminution in agmatine deamination. In this work, we have also demonstrated that pyridoxal 5-phosphate, Mg(2+) and Mn(2+) had no effect on putrescine production from agmatine. Results presented in this paper indicate differences in regulation mechanisms of agmatine deiminase pathway among P. aeruginosa PAO1 and L. hilgardii X(1)B, E. faecalis ATCC11700 and B. cereus CECT 148(T). These results are significant from two points of view, first food quality, and second the toxicological and microbiological aspects. It should be taken into account that putrescine, whose origin is still controversial, is quantitatively the main biogenic amine found in food.

  7. Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

    PubMed Central

    KAMBAYASHI, Satoshi; IGASE, Masaya; KOBAYASHI, Kosuke; KIMURA, Ayana; SHIMOKAWA MIYAMA, Takako; BABA, Kenji; NOGUCHI, Shunsuke; MIZUNO, Takuya; OKUDA, Masaru

    2015-01-01

    Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma. PMID:26050843

  8. Hypoxia-Inducible Factor-1α Target Genes Contribute to Retinal Neuroprotection

    PubMed Central

    Cheng, Lin; Yu, Honghua; Yan, Naihong; Lai, Kunbei; Xiang, Mengqing

    2017-01-01

    Hypoxia-inducible factor (HIF) is a transcription factor that facilitates cellular adaptation to hypoxia and ischemia. Long-standing evidence suggests that one isotype of HIF, HIF-1α, is involved in the pathogenesis of various solid tumors and cardiac diseases. However, the role of HIF-1α in retina remains poorly understood. HIF-1α has been recognized as neuroprotective in cerebral ischemia in the past two decades. Additionally, an increasing number of studies has shown that HIF-1α and its target genes contribute to retinal neuroprotection. This review will focus on recent advances in the studies of HIF-1α and its target genes that contribute to retinal neuroprotection. A thorough understanding of the function of HIF-1α and its target genes may lead to identification of novel therapeutic targets for treating degenerative retinal diseases including glaucoma, age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions. PMID:28289375

  9. Hypoxia-Inducible Factor (HIF) as a Target for Novel Therapies in Rheumatoid Arthritis

    PubMed Central

    Hua, Susan; Dias, Thilani H.

    2016-01-01

    Hypoxia is an important micro-environmental characteristic of rheumatoid arthritis (RA). Hypoxia-inducible factors (HIF) are key transcriptional factors that are highly expressed in RA synovium to regulate the adaptive responses to this hypoxic milieu. Accumulating evidence supports hypoxia and HIFs in regulating a number of important pathophysiological characteristics of RA, including synovial inflammation, angiogenesis, and cartilage destruction. Experimental and clinical data have confirmed the upregulation of both HIF-1α and HIF-2α in RA. This review will focus on the differential expression of HIFs within the synovial joint and its functional behavior in different cell types to regulate RA progression. Potential development of new therapeutic strategies targeting HIF-regulated pathways at sites of disease in RA will also be addressed. PMID:27445820

  10. Current advances in the novel functions of hypoxia-inducible factor and prolyl hydroxylase in invertebrates.

    PubMed

    Wang, L; Cui, S; Ma, L; Kong, L; Geng, X

    2015-12-01

    Oxygen is essential for aerobic life, and hypoxia has very severe consequences. Organisms need to overcome low oxygen levels to maintain biological functions during normal development and in disease states. The mechanism underlying the hypoxic response has been widely investigated in model animals such as Drosophila melanogaster and Caenorhabditis elegans. Hypoxia-inducible factor (HIF), a key gene product in the response to oxygen deprivation, is primarily regulated by prolyl hydroxylase domain enzymes (PHDs). However, recent findings have uncovered novel HIF-independent functions of PHDs. This review provides an overview of how invertebrates are able to sustain hypoxic damages, and highlights some recent discoveries in the regulation of cellular signalling by PHDs. Given that some core genes and major pathways are evolutionarily conserved, these research findings could provide insight into oxygen-sensitive signalling in mammals, and have biomedical implications for human diseases.

  11. Nap Interferes with Hypoxia-Inducible Factors and VEGF Expression in Retina of Diabetic Rats.

    PubMed

    D'Amico, Agata Grazia; Maugeri, Grazia; Bucolo, Claudio; Saccone, Salvatore; Federico, Concetta; Cavallaro, Sebastiano; D'Agata, Velia

    2017-02-01

    The retinal microvascular damage is a complication of diabetic retinopathy (DR). Hyperglycemia and hypoxia are responsible of aberrant vessel's proliferation. The cellular response to hypoxia is mediated through activation of hypoxia-inducible factors (HIFs). Among these, HIF-1α modulates expression of its target gene, VEGF, whose upregulation controls the angiogenic event during DR development. In a previous study, we have demonstrated that a small peptide, NAP, is able to protect retina from hyperglycemic insult. Here, we have demonstrated that its intraocular administration in a rat model of diabetic retinopathy has reduced expression of HIF-1α, HIF-2α, and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization study by confocal microscopy. Although these evidences need to be further deepened to understand the molecular mechanism involved in the protective NAP action, the present data suggest that this small peptide may be effective to prevent the development of this ocular pathology.

  12. Hypoxia induces unique proliferative response in adventitial fibroblasts by activating PDGFβ receptor-JNK1 signalling

    PubMed Central

    Panzhinskiy, Evgeniy; Zawada, W. Michael; Stenmark, Kurt R.; Das, Mita

    2012-01-01

    Aims Pulmonary hypertension (PH) is a devastating condition for which no disease-modifying therapies exist. PH is recognized as proliferative disease of the pulmonary artery (PA). In the experimental newborn calf model of hypoxia-induced PH, adventitial fibroblasts in the PA wall exhibit a heightened replication index. Because elevated platelet-derived growth factor β receptor (PDGFβ-R) signalling is associated with PH, we tested the hypothesis that the activation of PDGFβ-R contributes to fibroblast proliferation and adventitial remodelling in PH. Methods and results Newborn calves were exposed to either ambient air (PB = 640 mmHg) (Neo-C) or high altitude (PB = 445 mm Hg) (Neo-PH) for 2 weeks. PDGFβ-R phosphorylation was markedly elevated in PA adventitia of Neo-PH calves as well as in cultured PA fibroblasts isolated from Neo-PH animals. PDGFβ-R activation with PDGF-BB stimulated higher replication in Neo-PH cells compared with that of control fibroblasts. PDGF-BB-induced proliferation was dependent on reactive oxygen species generation and extracellular signal-regulated kinase1/2 activation in both cell populations; however, only Neo-PH cell division via PDGFβ-R activation displayed a unique dependence on c-Jun N-terminal kinase1 (JNK1) stimulation as the blockade of JNK1 with SP600125, a pharmacological antagonist of the JNK pathway, and JNK1-targeted siRNA selectively blunted Neo-PH cell proliferation. Conclusions Our data strongly suggest that hypoxia-induced modified cells engage the PDGFβ-R-JNK1 axis to confer distinctively heightened proliferation and adventitial remodelling in PH. PMID:22735370

  13. Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

    PubMed Central

    Zou, Dan; Liu, Peng; Chen, Ka; Xie, Qi; Liang, Xinyu; Bai, Qian; Zhou, Qicheng; Liu, Kai; Zhang, Ting; Zhu, Jundong; Mi, Mantian

    2015-01-01

    Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis. PMID:25919288

  14. Absence of the inflammasome adaptor ASC reduces hypoxia-induced pulmonary hypertension in mice.

    PubMed

    Cero, Fadila Telarevic; Hillestad, Vigdis; Sjaastad, Ivar; Yndestad, Arne; Aukrust, Pål; Ranheim, Trine; Lunde, Ida Gjervold; Olsen, Maria Belland; Lien, Egil; Zhang, Lili; Haugstad, Solveig Bjærum; Løberg, Else Marit; Christensen, Geir; Larsen, Karl-Otto; Skjønsberg, Ole Henning

    2015-08-15

    Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1β, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC(-/-), and NLRP3(-/-) mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC(-/-) mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P < 0.001), indicating a substantially reduced pulmonary hypertension in mice lacking ASC. Magnetic resonance imaging further supported these findings by demonstrating reduced right ventricular remodeling. RVSP of NLRP3(-/-) mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1β in WT and NLRP3(-/-) mice, this response was absent in ASC(-/-) mice. Moreover, ASC(-/-) mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension.

  15. Hypoxia inducible factor 1α contributes to regulation of autophagy in retinal detachment.

    PubMed

    Shelby, Shameka J; Angadi, Pavan S; Zheng, Qiong-Duon; Yao, Jingyu; Jia, Lin; Zacks, David N

    2015-08-01

    Photoreceptor (PR) cells receive oxygen and nutritional support from the underlying retinal pigment epithelium (RPE). Retinal detachment results in PR hypoxia and their time-dependent death. Detachment also activates autophagy within the PR, which serves to reduce the rate of PR apoptosis. In this study, we test the hypothesis that autophagy activation in the PR results, at least in part, from the detachment-induced activation of hypoxia-inducible factors (HIF). Retina-RPE separation was created in Brown-Norway rats and C57BL/6J mice by injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested and assayed for HIF protein levels. Cultured 661W photoreceptor cells were subjected to hypoxic conditions and assayed for induction of HIF and autophagy. The requirement of HIF-1α and HIF-2α in regulating photoreceptor autophagy was tested using siRNA in vitro and in vivo. We observed increased levels of HIF-1α and HIF-2α within 1 day post-detachment, as well as increased levels of BNIP3, a downstream target of HIF-1α that contributes to autophagy activation. Exposing 661W cells to hypoxia resulted in increased HIF-1α and HIF-2α levels and increase in conversion of LC3-I to LC3-II. Silencing of HIF-1α, but not HIF-2α, reduced the hypoxia-induced increase in LC3-II formation and increased cell death in 661W cells. Silencing of HIF-1α in rat retinas prevented the detachment-induced increase in BNIP3 and LC3-II, resulting in increased PR cell death. Our data support the hypothesis that HIF-1α, but not HIF-2α, serves as an early response signal to induce autophagy and reduce photoreceptor cell death.

  16. Low sodium intake does not impair renal compensation of hypoxia-induced respiratory alkalosis.

    PubMed

    Höhne, Claudia; Boemke, Willehad; Schleyer, Nora; Francis, Roland C; Krebs, Martin O; Kaczmarczyk, Gabriele

    2002-05-01

    Acute hypoxia causes hyperventilation and respiratory alkalosis, often combined with increased diuresis and sodium, potassium, and bicarbonate excretion. With a low sodium intake, the excretion of the anion bicarbonate may be limited by the lower excretion rate of the cation sodium through activated sodium-retaining mechanisms. This study investigates whether the short-term renal compensation of hypoxia-induced respiratory alkalosis is impaired by a low sodium intake. Nine conscious, tracheotomized dogs were studied twice either on a low-sodium (LS = 0.5 mmol sodium x kg body wt-1 x day-1) or high-sodium (HS = 7.5 mmol sodium x kg body wt-1 x day-1) diet. The dogs breathed spontaneously via a ventilator circuit during the experiments: first hour, normoxia (inspiratory oxygen fraction = 0.21); second to fourth hour, hypoxia (inspiratory oxygen fraction = 0.1). During hypoxia (arterial PO2 34.4 +/- 2.1 Torr), plasma pH increased from 7.37 +/- 0.01 to 7.48 +/- 0.01 (P < 0.05) because of hyperventilation (arterial PCO2 25.6 +/- 2.4 Torr). Urinary pH and urinary bicarbonate excretion increased irrespective of the sodium intake. Sodium excretion increased more during HS than during LS, whereas the increase in potassium excretion was comparable in both groups. Thus the quick onset of bicarbonate excretion within the first hour of hypoxia-induced respiratory alkalosis was not impaired by a low sodium intake. The increased sodium excretion during hypoxia seems to be combined with a decrease in plasma aldosterone and angiotensin II in LS as well as in HS dogs. Other factors, e.g., increased mean arterial blood pressure, minute ventilation, and renal blood flow, may have contributed.

  17. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  18. Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein

    PubMed Central

    Neumann, Paul; Patel, Tarun B.

    2017-01-01

    Receptor Tyrosine Kinase (RTK) signaling plays a major role in tumorigenesis and normal development. Sprouty2 (Spry2) attenuates RTK signaling and inhibits processes such as angiogenesis, cell proliferation, migration and survival, which are all upregulated in tumors. Indeed in cancers of the liver, lung, prostate and breast, Spry2 protein levels are markedly decreased correlating with poor patient prognosis and shorter survival. Thus, it is important to understand how expression of Spry2 is regulated. While prior studies have focused on the post-translation regulation of Spry2, very few studies have focused on the transcriptional regulation of SPRY2 gene. Here, we demonstrate that in the human hepatoma cell line, Hep3B, the transcription of SPRY2 is inhibited by the transcription regulating hypoxia inducible factors (HIFs). HIFs are composed of an oxygen regulated alpha subunit (HIF1α or HIF2α) and a beta subunit (HIF1β). Intriguingly, silencing of HIF1α and HIF2α elevates SPRY2 mRNA and protein levels suggesting HIFs reduce the transcription of the SPRY2 promoter. In silico analysis identified ten hypoxia response elements (HREs) in the proximal promoter and first intron of SPRY2. Using chromatin immunoprecipitation (ChIP), we show that HIF1α/2α bind near the putative HREs in the proximal promoter and intron of SPRY2. Our studies demonstrated that not only is the SPRY2 promoter methylated, but silencing HIF1α/2α reduced the methylation. ChIP assays also showed DNA methyltransferase1 (DNMT1) binding to the proximal promoter and first intron of SPRY2 and silencing HIF1α/2α decreased this association. Additionally, silencing of DNMT1 mimicked the HIF1α/2α silencing-mediated increase in SPRY2 mRNA and protein. While simultaneous silencing of HIF1α/2α and DNMT1 increased SPRY2 mRNA a little more, the increase was not additive suggesting a common mechanism by which DNMT1 and HIF1α/2α regulate SPRY2 transcription. Together these data suggest that the

  19. Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat.

    PubMed Central

    Lortie, M J; Novotny, W F; Peterson, O W; Vallon, V; Malvey, K; Mendonca, M; Satriano, J; Insel, P; Thomson, S C; Blantz, R C

    1996-01-01

    Until recently, conversion of arginine to agmatine by arginine decarboxylase (ADC) was considered important only in plants and bacteria. In the following, we demonstrate ADC activity in the membrane-enriched fraction of brain, liver, and kidney cortex and medulla by radiochemical assay. Diamine oxidase, an enzyme shown here to metabolize agmatine, was localized by immunohistochemistry in kidney glomeruli and other nonrenal cells. Production of labeled agmatine, citrulline, and ornithine from [3H]arginine was demonstrated and endogenous agmatine levels (10(-6)M) in plasma ultrafiltrate and kidney were measured by HPLC. Microperfusion of agmatine into renal interstitium and into the urinary space of surface glomeruli of Wistar-Frömter rats produced reversible increases in nephron filtration rate (SNGFR) and absolute proximal reabsorption (APR). Renal denervation did not alter SNGFR effects but prevented APR changes. Yohimbine (an alpha 2 antagonist) microperfusion into the urinary space produced opposite effects to that of agmatine. Microperfusion of urinary space with BU-224 (microM), a synthetic imidazoline2 (I2) agonist, duplicated agmatine effects on SNGFR but not APR whereas an I1 agonist had no effect. Agmatine effects on SNGFR and APR are not only dissociable but appear to be mediated by different mechanisms. The production and degradation of this biologically active substance derived from arginine constitutes a novel endogenous regulatory system in the kidney. PMID:8567962

  20. Agmatine protects against scopolamine-induced water maze performance impairment and hippocampal ERK and Akt inactivation.

    PubMed

    Moosavi, Maryam; Khales, Golnaz Yadollahi; Abbasi, Leila; Zarifkar, Asadollah; Rastegar, Karim

    2012-04-01

    Cholinergic brain activity plays a significant role in memory. Scopolamine a muscarinic cholinergic antagonist is known to induce impairment in Morris water maze performance, the task which is mainly dependent on the hippocampus. It is suggested that hippocampal ERK and Akt activation play roles in synaptic plasticity and some types of learning and memory. Agmatine, a polyamine derived from l-arginine decarboxylation, is recently shown to exert some neuroprotective effects. This study was aimed to investigate if agmatine could reverse scopolamine-induced memory impairment and possible hippocampal ERK and Akt activity alteration. Adult male Sprague-Dawley rats weighing 200-250 g were randomly assigned into 5 groups. The animals were trained for 3 days in Morris water maze and in day 4 their memory retention was assessed in probe trial which was consisted of a 60 s trial with no platform. Scopolamine (1 mg/kg/ip) or saline were injected 30 min and agmatine (20 or 40 mg/kg/ip) was administered 60 min before each session. The hippocampi were isolated after behavioral studies and western blotting studies on hippocampal lysates were done to determine the levels of activated ERK and Akt. Scopolamine treatment not only impaired water maze learning and memory, but also decreased the amount of phosphorylated (activated) ERK and Akt. Agmatine pre-treatment prevented both the learning impairment and hippocampal ERK and Akt inactivation induced by scopolamine. It seems that agmatine may act as a candidate substance against amnesia.

  1. Protective effect of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride on hypoxia-induced toxicity by suppressing microglial activation in BV-2 cells

    PubMed Central

    Kim, Jiae; Kim, Su-Min; Na, Jung-Min; Hahn, Hoh-Gyu; Cho, Sung-Woo; Yang, Seung-Ju

    2016-01-01

    We recently reported the anti-inflammatory effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on the ATP-induced activation of the NFAT and MAPK pathways through the P2X7 receptor in microglia. To further investigate the underlying mechanism of KHG26792, we studied its protective effects on hypoxia-induced toxicity in microglia. The administration of KHG26792 significantly reduced the hypoxia-induced expression and activity of caspase-3 in BV-2 microglial cells. KHG26792 also reduced hypoxia-induced inducible nitric oxide synthase protein expression, which correlated with reduced nitric oxide accumulation. In addition, KHG26792 attenuated hypoxia-induced protein nitration, reactive oxygen species production, and NADPH oxidase activity. These effects were accompanied by the suppression of hypoxia-induced protein expression of hypoxia-inducible factor 1-alpha and NADPH oxidase-2. Although the clinical relevance of our findings remains to be determined, these data results suggest that KHG26792 prevents hypoxia-induced toxicity by suppressing microglial activation. PMID:27756444

  2. Agmatine, a metabolite of L-arginine, reverses scopolamine-induced learning and memory impairment in rats.

    PubMed

    Utkan, Tijen; Gocmez, Semil S; Regunathan, Soundararajan; Aricioglu, Feyza

    2012-10-01

    Agmatine (l-amino-4-guanidino-butane), a metabolite of L-arginine through the action of arginine decarboxylase, is a novel neurotransmitter. In the present study, effects of agmatine on cognitive functions have been evaluated by using one trial step-down passive avoidance and three panel runway task. Agmatine (20, 40, 80 mg/kg i.p.) was administered either in the presence or absence of a cholinergic antagonist, scopolamine (1 mg/kg i.p.). Scopolamine significantly impaired learning and memory in both passive avoidance and three panel runway test. Agmatine did not affect emotional learning, working and reference memory but significantly improved scopolamine-induced impairment of learning and memory in a dose dependent manner. Our results indicate that agmatine, as an endogenous substance, may have an important role in modulation of learning and memory functions.

  3. Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

    PubMed Central

    Lee, H J; Ryu, J M; Jung, Y H; Lee, K H; Kim, D I; Han, H J

    2016-01-01

    Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation. PMID:27010859

  4. Agmatine, by Improving Neuroplasticity Markers and Inducing Nrf2, Prevents Corticosterone-Induced Depressive-Like Behavior in Mice.

    PubMed

    Freitas, Andiara E; Egea, Javier; Buendia, Izaskun; Gómez-Rangel, Vanessa; Parada, Esther; Navarro, Elisa; Casas, Ana Isabel; Wojnicz, Aneta; Ortiz, José Avendaño; Cuadrado, Antonio; Ruiz-Nuño, Ana; Rodrigues, Ana Lúcia S; Lopez, Manuela G

    2016-07-01

    Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (-/-)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine's ability to produce an antidepressant-like effect was abolished in Nrf2 (-/-) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action.

  5. Chronic nicotine blunts hypoxic sensitivity in perinatal rat adrenal chromaffin cells via upregulation of KATP channels: role of alpha7 nicotinic acetylcholine receptor and hypoxia-inducible factor-2alpha.

    PubMed

    Buttigieg, Josef; Brown, Stephen; Holloway, Alison C; Nurse, Colin A

    2009-06-03

    Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K(ATP) channels via stimulation of alpha7 nicotinic ACh receptors (AChRs). These K(ATP) channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K(+) current, membrane depolarization, rise in cytosolic Ca(2+), and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K(ATP) channels, was present. Both K(ATP) current density and K(ATP) channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), suggesting involvement of the HIF pathway. The therapeutic potential of K(ATP) channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

  6. Basal and therapy-driven hypoxia-inducible factor-1α confers resistance to endocrine therapy in estrogen receptor-positive breast cancer.

    PubMed

    Jia, Xiaoqing; Hong, Qi; Lei, Li; Li, Daqiang; Li, Jianwei; Mo, Miao; Wang, Yujie; Shao, Zhimin; Shen, Zhenzhou; Cheng, Jingyi; Liu, Guangyu

    2015-04-20

    Resistance is an obstacle to endocrine therapy for breast cancer. We measured levels of hypoxia-inducible factor (HIF)-1α in 52 primary breast cancer patients before and after receiving neoadjuvant endocrine therapy with letrozole for at least 3 months. Pre-treatment levels of HIF-1α were associated with negative clinical outcome. Furthermore, levels of HIF-1α were increased in post-treatment residual tumors compared with those in pre-treatment biopsy samples. In animal studies, xenografts stably expressing HIF-1α were resistant to endocrine therapy with fulvestrant compared with the effects in control xenografts. Additionally, HIF-1α transcription was inhibited by zoledronic acid, a conventional drug for the treatment of postmenopausal osteoporosis, and was accompanied by a marked inhibition of the RAS/MAPK/ERK1/2 pathway. HIF-1α is a determinant of resistance to endocrine therapy and should be considered as a potential therapeutic target for overcoming endocrine resistance in estrogen receptor (ER)-positive breast cancer. In addition, zoledronic acid may overcome endocrine resistance in ER-positive human breast cancer by targeting HIF-1α transcription through inhibition of the RAS/MAPK/ERK1/2 pathway. Clinical studies on the administration of zoledronic acid as a second line treatment in patients who failed endocrine therapy should be considered to improve therapeutic outcomes in breast cancer patients.

  7. Hypoxia-Inducible Histone Lysine Demethylases: Impact on the Aging Process and Age-Related Diseases

    PubMed Central

    Salminen, Antero; Kaarniranta, Kai; Kauppinen, Anu

    2016-01-01

    Hypoxia is an environmental stress at high altitude and underground conditions but it is also present in many chronic age-related diseases, where blood flow into tissues is impaired. The oxygen-sensing system stimulates gene expression protecting tissues against hypoxic insults. Hypoxia stabilizes the expression of hypoxia-inducible transcription factor-1α (HIF-1α), which controls the expression of hundreds of survival genes related to e.g. enhanced energy metabolism and autophagy. Moreover, many stress-related signaling mechanisms, such as oxidative stress and energy metabolic disturbances, as well as the signaling cascades via ceramide, mTOR, NF-κB, and TGF-β pathways, can also induce the expression of HIF-1α protein to facilitate cell survival in normoxia. Hypoxia is linked to prominent epigenetic changes in chromatin landscape. Screening studies have indicated that the stabilization of HIF-1α increases the expression of distinct histone lysine demethylases (KDM). HIF-1α stimulates the expression of KDM3A, KDM4B, KDM4C, and KDM6B, which enhance gene transcription by demethylating H3K9 and H3K27 sites (repressive epigenetic marks). In addition, HIF-1α induces the expression of KDM2B and KDM5B, which repress transcription by demethylating H3K4me2,3 sites (activating marks). Hypoxia-inducible KDMs support locally the gene transcription induced by HIF-1α, although they can also control genome-wide chromatin landscape, especially KDMs which demethylate H3K9 and H3K27 sites. These epigenetic marks have important role in the control of heterochromatin segments and 3D folding of chromosomes, as well as the genetic loci regulating cell type commitment, proliferation, and cellular senescence, e.g. the INK4 box. A chronic stimulation of HIF-1α can provoke tissue fibrosis and cellular senescence, which both are increasingly present with aging and age-related diseases. We will review the regulation of HIF-1α-dependent induction of KDMs and clarify their role in

  8. HIF-1α Inhibition Sensitized Pituitary Adenoma Cells to Temozolomide by Regulating Presenilin 1 Expression and Autophagy.

    PubMed

    Kun, Zhang; Yuling, Yang; Dongchun, Wang; Bingbing, Xie; Xiaoli, Li; Bin, Xu

    2016-12-01

    Pituitary adenomas usually develop temozolomide resistance, which could compromise the anticancer effects of temozolomide. Suppression of hypoxia-inducible factor 1α has been shown to sensitize glioblastoma cells to temozolomide treatment according to previous reports. However, whether and how the suppression of hypoxia-inducible factor 1α could sensitize pituitary adenomas to temozolomide treatment are still poorly understood. In the present study, using hypoxia-inducible factor 1α knockdown strategy, we demonstrated for the first time that hypoxia-inducible factor 1α knockdown could inhibit temozolomide-induced autophagy in rat pituitary adenoma GH3 cells and thus increase antitumor efficacy of temozolomide. Furthermore, we found hypoxia-inducible factor 1α knockdown could block autophagy process through neutralizing lysosomal pH value but not inhibiting autophagy induction. Finally, we found hypoxia-inducible factor 1α could regulate lysosomal pH value through regulating full length presenilin 1 expression, and exogenous reexpression of presenilin 1could restore lysosome acidic levels. Our data indicated hypoxia-inducible factor 1α knockdown could be a potential approach to improve the efficacy of temozolomide therapy for pituitary adenomas.

  9. Excess glucose induces hypoxia-inducible factor-1α in pancreatic cancer cells and stimulates glucose metabolism and cell migration

    PubMed Central

    Liu, Zhiwen; Jia, Xiaohui; Duan, Yijie; Xiao, Huijie; Sundqvist, Karl-Gösta; Permert, Johan; Wang, Feng

    2013-01-01

    Pancreatic cancer patients frequently show hyperglycemia, but it is uncertain whether hyperglycemia stimulates pancreatic cancer cells. We have investigated whether excess glucose induces hypoxia-inducible factor-1α (HIF-1α) and stimulates glucose metabolism and cell migration in pancreatic cancer cells. We studied wild-type (wt) MiaPaCa2 pancreatic cancer cells and a MiaPaCa2 subline (namely si-MiaPaCa2) that had HIF-1α-specific small interfering RNA. Wt-MiaPaCa2 cells are known to be HIF-1α-positive in hypoxia and HIF-1α-negative in normoxia, whereas si-MiaPaCa2 cells are devoid of HIF-1α in both normoxia and hypoxia. We incubated these cells with different amounts of glucose and determined HIF-1α mRNA and protein by real-time polymerase chain reaction and western blotting. We determined glucose consumption, lactate production and intracellular hexokinase-II and ATP to assess glucose metabolisms and determined pyruvate dehydrogenase kinase-1, reactive oxygen species and fumarate to assess mitochondrial activities. Further, we studied cell migration using a Boyden chamber. Excess glucose (16.7−22.2mM) increased HIF-1α in hypoxic wt-MiaPaCa2 cells. HIF-1α expression increased ATP contents and inhibited mitochondrial activities. Extracellular glucose and hypoxia stimulated glucose metabolisms independent of HIF-1α. Excess glucose stimulated the migration of wt- and si-MiaPaCa2 cells in both normoxia and hypoxia. Thus, glucose stimulated cell migration independent of HIF-1α. Nevertheless, hypoxic wt-MiaPaCa2 cells showed greater migrating ability than their si-MiaPaCa2 counterparts. We conclude that (1) excess glucose increases HIF-1α and ATP in hypoxic wt-MiaPaCa2 cells, (2) extracellular glucose and hypoxia regulate glucose metabolisms independent of HIF-1α and (3) glucose stimulates cell migration by mechanisms that are both dependent on HIF-1α and independent of it. PMID:23377827

  10. Epstein-Barr virus latent membrane protein 1 induces synthesis of hypoxia-inducible factor 1 alpha.

    PubMed

    Wakisaka, Naohiro; Kondo, Satoru; Yoshizaki, Tomokazu; Murono, Shigeyuki; Furukawa, Mitsuru; Pagano, Joseph S

    2004-06-01

    Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1 alpha and HIF-1 beta that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1 alpha is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1 alpha protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1 alpha by LMP1. Catalase, an H(2)O(2) scavenger, strongly suppresses LMP1-induced production of H(2)O(2), which results in a decrease in the expression of HIF-1 alpha induced by LMP1. Inhibition of the NF-kappa B, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1 alpha expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1 alpha protein expression, which is controlled by p42/p44 MAPK activity and H(2)O(2). The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1 alpha along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.

  11. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    SciTech Connect

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  12. Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

    PubMed

    Bouquerel, P; Gstalder, C; Müller, D; Laurent, J; Brizuela, L; Sabbadini, R A; Malavaud, B; Pyronnet, S; Martineau, Y; Ader, I; Cuvillier, O

    2016-03-14

    The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

  13. NFATc3 Mediates Chronic Hypoxia-induced Pulmonary Arterial Remodeling with α-Actin Up-regulation

    PubMed Central

    de Frutos, S.; Spangler, R.; Alò, D.; González Bosc, L. V.

    2009-01-01

    Physiological responses to chronic hypoxia include polycythemia, pulmonary arterial remodeling and vasoconstriction. Chronic hypoxia causes pulmonary arterial hypertension leading to right ventricular hypertrophy and heart failure. During pulmonary hypertension, pulmonary arteries exhibit increased expression of smooth muscle-α-actin and -myosin heavy chain. NFATc3 (nuclear factor of activated T cells isoform c3), which is a Ca2+-dependent transcription factor, has been recently linked to smooth muscle phenotypic maintenance through the regulation of the expression of α-actin. The aim of this study was to determine if: a) NFATc3 is expressed in murine pulmonary arteries, b) hypoxia induces NFAT activation, c) NFATc3 mediates the up-regulation of α-actin during chronic hypoxia, and d) NFATc3 is involved in chronic hypoxia-induced pulmonary vascular remodeling. NFATc3 transcript and protein were found in pulmonary arteries. NFAT-luciferase reporter mice were exposed to normoxia (630 torr) or hypoxia (380 torr) for 2, 7 or 21 days. Exposure to hypoxia elicited a significant increase in luciferase activity and pulmonary arterial smooth muscle nuclear NFATc3 localization, demonstrating NFAT activation. Hypoxia induced up-regulation of α-actin and was prevented by the calcineurin/NFAT inhibitor, cyclosporin A (25 mg/Kg/day s.c.). In addition, NFATc3 knockout mice did not showed increased α-actin levels and arterial wall thickness after hypoxia. These results strongly suggest that NFATc3 plays a role in the chronic hypoxia-induced vascular changes that underlie pulmonary hypertension. PMID:17403661

  14. Stabilization of hypoxia-inducible factor-1α in buffer containing cobalt chloride for Western blot analysis.

    PubMed

    Srinivasan, Sathyanarayanan; Dunn, Jeff F

    2011-09-01

    Hypoxia-inducible factor-1α (HIF-1α) is a widely studied protein with significant biomedical impact. Care is needed to stabilize HIF-1α protein during sample preparation for Western blot analysis due to its rapid degradation in the presence of oxygen. Enzyme inhibitor cocktails can be complex and expensive. We present a protease inhibitor-free buffer, containing cobalt chloride, which is effective at stabilizing HIF-1α, while being inexpensive, straightforward, and convenient, and has potential for widespread application.

  15. Prediction of Susceptibility to Acute Mountain Sickness Using Hypoxia-Induced Intrapulmonary Arteriovenous Shunt and Intracardiac Shunt Fractions

    DTIC Science & Technology

    2013-10-01

    TITLE: Prediction of Susceptibility to Acute Mountain Sickness Using Hypoxia-Induced Intrapulmonary Arteriovenous Shunt and Intracardiac Shunt...01October2012-30September2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prediction of Susceptibility to Acute Mountain Sickness Using...following organizations have approved our protocol: a) Sacred Heart Medical Center IRB, the review board for Oregon Heart and Vascular Institute; b) Oregon

  16. A Membrane Protease Regulates Energy Production in Macrophages by Activating Hypoxia-inducible Factor-1 via a Non-proteolytic Mechanism*

    PubMed Central

    Sakamoto, Takeharu; Seiki, Motoharu

    2010-01-01

    Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism. PMID:20663879

  17. Long-term divergent tidal flat benthic community recovery following hypoxia-induced mortality.

    PubMed

    Van Colen, C; Montserrat, F; Vincx, M; Herman, P M J; Ysebaert, T; Degraer, S

    2010-02-01

    Macrobenthos recovery after hypoxia-induced mass mortality was assessed in an estuarine tidal mudflat during 3 years. During the first 2 years, a Pearson-Rosenberg type of community recovery took place along with the improving bottom water oxygen conditions. After 3 months, spionid polychaetes became superabundant (i.e. opportunistic peak), followed rapidly by a steep decline (i.e. ecotone point). Subsequently, a moderate increase in species richness and a steep increase in biomass, related to the growth of long-lived species occurred (i.e. transition region). Afterwards, however, the recovering community diverged again from the ambient, undisturbed, sediments due to enhanced recruitment success of long-lived species presumably resulting from the lowered interference from bioturbation during early recovery stages in the disturbed plots. Hence, despite early community recovery may be more or less deterministic, lagged divergent community reassembling may occur at the longer-term, thereby contributing to benthos patchiness in areas which are frequently subjected to disturbances.

  18. Hypoxia inducible factor: a potential prognostic biomarker in oral squamous cell carcinoma.

    PubMed

    Qian, Jiang; Wenguang, Xu; Zhiyong, Wang; Yuntao, Zou; Wei, Han

    2016-08-01

    Oral squamous cell carcinoma (OSCC) is the most common oral cancer. Hypoxia inducible factor (HIF) is involved in many malignant tumors' growth and metastasis and upregulated by hypoxia, including oral cancer. Many studies have studied about the prognostic value of HIF expression in OSCC; however, they do not get the consistent results. Therefore, this study explored the correlation between the HIF expression and the prognosis of OSCC. It conducted a meta-analysis of relevant publications searched in the Web of Science, PubMed, and ISI Web of Knowledge databases. Totally, this study identified 12 relevant articles reporting a total of 1112 patients. This analysis revealed a significant association between increased risk of mortality (RR = 1.20; 95 % CI 0.74-1.95; I (2) 85.4 %) and overexpression of HIFs. Furthermore, different HIF isoforms were associated with overall survival [HIF-1α (RR = 1.18; 95 % CI 0.66-2.11; I (2) 87.2 %) and HIF-2α (RR = 1.40; 95 % CI 0.93-2.09; I(2) 0.0 %)]. These results show that overexpression of HIFs, regardless of whether the HIF-1α or HIF-2α isoforms are overexpressed is significantly associated with increased risk of mortality in OSCC patients. In this study, the funnel is symmetric, suggesting existed no publication bias.

  19. Hypoxia-induced angiogenesis is increased by the controlled release of deferoxiamine from gelatin hydrogels.

    PubMed

    Saito, Takashi; Tabata, Yasuhiko

    2014-08-01

    The objective of this study is to design biodegradable hydrogels for the controlled release of deferoxiamine (DFO) and evaluate their biological activity. When the DFO was added to human umbilical vein endothelial cells cultured in 5.0% O2, the level of hypoxia-inducible factor-1α and vascular endothelial growth factor significantly increased compared with that without DFO. The expression of angiogenesis-related genes was accordingly increased by the DFO addition. An aqueous solution of mixed gelatin and DFO was freeze-dried, and dehydrothermally treated at 140°C for 24h to prepare a gelatin hydrogel incorporating DFO. In the release test with phosphate-buffered saline solution (PBS) at 37°C, an initial DFO release of 60% was observed, followed by no release. When placed in PBS containing collagenase, the hydrogel was enzymatically degraded with time, and consequently released DFO in a degradation-dependent manner. After the hydrogel incorporating DFO was injected intramuscularly into a mouse model of hind limb ischemia, the number of new blood vessels formed was significantly higher than that with free DFO and DFO-free hydrogel. It is concluded that the DFO-containing hydrogel shows promising for inducing angiogenesis locally.

  20. Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α in asthenozoospermia.

    PubMed

    Salvolini, Eleonora; Buldreghini, Eddi; Lucarini, Guendalina; Vignini, Arianna; Giulietti, Alessia; Lenzi, Andrea; Mazzanti, Laura; Di Primio, Roberto; Balercia, Giancarlo

    2014-11-01

    Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.

  1. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

    SciTech Connect

    Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun . E-mail: dli2@slu.edu

    2006-11-01

    Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), {delta}p85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.

  2. Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

    PubMed Central

    Kribben, A; Wieder, E D; Wetzels, J F; Yu, L; Gengaro, P E; Burke, T J; Schrier, R W

    1994-01-01

    The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases. Images PMID:8182125

  3. Hypoxia-inducible factor 1 is required for remote ischemic preconditioning of the heart.

    PubMed

    Cai, Zheqing; Luo, Weibo; Zhan, Huiwang; Semenza, Gregg L

    2013-10-22

    Both preclinical and clinical studies suggest that brief cycles of ischemia and reperfusion in the arm or leg may protect the heart against injury following prolonged coronary artery occlusion and reperfusion, a phenomenon known as remote ischemic preconditioning. Recent studies in mice indicate that increased plasma interleukin-10 (IL-10) levels play an important role in remote ischemic preconditioning induced by clamping the femoral artery for 5 min followed by 5 min of reperfusion for a total of three cycles. In this study, we demonstrate that remote ischemic preconditioning increases plasma IL-10 levels and decreases myocardial infarct size in wild-type mice but not in littermates that are heterozygous for a knockout allele at the locus encoding hypoxia-inducible factor (HIF) 1α. Injection of a recombinant adenovirus encoding a constitutively active form of HIF-1α into mouse hind limb muscle was sufficient to increase plasma IL-10 levels and decrease myocardial infarct size. Exposure of C2C12 mouse myocytes to cyclic hypoxia and reoxygenation rapidly increased levels of IL-10 mRNA, which was blocked by administration of the HIF-1 inhibitor acriflavine or by expression of short hairpin RNA targeting HIF-1α or HIF-1β. Chromatin immunoprecipitation assays demonstrated that binding of HIF-1 to the Il10 gene was induced when myocytes were subjected to cyclic hypoxia and reoxygenation. Taken together, these data indicate that HIF-1 activates Il10 gene transcription and is required for remote ischemic preconditioning.

  4. Hypoxia-inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia.

    PubMed

    Fang, Hsin-Yu; Hughes, Russell; Murdoch, Craig; Coffelt, Seth B; Biswas, Subhra K; Harris, Adrian L; Johnson, Randall S; Imityaz, Hongxia Z; Simon, M Celeste; Fredlund, Erik; Greten, Florian R; Rius, Jordi; Lewis, Claire E

    2009-07-23

    Ischemia exists in many diseased tissues, including arthritic joints, atherosclerotic plaques, and malignant tumors. Macrophages accumulate in these sites and up-regulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18 hours. For example, they were seen to up-regulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, vascular endothelial growth factor A, interleukin (IL)-1beta and IL-8, adrenomedullin, CXCR4, and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the nuclear factor-kappaB (NF-kappaB) signaling pathway. We then used both genetic and pharmacologic methods to manipulate the levels of HIFs-1alpha and 2alpha or NF-kappaB in primary macrophages to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIF-1 and -2, but not NF-kappaB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues, such as malignant tumors.

  5. Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

    PubMed Central

    Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression. PMID:25978439

  6. Hypoxia-induced retinal neovascularization in zebrafish embryos: a potential model of retinopathy of prematurity.

    PubMed

    Wu, Yu-Ching; Chang, Chao-Yuan; Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.

  7. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    NASA Astrophysics Data System (ADS)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  8. Regulation of the Drosophila hypoxia-inducible factor alpha Sima by CRM1-dependent nuclear export.

    PubMed

    Romero, Nuria M; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

    2008-05-01

    Hypoxia-inducible factor alpha (HIF-alpha) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-alpha protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia.

  9. Hypoxia Inducible Factor Pathway and Physiological Adaptation: A Cell Survival Pathway?

    PubMed

    Kumar, Hemant; Choi, Dong-Kug

    2015-01-01

    Oxygen homeostasis reflects the constant body requirement to generate energy. Hypoxia (0.1-1% O2), physioxia or physoxia (∼1-13%), and normoxia (∼20%) are terms used to define oxygen concentration in the cellular environment. A decrease in oxygen (hypoxia) or excess oxygen (hyperoxia) could be deleterious for cellular adaptation and survival. Hypoxia can occur under both physiological (e.g., exercise, embryonic development, underwater diving, or high altitude) and pathological conditions (e.g., inflammation, solid tumor formation, lung disease, or myocardial infarction). Hypoxia plays a key role in the pathophysiology of heart disease, cancers, stroke, and other causes of mortality. Hypoxia inducible factor(s) (HIFs) are key oxygen sensors that mediate the ability of the cell to cope with decreased oxygen tension. These transcription factors regulate cellular adaptation to hypoxia and protect cells by responding acutely and inducing production of endogenous metabolites and proteins to promptly regulate metabolic pathways. Here, we review the role of the HIF pathway as a metabolic adaptation pathway and how this pathway plays a role in cell survival. We emphasize the roles of the HIF pathway in physiological adaptation, cell death, pH regulation, and adaptation during exercise.

  10. Hypoxia inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia

    PubMed Central

    Fang, Hsin-Yu; Hughes, Russell; Murdoch, Craig; Coffelt, Seth; Biswas, Subhra K.; Harris, Adrian L.; Johnson, Randall S.; Imityaz, Hongxia Z.; Simon, M. Celeste; Fredlund, Erik; Greten, Florian; Rius, Jordi; Lewis, Claire E.

    2010-01-01

    Ischemia exists in many diseased tissues including arthritic joints, atherosclerotic plaques and malignant tumors. Macrophages accumulate in these sites and upregulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18h. For example, they were seen to upregulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, VEGFA, interleukins 1β and 8, adrenomedullin, CXCR4 and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the NF-κB signalling pathway. We then used both genetic and pharmacological methods to manipulate the levels of HIFs 1α and 2α or NF-κB in primary macrophages in order to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIFs 1 and 2, but not NF-κB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues like malignant tumors. PMID:19454749

  11. Molecular Characterisation, Evolution and Expression of Hypoxia-Inducible Factor in Aurelia sp.1

    PubMed Central

    Wang, Guoshan; Yu, Zhigang; Zhen, Yu; Mi, Tiezhu; Shi, Yan; Wang, Jianyan; Wang, Minxiao; Sun, Song

    2014-01-01

    The maintenance of physiological oxygen homeostasis is mediated by hypoxia-inducible factor (HIF), a key transcriptional factor of the PHD-HIF system in all metazoans. However, the molecular evolutionary origin of this central physiological regulatory system is not well characterized. As the earliest eumetazoans, Cnidarians can be served as an interesting model for exploring the HIF system from an evolutionary perspective. We identified the complete cDNA sequence of HIF-1α (ASHIF) from the Aurelia sp.1, and the predicted HIF-1α protein (pASHIF) was comprised of 674 amino acids originating from 2,025 bp nucleotides. A Pairwise comparison revealed that pASHIF not only possessed conserved basic helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains but also contained the oxygen dependent degradation (ODD) and the C-terminal transactivation domains (C-TAD), the key domains for hypoxia regulation. As indicated by sequence analysis, the ASHIF gene contains 8 exons interrupted by 7 introns. Western blot analysis indicated that pASHIF that existed in the polyps and medusa of Aurelia. sp.1 was more stable for a hypoxic response than normoxia. PMID:24926666

  12. Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1.

    PubMed

    Abán, C; Martinez, N; Carou, C; Albamonte, I; Toro, A; Seyahian, A; Franchi, A; Leguizamón, G; Trigubo, D; Damiano, A; Farina, M

    2016-10-01

    During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl2), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl2 incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process.

  13. Hypoxia-inducible factor-1 facilitates cervical cancer progression in human papillomavirus type 16 transgenic mice.

    PubMed

    Lu, Zhi Hong; Wright, Jason D; Belt, Brian; Cardiff, Robert D; Arbeit, Jeffrey M

    2007-08-01

    Advanced cervical cancer remains a vexing clinical challenge despite screening programs. Many of these cancers are hypoxic, and expression of the alpha subunit of the major regulator of the hypoxic cellular response, the transcription factor hypoxia-inducible factor-1 (HIF-1), is correlated with poor prognosis. Here, we tested a functional role for HIF-1alpha in pathogenesis of cervical cancer in estrogen-treated transgenic mice. Double-transgenic (DTG) mice developed locally invasive cervical cancers 70 times larger than K14-HPV16 mice. In vivo bromodeoxyuridine incorporation was elevated in DTG cancers without a significant increase in apoptosis. HIF-1alpha gain of function did not up-regulate canonical HIF-1 targets in premalignant DTG cervices, in contrast to elevation of these targets in K14-HIF-1alpha transgenic cervices. The DTG transcriptional signature included up-regulation of mRNAs encoding cytokines and chemokines, immune signaling molecules, extracellular proteases, and cell motility factors, as well as reduced expression of cell adhesion and epithelial differentiation genes. Importantly, a set of gene markers derived from the DTG transcriptome predicted cervical cancer progression in patients. This study suggests a novel paradigm for HIF-1 function evident in multistage carcinogenesis as opposed to established malignancies, including interaction with viral oncogenes to induce multiple genomic networks in premalignancy that fosters the development of advanced cervical cancer.

  14. Role of hypoxia-inducible factor 1{alpha} in modulating cobalt-induced lung inflammation.

    PubMed

    Saini, Yogesh; Kim, Kyung Y; Lewandowski, Ryan; Bramble, Lori A; Harkema, Jack R; Lapres, John J

    2010-02-01

    Hypoxia plays an important role in development, cellular homeostasis, and pathological conditions, such as cancer and stroke. There is also growing evidence that hypoxia is an important modulator of the inflammatory process. Hypoxia-inducible factors (HIFs) are a family of proteins that regulate the cellular response to oxygen deficit, and loss of HIFs impairs inflammatory cell function. There is little known, however, about the role of epithelial-derived HIF signaling in modulating inflammation. Cobalt is capable of eliciting an allergic response and promoting HIF signaling. To characterize the inflammatory function of epithelial-derived HIF in response to inhaled cobalt, a conditional lung-specific HIF1alpha, the most ubiquitously expressed HIF, deletion mouse, was created. Control mice showed classic signs of metal-induced injury following cobalt exposure, including fibrosis and neutrophil infiltration. In contrast, HIF1alpha-deficient mice displayed a Th2 response that resembled asthma, including increased eosinophilic infiltration, mucus cell metaplasia, and chitinase-like protein expression. The results suggest that epithelial-derived HIF signaling has a critical role in establishing a tissue's inflammatory response, and compromised HIF1alpha signaling biases the tissue towards a Th2-mediated reaction.

  15. Chronic intermittent hypoxia induces cardiac inflammation and dysfunction in a rat obstructive sleep apnea model

    PubMed Central

    Wei, Qin; Bian, Yeping; Yu, Fuchao; Zhang, Qiang; Zhang, Guanghao; Li, Yang; Song, Songsong; Ren, Xiaomei; Tong, Jiayi

    2016-01-01

    Abstract Chronic intermittent hypoxia is considered to play an important role in cardiovascular pathogenesis during the development of obstructive sleep apnea (OSA). We used a well-described OSA rat model induced with simultaneous intermittent hypoxia. Male Sprague Dawley rats were individually placed into plexiglass chambers with air pressure and components were electronically controlled. The rats were exposed to intermittent hypoxia 8 hours daily for 5 weeks. The changes of cardiac structure and function were examined by ultrasound. The cardiac pathology, apoptosis, and fibrosis were analyzed by H&E staining, TUNNEL assay, and picosirius staining, respectively. The expression of inflammation and fibrosis marker genes was analyzed by quantitative real-time PCR and Western blot. Chronic intermittent hypoxia/low pressure resulted in significant increase of left ventricular internal diameters (LVIDs), end-systolic volume (ESV), end-diastolic volume (EDV), and blood lactate level and marked reduction in ejection fraction and fractional shortening. Chronic intermittent hypoxia increased TUNNEL-positive myocytes, disrupted normal arrangement of cardiac fibers, and increased Sirius stained collagen fibers. The expression levels of hypoxia induced factor (HIF)-1α, NF-kB, IL-6, and matrix metallopeptidase 2 (MMP-2) were significantly increased in the heart of rats exposed to chronic intermittent hypoxia. In conclusion, the left ventricular function was adversely affected by chronic intermittent hypoxia, which is associated with increased expression of HIF-1α and NF-kB signaling molecules and development of cardiac inflammation, apoptosis and fibrosis. PMID:27924067

  16. Beyond oxygen: complex regulation and activity of hypoxia inducible factors in pregnancy

    PubMed Central

    Pringle, K.G.; Kind, K.L.; Sferruzzi-Perri, A.N.; Thompson, J.G.; Roberts, C.T.

    2010-01-01

    In the first trimester the extravillous cytotrophoblast cells occlude the uterine spiral arterioles creating a low oxygen environment early in pregnancy, which is essential for pregnancy success. Paradoxically, shallow trophoblast invasion and defective vascular remodelling of the uterine spiral arteries in the first trimester may result in impaired placental perfusion and chronic placental ischemia and hypoxia later in gestation leading to adverse pregnancy outcomes. The hypoxia inducible factors (HIFs) are key mediators of the response to low oxygen. We aimed to elucidate mechanisms of regulation of HIFs and the role these may play in the control of placental differentiation, growth and function in both normal and pathological pregnancies. The Pubmed database was consulted for identification of the most relevant published articles. Search terms used were oxygen, placenta, trophoblast, pregnancy, HIF and hypoxia. The HIFs are able to function throughout all aspects of normal and abnormal placental differentiation, growth and function; during the first trimester (physiologically low oxygen), during mid-late gestation (where there is adequate supply of blood and oxygen to the placenta) and in pathological pregnancies complicated by placental hypoxia/ischemia. During normal pregnancy HIFs may respond to complex alterations in oxygen, hormones, cytokines and growth factors to regulate placental invasion, differentiation, transport and vascularization. In the ever-changing environment created during pregnancy, the HIFs appear to act as key mediators of placental development and function and thereby are likely to be important contributors to both normal and adverse pregnancy outcomes. PMID:19926662

  17. Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells.

    PubMed

    Samanta, Debangshu; Gilkes, Daniele M; Chaturvedi, Pallavi; Xiang, Lisha; Semenza, Gregg L

    2014-12-16

    Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER(+)/PR(+) breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.

  18. Hypoxia-inducible factor 1–mediated characteristic features of cancer cells for tumor radioresistance

    PubMed Central

    Harada, Hiroshi

    2016-01-01

    Tumor hypoxia has been attracting increasing attention in the fields of radiation biology and oncology since Thomlinson and Gray detected hypoxic cells in malignant solid tumors and showed that they exert a negative impact on the outcome of radiation therapy. This unfavorable influence has, at least partly, been attributed to cancer cells acquiring a radioresistant phenotype through the activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). On the other hand, accumulating evidence has recently revealed that, even though HIF-1 is recognized as an important regulator of cellular adaptive responses to hypoxia, it may not become active and induce tumor radioresistance under hypoxic conditions only. The mechanisms by which HIF-1 is activated in cancer cells not only under hypoxic conditions, but also under normoxic conditions, through cancer-specific genetic alterations and the resultant imbalance in intermediate metabolites have been summarized herein. The relevance of the HIF-1–mediated characteristic features of cancer cells, such as the production of antioxidants through reprogramming of the glucose metabolic pathway and cell cycle regulation, for tumor radioresistance has also been reviewed. PMID:26983985

  19. Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells.

    PubMed

    Huang, Guichun; Tao, Leilei; Shen, Sunan; Chen, Longbang

    2016-06-02

    Tumor hypoxia is one of the important features of lung adenocarcinoma. Chemokines might mediate the effects caused by tumor hypoxia. As confirmed in tumor tissue and serum of patients, CC chemokine 28 (CCL28) was the only hypoxia induced chemokine in lung adenocarcinoma cells. CCL28 could promote tube formation, migration and proliferation of endothelial cells. In addition, angiogenesis was promoted by CCL28 in the chick chorioallantoic membrane and matrigel implanted in dorsal back of athymic nude mice (CByJ.Cg-Foxn1(nu)/J). Tumors formed by lung adenocarcinoma cells with high expression of CCL28 grew faster and had a higher vascular density, whereas tumor formation rate of lung adenocarcinoma cells with CCL28 expression knockdown was quite low and had a lower vascular density. CCR3, receptor of CCL28, was highly expressed in vascular endothelial cells in lung adenocarcinoma when examining by immunohistochemistry. Further signaling pathways in endothelial cells, modulated by CCL28, were analyzed by Phosphorylation Antibody Array. CCL28/CCR3 signaling pathway could bypass that of VEGF/VEGFR on the levels of PI3K-Akt, p38 MAPK and PLC gamma. The effects could be neutralized by antibody against CCR3. In conclusion, CCL28, as a chemokine induced by tumor hypoxia, could promote angiogenesis in lung adenocarcinoma through targeting CCR3 on microvascular endothelial cells.

  20. Hypoxia-Inducible Factor-1α Expression in Indonesian Laryngeal Squamous Cell Carcinoma Patients

    PubMed Central

    Priyanto, Priyanto; Indrasari, Sagung Rai

    2016-01-01

    Objectives. This research aimed to determine the association between hypoxia-inducible factor-1α (HIF-1α) expression and laryngeal squamous cell carcinoma clinical stage. Methods. We retrospectively analyzed paraffin-embedded tissue from 47 laryngeal squamous cell carcinoma (LSCC) patients from 2011 to 2014. HIF-1α expression was analyzed by immunohistochemistry using an anti-HIF-1α mouse monoclonal antibody. The association between HIF-1α expression and clinical stage was analyzed using the chi square test. Results. The glottis was the predominant site of laryngeal squamous cell carcinoma occurrence, and 43/47 (91.5%) patients presented at an advanced stage. Of the advanced stage patients, 27/43 stained positive for HIF-1α expression and 16/43 stained negative. Of the early stage patients, 2/4 stained positive for HIF-1α expression and 2/4 stained negative. Statistical analysis did not demonstrate significant association of HIF-1α expression. Conclusion. There was no statistically significant association between HIF-1α expression and the clinical stage or histological differentiation of LSCC. PMID:27882053

  1. Hypoxia inducible factor-1: regulation by nitric oxide in posthypoxic microvascular endothelium.

    PubMed

    Natarajan, Ramesh; Jones, Drew G; Fisher, Bernard J; Wallace, Timothy J; Ghosh, Shobha; Fowler, Alpha A

    2005-10-01

    Microvascular endothelial cells provide a critical regulatory interface between blood constituents and tissue. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for expression of hypoxia-dependent genes. We employed a model of hypoxia and reoxygenation (H/R) using the dermal microvascular endothelial cell line HMEC-1 to examine the effects of altered oxygen concentrations on microvascular HIF-1 expression and nitric oxide (NO) formation. Hypoxia increased inducible NO synthase (iNOS) mRNA in a time-dependent manner in HMEC-1. However, endothelial NO synthase mRNA progressively declined during hypoxia. H/R promoted significant increases in cellular nitrite levels that were significantly abrogated by the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine, di hy drochloride. Exogenous NO promoted stabilization of the alpha subunit of HIF-1 and produced functional DNA binding. Exposure of HMEC-1 to H/R resulted in previously unrecognized biphasic HIF-1alpha stabilization during reoxygenation. When the iNOS gene was silenced through the use of iNOS-specific small interfering RNA, HIF-1alpha stabilization and HIF-1 activation were dramatically diminished, suggesting that inducible NOS-derived NO is a key factor sustaining HIF-1 activation during both hypoxia and reoxygenation.

  2. Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit.

    PubMed

    Pugh, C W; O'Rourke, J F; Nagao, M; Gleadle, J M; Ratcliffe, P J

    1997-04-25

    Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

  3. Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration

    PubMed Central

    Kurihara, Toshihide; Westenskow, Peter D; Gantner, Marin L; Usui, Yoshihiko; Schultz, Andrew; Bravo, Stephen; Aguilar, Edith; Wittgrove, Carli; Friedlander, Mollie SH; Paris, Liliana P; Chew, Emily; Siuzdak, Gary; Friedlander, Martin

    2016-01-01

    Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies. DOI: http://dx.doi.org/10.7554/eLife.14319.001 PMID:26978795

  4. Failure to prolyl hydroxylate hypoxia-inducible factor α phenocopies VHL inactivation in vivo

    PubMed Central

    Kim, William Y; Safran, Michal; Buckley, Marshall R M; Ebert, Benjamin L; Glickman, Jonathan; Bosenberg, Marcus; Regan, Meredith; Kaelin, William G

    2006-01-01

    Many functions have been assigned to the von Hippel-Lindau tumor suppressor gene product (pVHL), including targeting the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for destruction. The binding of pVHL to HIFα requires that HIFα be hydroxylated on one of two prolyl residues. We introduced HIF1α and HIF2α variants that cannot be hydroxylated on these sites into the ubiquitously expressed ROSA26 locus along with a Lox-stop-Lox cassette that renders their expression Cre-dependent. Expression of the HIF2α variant in the skin and liver induced changes that were highly similar to those seen when pVHL is lost in these organs. Dual expression of the HIF1α and HIF2α variants in liver, however, more closely phenocopied the changes seen after pVHL inactivation than did the HIF2α variant alone. Moreover, gene expression profiling confirmed that the genes regulated by HIF1α and HIF2α in the liver are overlapping but non-identical. Therefore, the pathological changes caused by pVHL inactivation in skin and liver are due largely to dysregulation of HIF target genes. PMID:16977322

  5. Hypoxia inducible factors are dispensable for myeloid cell migration into the inflamed mouse eye

    PubMed Central

    Gardner, Peter J.; Liyanage, Sidath E.; Cristante, Enrico; Sampson, Robert D.; Dick, Andrew D.; Ali, Robin R.; Bainbridge, James W.

    2017-01-01

    Hypoxia inducible factors (HIFs) are ubiquitously expressed transcription factors important for cell homeostasis during dynamic oxygen levels. Myeloid specific HIFs are crucial for aspects of myeloid cell function, including their ability to migrate into inflamed tissues during autoimmune disease. This contrasts with the concept that accumulation of myeloid cells at ischemic and hypoxic sites results from a lack of chemotactic responsiveness. Here we seek to address the role of HIFs in myeloid trafficking during inflammation in a mouse model of human uveitis. We show using mice with myeloid-specific Cre-deletion of HIFs that myeloid HIFs are dispensable for leukocyte migration into the inflamed eye. Myeloid-specific deletion of Hif1a, Epas1, or both together, had no impact on the number of myeloid cells migrating into the eye. Additionally, stabilization of HIF pathways via deletion of Vhl in myeloid cells had no impact on myeloid trafficking into the inflamed eye. Finally, we chemically induce hypoxemia via hemolytic anemia resulting in HIF stabilization within circulating leukocytes to demonstrate the dispensable role of HIFs in myeloid cell migration into the inflamed eye. These data suggest, contrary to previous reports, that HIF pathways in myeloid cells during inflammation and hypoxia are dispensable for myeloid cell tissue trafficking. PMID:28112274

  6. Hypoxia-Inducible Factors: Mediators of Cancer Progression; Prognostic and Therapeutic Targets in Soft Tissue Sarcomas

    PubMed Central

    Sadri, Navid; Zhang, Paul J.

    2013-01-01

    Soft-tissue sarcomas remain aggressive tumors that result in death in greater than a third of patients due to either loco-regional recurrence or distant metastasis. Surgical resection remains the main choice of treatment for soft tissue sarcomas with pre- and/or post-operational radiation and neoadjuvant chemotherapy employed in more advanced stage disease. However, in recent decades, there has been little progress in the average five-year survival for the majority of patients with high-grade soft tissue sarcomas, highlighting the need for improved targeted therapeutic agents. Clinical and preclinical studies demonstrate that tumor hypoxia and up-regulation of hypoxia-inducible factors (HIFs) is associated with decreased survival, increased metastasis, and resistance to therapy in soft tissue sarcomas. HIF-mediated gene expression regulates many critical aspects of tumor biology, including cell survival, metabolic programming, angiogenesis, metastasis, and therapy resistance. In this review, we discuss HIFs and HIF-mediated genes as potential prognostic markers and therapeutic targets in sarcomas. Many pharmacological agents targeting hypoxia-related pathways are in development that may hold therapeutic potential for treating both primary and metastatic sarcomas that demonstrate increased HIF expression. PMID:24216979

  7. Macrophage Migration Inhibitory Factor Activates Hypoxia-Inducible Factor in a p53-Dependent Manner

    PubMed Central

    Oda, Seiko; Oda, Tomoyuki; Nishi, Kenichiro; Takabuchi, Satoshi; Wakamatsu, Takuhiko; Tanaka, Tomoharu; Adachi, Takehiko; Fukuda, Kazuhiko; Semenza, Gregg L.; Hirota, Kiichi

    2008-01-01

    Background Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner. Methods/Principal Findings The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly. Conclusion/Significance MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF. PMID:18493321

  8. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  9. Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells.

    PubMed

    Qiao, N; Xu, C; Zhu, Y-X; Cao, Y; Liu, D-C; Han, X

    2015-02-19

    Hypoxia complicates islet isolation for transplantation and may contribute to pancreatic β-cell failure in type 2 diabetes. Pancreatic β-cells are susceptible to hypoxia-induced apoptosis. Severe hypoxic conditions during the immediate post-transplantation period are a main non-immune factor leading to β-cell death and islet graft failure. In this study, we identified the transcription factor Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells. Hypoxia regulates Ets-1 at multiple levels according to the degree of β-cell oxygen deprivation. Moderate hypoxia promotes Ets-1 gene transcription, whereas severe hypoxia promotes its transactivation activity, as well as its ubiquitin-proteasome mediated degradation. This degradation causes a relative insufficiency of Ets-1 activity, and limits the transactivation effect of Ets-1 on downstream hypoxic-inducible genes and its anti-apoptotic function. Overexpression of ectopic Ets-1 in MIN6 and INS-1 cells protects them from severe hypoxia-induced apoptosis in a mitochondria-dependent manner, confirming that a sufficient amount of Ets-1 activity is critical for protection of pancreatic β-cells against hypoxic injury. Targeting Ets-1 expression may be a useful strategy for islet graft protection during the immediate post-transplantation period.

  10. Salidroside attenuates chronic hypoxia-induced pulmonary hypertension via adenosine A2a receptor related mitochondria-dependent apoptosis pathway.

    PubMed

    Huang, Xiaoying; Zou, Lizhen; Yu, Xiaoming; Chen, Mayun; Guo, Rui; Cai, Hui; Yao, Dan; Xu, Xiaomei; Chen, Yanfan; Ding, Cheng; Cai, Xueding; Wang, Liangxing

    2015-05-01

    Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Salidroside, an active ingredient isolated from Rhodiola rosea is proposed to exert protective effects against PAH. However, the function of salidroside in PAH has not been investigated systematically and the underlying mechanisms are not clear. To investigate the effects of salidroside on PAH, the mice in chronic hypoxia model of PAH were given by an increasing concentration of salidroside (0, 16 mg/kg, 32 mg/kg, and 64 mg/kg). After salidroside treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling were attenuated, suggesting a protective role played by salidroside in PAH. To explore the potential mechanisms, the apoptosis of PASMCs after salidroside treatment under hypoxia conditions were determined in vivo and in vitro, and also the mitochondria-dependent apoptosis factors, Bax, Bcl-2, cytochrome C, and caspase 9 were examined. The results revealed that salidroside reversed hypoxia-induced cell apoptosis resistance at least partially via a mitochondria-dependent pathway. In addition, salidroside upregulated the expression of adenosine A2a receptor (A2aR) in lung tissues of mice and in PASMCs in vitro after hypoxia exposure. Combined the evidence above, we conclude that salidroside can attenuate chronic hypoxia-induced PAH by promoting PASMCs apoptosis via an A2aR related mitochondria dependent pathway.

  11. TERT alleviates irradiation-induced late rectal injury by reducing hypoxia-induced ROS levels through the activation of NF-κB and autophagy

    PubMed Central

    Liu, Qi; Sun, Yong; Lv, Yuefeng; Le, Ziyu; Xin, Yuhu; Zhang, Ping; Liu, Yong

    2016-01-01

    The hypoxic microenvironment which is present following irradiation has been proven to promote radiation-induced injury to normal tissues. Previous studies have demonstrated that telomerase reverse transcriptase (TERT) is regulated by hypoxia, and that it plays a protective role in the process of wound repair. However, its effects on radiation-induced injury remain unclear. In this study, we examined the effects of human TERT on irradiation-induced late rectal injury in fibroblasts under hypoxic conditions. We also performed in vivo experiments. The rectums of 5-week-old female C57BL/6N mice were irradiated locally with a single dose of 25 Gy. We then examined the fibrotic changes using hematoxylin and eosin staining, and Masson's staining. The expression of hypoxia inducible factor-1α (HIF-1α) and TERT was analyzed by immunohistochemistry. In in vitro experiments, apoptosis, reactive oxygen species (ROS) production and the autophagy level induced by exposure to hypoxia were assayed in fibroblasts. The association between TERT, nuclear factor-κB (NF-κB) and the autophagy level was examined by western blot analysis. The antioxidant effects of TERT were examined on the basis of the ratio of glutathione to glutathione disulfide (GSH/GSSG) and mitochondrial membrane potential. Rectal fibrosis was induced significantly at 12 weeks following irradiation. The HIF-1α and TERT expression levels increased in the fibrotic region. The TERT-overexpressing fibroblasts (transfected with an hTERT-expressing lentiviral vector) exhibited reduced apoptosis, reduced ROS production, a higher autophagy level, a higher GSH/GSSG ratio and stable mitochondrial membrane potential compared with the fibroblasts in which TERT had been silenced by siRNA. NF-κB was activated by TERT, and the inhibition of TERT reduced the autophagy level in the fibroblasts. These results demonstrate that TERT decreases cellular ROS production, while maintaining mitochondrial function and protecting the

  12. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells.

    PubMed

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl(2)), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl(2) treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl(2) administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl(2)-induced reactive oxygen species (ROS) formation and completely negated CoCl(2)-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl(2) administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl(2) increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl(2)-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption.

  13. Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state.

    PubMed

    Park, In-Ho; Kim, Kwang-Ho; Choi, Hyun-Kyung; Shim, Jae-Seung; Whang, Soo-Young; Hahn, Sang June; Kwon, Oh-Joo; Oh, Il-Hoan

    2013-09-27

    With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

  14. Ethanol enhances tumor angiogenesis in vitro induced by low-dose arsenic in colon cancer cells through hypoxia-inducible factor 1 alpha pathway.

    PubMed

    Wang, Lei; Son, Young-Ok; Ding, Songze; Wang, Xin; Hitron, John Andrew; Budhraja, Amit; Lee, Jeong-Chae; Lin, Qinchen; Poyil, Pratheeshkumar; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2012-12-01

    Health effects due to environmental exposure to arsenic are a major global health concern. Arsenic has been known to induce carcinogenesis and enhance tumor development via complex and unclear mechanism. Ethanol is also a well-established risk factor for many malignancies. However, little is known about the effects of coexposure to arsenic and ethanol in tumor development. In this study, we investigate the signaling and angiogenic effect of coexposure of arsenic and ethanol on different colon cancer cell lines. Results show that ethanol markedly enhanced arsenic-induced tumor angiogenesis in vitro. These responses are related to intracellular reactive oxygen species (ROS) generation, NADPH oxidase activation, and upregulation of PI3K/Akt and hypoxia-inducible factor 1 alpha (HIF-1α) signaling. We have also found that ethanol increases the arsenic-induced expression and secretion of angiogenic signaling molecules such as vascular endothelial growth factor, which further confirmed the above observation. Antioxidant enzymes inhibited arsenic/ethanol-induced tumor angiogenesis, demonstrating that the responsive signaling pathways of coexposure to arsenic and ethanol are related to ROS generation. We conclude that ethanol is able to enhance arsenic-induced tumor angiogenesis in colorectal cancer cells via the HIF-1α pathway. These results indicate that alcohol consumption should be taken into consideration in the investigation of arsenic-induced carcinogenesis in arsenic-exposed populations.

  15. Regulatory effect of hypoxia-inducible factor-1α on hCG-stimulated endothelin-2 expression in granulosa cells from the PMSG-treated rat ovary.

    PubMed

    Zhang, Jisen; Zhang, Zhenghong; Wu, Yanqing; Chen, Liyun; Luo, Qianping; Chen, Jiajie; Huang, Xiaohong; Cheng, Yong; Wang, Zhengchao

    2012-01-01

    Endothelin (ET)-2 plays a crucial role in ovarian ovulation in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of ET-2 gene in response to hCG in rat ovarian granulosa cells (GCs) during gonadotropin-induced superovulation. By real-time RT-PCR analysis, ET-2 mRNA expression was found to significantly increase in cultured ovarian GCs after treatment with hCG, or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while this increased ET-2 mRNA expression could also be blocked by ferrous ammonium sulfate (FAS) under human chorionic gonadotropin (hCG) treatment. Further analysis also found that these changes of ET-2 mRNA were consistent with HIF-1α expression or HIF-1 activity, and HIF-1α inhibitor echinomycin inhibited ovulation in rats. Taken together, these results indicate that ET-2 is transcriptionally activated by hCG through HIF-1α-mediated mechanism in GCs. This HIF-1α-induced transcriptional activation may be one of the important mechanisms mediating the increase of ET-2 expression in GCs during the gonadotropin-induced mammalian ovulatory process in vivo.

  16. Loss of von Hippel-Lindau Protein (VHL) Increases Systemic Cholesterol Levels through Targeting Hypoxia-Inducible Factor 2α and Regulation of Bile Acid Homeostasis

    PubMed Central

    Ramakrishnan, Sadeesh K.; Taylor, Matthew; Qu, Aijuan; Ahn, Sung-Hoon; Suresh, Madathilparambil V.; Raghavendran, Krishnan; Gonzalez, Frank J.

    2014-01-01

    Cholesterol synthesis is a highly oxygen-dependent process. Paradoxically, hypoxia is correlated with an increase in cellular and systemic cholesterol levels and risk of cardiovascular diseases. The mechanism for the increase in cholesterol during hypoxia is unclear. Hypoxia signaling is mediated through hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. The present study demonstrates that activation of HIF signaling in the liver increases hepatic and systemic cholesterol levels due to a decrease in the expression of cholesterol hydroxylase CYP7A1 and other enzymes involved in bile acid synthesis. Specifically, activation of hepatic HIF-2α (but not HIF-1α) led to hypercholesterolemia. HIF-2α repressed the circadian expression of Rev-erbα, resulting in increased expression of E4BP4, a negative regulator of Cyp7a1. To understand if HIF-mediated decrease in bile acid synthesis is a physiologically relevant pathway by which hypoxia maintains or increases systemic cholesterol levels, two hypoxic mouse models were assessed, an acute lung injury model and mice exposed to 10% O2 for 3 weeks. In both models, cholesterol levels increased with a concomitant decrease in expression of genes involved in bile acid synthesis. The present study demonstrates that hypoxic activation of hepatic HIF-2α leads to an adaptive increase in cholesterol levels through inhibition of bile acid synthesis. PMID:24421394

  17. Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species.

    PubMed

    Goyal, Parag; Weissmann, Norbert; Grimminger, Friedrich; Hegel, Cornelia; Bader, Lucius; Rose, Frank; Fink, Ludger; Ghofrani, Hossein A; Schermuly, Ralph T; Schmidt, Harald H H W; Seeger, Werner; Hänze, Jörg

    2004-05-15

    Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.

  18. Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway

    PubMed Central

    Zhu, Chengxing; Yu, Jiong; Pan, Qiaoling; Yang, Jinfeng; Hao, Guangshu; Wang, Yingjie; Li, Lanjuan; Cao, Hongcui

    2016-01-01

    Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway. In contrast, these genes were down-regulated in the HIF-2α-silenced hPMSCs. After adding the MAPK/ERK inhibitor PD0325901, cell growth and the expression of CCND1 and MYC were inhibited. Furthermore, the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) showed that HIF-2α bound to the MAPK3 (ERK1) promoter, indicative of its direct regulation of MAPK/ERK components at the transcriptional level during hPMSC expansion. Taken together, our results suggest that HIF-2α facilitated the preservation of hPMSC stemness and promoted their proliferation by regulating CCND1 and MYC through the MAPK/ERK signaling pathway. PMID:27765951

  19. PEDF and PEDF-derived peptide 44mer protect cardiomyocytes against hypoxia-induced apoptosis and necroptosis via anti-oxidative effect.

    PubMed

    Gao, Xiao; Zhang, Hao; Zhuang, Wei; Yuan, Guangda; Sun, Teng; Jiang, Xia; Zhou, Zhongxin; Yuan, Honghua; Zhang, Zhongming; Dong, Hongyan

    2014-07-11

    Pigment epithelium-derived factor (PEDF) has many biological activities. But it's not known whether PEDF and its functional peptides could protect against hypoxia-induced cell death and the mechanisms are still unclear. We used cultured H9c2 cells and primary cardiomyocytes to show that apoptosis and necroptosis were significantly increased after hypoxia. Both PEDF and its fuctional peptides 44mer reduced apoptosis and necroptosis rates and inhibited the expression of cleaved caspase 3 and receptor-interacting protein 3 (RIP3). Furthermore, PEDF and 44mer could up-regulate super oxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels, promote clearing of reactive oxygen species (ROS) and malondialdehyde (MDA). While, 34mer, another functional peptides had no effect on cell apoptosis and necroptosis. Hereby this is the first evidence that PEDF and its functional peptide 44mer protect cultured H9c2 cells and primary cardiomyocytes against apoptosis and necroptosis under hypoxic condition via the anti-oxidative mechanism.

  20. Effects of Tetrahydrocurcumin on Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor Expression in Cervical Cancer Cell-Induced Angiogenesis in Nude Mice

    PubMed Central

    Yoysungnoen, Bhornprom; Patumraj, Suthiluk; Changtam, Chatchawan

    2015-01-01

    Tetrahydrocurcumin (THC), one of the important in vivo metabolites of curcumin, inhibits tumor angiogenesis. Its effects on angiogenesis in cervical cancer- (CaSki-) implanted nude mice and its mechanisms on hypoxia-inducible factor-1α and vascular endothelial growth factor expression were investigated. Female BALB/c nude mice were divided into control (CON) and CaSki-implanted groups (CaSki group). One month after the injection with cervical cancer cells, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. The microvascular density (MVD) was evaluated using the CD31 expression. VEGF, VEGFR-2, and HIF-1α expression were also detected by immunohistochemistry. The MVD in CaSki + vehicle group was significantly increased compared to the CON + vehicle group. Interestingly, when treated with THC at all doses, the CaSki group showed a significant smaller number of the MVD. The CaSki + vehicle group also showed significantly increased VEGF, VEGFR-2, and HIF-1α expressions, but they were downregulated when mice were treated with THC at all doses. THC demonstrated an inhibitory effect against tumor angiogenesis in CaSki-implanted nude mice model. This effect is likely to be mediated by the downregulation of HIF-1-α, VEGF expression, and its receptor. THC could be developed into a promising agent for cancer therapy in the future. PMID:25789317

  1. Effects of tetrahydrocurcumin on hypoxia-inducible factor-1α and vascular endothelial growth factor expression in cervical cancer cell-induced angiogenesis in nude mice.

    PubMed

    Yoysungnoen, Bhornprom; Bhattarakosol, Parvapan; Patumraj, Suthiluk; Changtam, Chatchawan

    2015-01-01

    Tetrahydrocurcumin (THC), one of the important in vivo metabolites of curcumin, inhibits tumor angiogenesis. Its effects on angiogenesis in cervical cancer- (CaSki-) implanted nude mice and its mechanisms on hypoxia-inducible factor-1α and vascular endothelial growth factor expression were investigated. Female BALB/c nude mice were divided into control (CON) and CaSki-implanted groups (CaSki group). One month after the injection with cervical cancer cells, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. The microvascular density (MVD) was evaluated using the CD31 expression. VEGF, VEGFR-2, and HIF-1α expression were also detected by immunohistochemistry. The MVD in CaSki + vehicle group was significantly increased compared to the CON + vehicle group. Interestingly, when treated with THC at all doses, the CaSki group showed a significant smaller number of the MVD. The CaSki + vehicle group also showed significantly increased VEGF, VEGFR-2, and HIF-1α expressions, but they were downregulated when mice were treated with THC at all doses. THC demonstrated an inhibitory effect against tumor angiogenesis in CaSki-implanted nude mice model. This effect is likely to be mediated by the downregulation of HIF-1-α, VEGF expression, and its receptor. THC could be developed into a promising agent for cancer therapy in the future.

  2. Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR- and hypoxia-induced factor 1α-mediated signaling.

    PubMed

    Wang, Kui; Liu, Rui; Li, Jingyi; Mao, Jiali; Lei, Yunlong; Wu, Jinhua; Zeng, Jun; Zhang, Tao; Wu, Hong; Chen, Lijuan; Huang, Canhua; Wei, Yuquan

    2011-09-01

    Quercetin, a dietary antioxidant present in fruits and vegetables, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing cell cycle arrest and promoting apoptotic cell death. In this study, we examined the biological activities of quercetin against gastric cancer. Our studies demonstrated that exposure of gastric cancer cells AGS and MKN28 to quercetin resulted in pronounced pro-apoptotic effect through activating the mitochondria pathway. Meanwhile, treatment with quercetin induced appearance of autophagic vacuoles, formation of acidic vesicular organelles (AVOs), conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes as well as activation of autophagy genes, suggesting that quercetin initiates the autophagic progression in gastric cancer cells. Furthermore, either administration of autophagic inhibitor chloroquine or selective ablation of atg5 or beclin 1 using small interfering RNA (siRNA) could augment quercetin-induced apoptotic cell death, suggesting that autophagy plays a protective role against quercetin-induced apoptosis. Moreover, functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling. Finally, a xenograft model provided additional evidence for occurrence of quercetin-induced apoptosis and autophagy in vivo. Together, our studies provided new insights regarding the biological and anti-proliferative activities of quercetin against gastric cancer, and may contribute to rational utility and pharmacological study of quercetin in future anti-cancer research.

  3. Integrative genomics reveals hypoxia inducible genes that are associated with a poor prognosis in neuroblastoma patients

    PubMed Central

    Kao, Clara; Hernandez, Kyle M.; DeWane, Gillian; Salwen, Helen R.; Chlenski, Alexandre; Dobratic, Marija; Mariani, Christopher J.; Godley, Lucy A.; Prabhakar, Nanduri; White, Kevin; Stranger, Barbara E.; Cohn, Susan L.

    2016-01-01

    Neuroblastoma is notable for its broad spectrum of clinical behavior ranging from spontaneous regression to rapidly progressive disease. Hypoxia is well known to confer a more aggressive phenotype in neuroblastoma. We analyzed transcriptome data from diagnostic neuroblastoma tumors and hypoxic neuroblastoma cell lines to identify genes whose expression levels correlate with poor patient outcome and are involved in the hypoxia response. By integrating a diverse set of transcriptome datasets, including those from neuroblastoma patients and neuroblastoma derived cell lines, we identified nine genes (SLCO4A1, ENO1, HK2, PGK1, MTFP1, HILPDA, VKORC1, TPI1, and HIST1H1C) that are up-regulated in hypoxia and whose expression levels are correlated with poor patient outcome in three independent neuroblastoma cohorts. Analysis of 5-hydroxymethylcytosine and ENCODE data indicate that at least five of these nine genes have an increase in 5-hydroxymethylcytosine and a more open chromatin structure in hypoxia versus normoxia and are putative targets of hypoxia inducible factor (HIF) as they contain HIF binding sites in their regulatory regions. Four of these genes are key components of the glycolytic pathway and another three are directly involved in cellular metabolism. We experimentally validated our computational findings demonstrating that seven of the nine genes are significantly up-regulated in response to hypoxia in the four neuroblastoma cell lines tested. This compact and robustly validated group of genes, is associated with the hypoxia response in aggressive neuroblastoma and may represent a novel target for biomarker and therapeutic development. PMID:27765905

  4. Altered expression of hypoxia-Inducible factor-1α participates in the epileptogenesis in animal models.

    PubMed

    Li, Jie; Jiang, Guohui; Chen, Yalan; Chen, Ling; Li, Zengyou; Wang, Zhihua; Wang, Xuefeng

    2014-09-01

    Although epilepsy is a common neurological disorder, its mechanism(s) are still not completely understood. Hypoxia can lead to neuronal cell death and angiogenesis, and the same mechanisms were also found in epilepsy. Hypoxia-inducible factor-1α (HIF-1α) is an important transcription protein that regulates gene expression in the brain and other tissues in response to decreases in oxygen availability. However, little is known regarding the expression of HIF-1α in the epileptic brain and whether HIF-1α interventions affect the epileptic process. The aims of this study are to investigate the expression profile of HIF-1α in rat models and to explore the role of HIF-1α in epilepsy. We performed Western blots and immunofluorescence in a lithium-pilocarpine rat epilepsy model. To determine the role of HIF-1α in epilepsy, we used the HIF-1α agonist DMOG and inhibitor KC7F2 to detect changes in the animal behavior in pentylenetetrazole (PTZ) and lithium-pilocarpine epilepsy models. The expression of HIF-1α was significantly increased after pilocarpine-induced status epilepticus. DMOG significantly prolonged the latent period in the PTZ kindling model and decreased the rate of spontaneous recurrent seizures during the chronic stage in the lithium-pilocarpine model. Conversely, the inhibitor KC7F2 produced an opposite behavioral change. Interestingly, both KC7F2 and DMOG had no effect on the acute stage of pilocarpine model and PTZ convulsive model. Our study suggests that upregulated HIF-1α may be involved in the process of epileptogenesis but not in the acute stage of epilepsy. The modulation of HIF-1α may offer a novel therapeutic target in epilepsy.

  5. Physiological and Therapeutic Vascular Remodeling Mediated by Hypoxia-Inducible Factor 1

    NASA Astrophysics Data System (ADS)

    Sarkar, Kakali; Semenza, Gregg L.

    Angiogenesis along with arteriogenesis and vasculogenesis is a fundamental process in ischemic repair in adult animals including humans. Hypoxia-inducible factor 1 (HIF-1) plays a central role in mediating adaptive responses to hypoxia/ischemia by expressing angiogenic cytokines/growth factors and their cognate receptors. Angiogenic growth factors are the homing signal for circulating angiogenic cells (CACs), which are mobilized to peripheral blood from bone marrow, recruited to target tissues, and promote vascularization. Impairment of HIF-1-mediated gene transcription contributes to the impaired vascular responses in peripheral vascular disease that are associated with aging and diabetes. Promoting neovascularization in ischemic tissues is a promising strategy for the treatment of peripheral vascular disease when surgical or catheter-based revascularization is not possible. Intramuscular injection of an adenovirus encoding a constitutively active form of HIF-1α (AdCA5), into the ischemic limb of diabetic mice increases the recovery of limb perfusion and function, rescues the diabetes-associated impairment of CACs, and increases vascularization. Administration of AdCA5 overcomes the effect of aging on recovery of blood flow in middle-aged mice following femoral artery ligation in a mouse model of age-dependent critical limb ischemia. Intramuscular injection of AdCA5 along with intravenous injection of bone-marrow-derived angiogenic cells cultured in the presence of prolyl-4-hydroxylase inhibitor dimethyloxalylglycine, increases blood flow and limb salvage in old mice following femoral artery ligation. HIF-1α gene therapy increases homing of bone-marrow-derived cells, whereas induction of HIF-1 in these cells increases their retention in the ischemic tissue by increasing their adhesion to endothelium leading to synergistic effects of combined therapy on improving blood flow.

  6. Immunohistochemical detection of hypoxia-inducible factor-1alpha in human renal allograft biopsies.

    PubMed

    Rosenberger, Christian; Pratschke, Johann; Rudolph, Birgit; Heyman, Samuel N; Schindler, Ralf; Babel, Nina; Eckardt, Kai-Uwe; Frei, Ulrich; Rosen, Seymour; Reinke, Petra

    2007-01-01

    Although it generally is accepted that renal hypoxia may occur in various situations after renal transplantation, direct evidence for such hypoxia is lacking, and possible implications on graft pathophysiology remain obscure. Hypoxia-inducible factors (HIF) are regulated at the protein level by oxygen-dependent enzymes and, hence, allow for tissue hypoxia detection. With the use of high-amplification HIF-1alpha immunohistochemistry in renal biopsies, hypoxia is shown at specific time points after transplantation with clinicohistologic correlations. Immediately after engraftment, in primarily functioning grafts, abundant HIF-1alpha is present and correlates with cold ischemic time >15 h and/or graft age >50 yr (P < 0.04). In contrast, a low HIF-1alpha score correlates with primary nonfunction, likely reflecting loss of oxygen consumption for tubular transport. Protocol biopsies at 2 wk show widespread HIF-1alpha induction, irrespective of histology. Beyond 3 mo, both protocol biopsies and indicated biopsies are virtually void of HIF-1alpha, with the only exception being clinical/subclinical rejection. HIF-derived transcriptional adaptation to hypoxia may counterbalance, at least partly, the negative impact of cold preservation and warm reflow injury. Transient hypoxia at 2 wk may be induced by hyperfiltration, hypertrophy, calcineurin inhibitor-induced toxicity, or a combination of these. Lack of detectable HIF-1alpha at 3 mo and beyond suggests that at this time point, graft oxygen homeostasis occurs. The strong correlation between hypoxia and clinical/subclinical rejection in long-term grafts suggests that hypoxia is involved in such graft dysfunction, and HIF-1alpha immunohistochemistry could enhance the specific diagnosis of acute rejection.

  7. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle.

    PubMed

    Takahashi, Natsumi; Davy, Philip M C; Gardner, Lauren H; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

  8. Coiled-coil coactivators play a structural role mediating interactions in hypoxia-inducible factor heterodimerization.

    PubMed

    Guo, Yirui; Scheuermann, Thomas H; Partch, Carrie L; Tomchick, Diana R; Gardner, Kevin H

    2015-03-20

    The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. These findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.

  9. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism.

    PubMed

    Elks, Philip M; Brizee, Sabrina; van der Vaart, Michiel; Walmsley, Sarah R; van Eeden, Fredericus J; Renshaw, Stephen A; Meijer, Annemarie H

    2013-01-01

    Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic

  10. Hypoxia-induced tetraploidisation of a diploid human melanoma cell line in vitro.

    PubMed Central

    Rofstad, E. K.; Johnsen, N. M.; Lyng, H.

    1996-01-01

    Many human tumours are hyperdiploid, particularly in advanced stages of growth. The purpose of the present work was to investigate whether exposure to hypoxia followed by reoxygenation might induce hyperploidisation of diploid human tumour cells in vitro. The investigation was performed by using the diploid melanoma cell line BEX-c (median chromosome number, 46; DNA index, 1.10 +/- 0.04) as test line and the hyperdiploid melanoma cell line SAX-c (median chromosome number, 61; DNA index, 1.42 +/- 0.03) as control line. Cell cultures kept in glass dishes in air-tight steel chambers were exposed to hypoxia (O2 concentrations < 10 p.p.m. or < 100 p.p.m.) at 37 degrees C for 24 h. DNA content was measured by flow cytometry. Metaphase spreads banded with trypsin-Versene-Giemsa were examined to determine the number of chromosomes per cell. An electronic particle counter was used to measure cell volume. The expression of p53 and pRb was studied by Western blot analysis. Transient exposure to hypoxia was found to induce a doubling of the number of chromosomes in BEX-c but not in SAX-c. The fraction of the BEX-c metaphase spreads with 92 chromosomes was approximately 10% at 18 h after reoxygenation, decreased to approximately 2% at 7 days after reoxygenation and then increased gradually with time. The whole cell population became tetraploid within 25 weeks. BEX-c and SAX-c behaved differently during the 24 h hypoxia exposure. Cell volume and fraction of cells in G2 + M increased with time in BEX-c but remained essentially unchanged in SAX-c. On the other hand, the expression of p53 and pRb was similar for the two lines; hypoxia induced increased expression of p53 and hypophosphorylation of pRb. Images Figure 5 PMID:8763866

  11. VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting.

    PubMed

    Oladipupo, Sunday; Hu, Song; Kovalski, Joanna; Yao, Junjie; Santeford, Andrea; Sohn, Rebecca E; Shohet, Ralph; Maslov, Konstantin; Wang, Lihong V; Arbeit, Jeffrey M

    2011-08-09

    Although our understanding of the molecular regulation of adult neovascularization has advanced tremendously, vascular-targeted therapies for tissue ischemia remain suboptimal. The master regulatory transcription factors of the hypoxia-inducible factor (HIF) family are attractive therapeutic targets because they coordinately up-regulate multiple genes controlling neovascularization. Here, we used an inducible model of epithelial HIF-1 activation, the TetON-HIF-1 mouse, to test the requirement for VEGF in HIF-1 mediated neovascularization. TetON-HIF-1, K14-Cre, and VEGF(flox/flox) alleles were combined to create TetON-HIF-1:VEGF(Δ) mice to activate HIF-1 and its target genes in adult basal keratinocytes in the absence of concomitant VEGF. HIF-1 induction failed to produce neovascularization in TetON-HIF-1:VEGF(Δ) mice despite robust up-regulation of multiple proangiogenic HIF targets, including PlGF, adrenomedullin, angiogenin, and PAI-1. In contrast, endothelial sprouting was preserved, enhanced, and more persistent, consistent with marked reduction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic microscopy, which provides noninvasive, label-free, high resolution, and wide-field vascular imaging, revealed the absence of both capillary expansion and arteriovenous remodeling in serially imaged individual TetON-HIF-1:VEGF(Δ) mice. Impaired TetON-HIF-1:VEGF(Δ) neovascularization could be partially rescued by 12-O-tetradecanoylphorbol-13-acetate skin treatment. These data suggest that therapeutic angiogenesis for ischemic cardiovascular disease may require treatment with both HIF-1 and VEGF.

  12. Disrupting Hypoxia-Induced Bicarbonate Transport Acidifies Tumor Cells and Suppresses Tumor Growth.

    PubMed

    McIntyre, Alan; Hulikova, Alzbeta; Ledaki, Ioanna; Snell, Cameron; Singleton, Dean; Steers, Graham; Seden, Peter; Jones, Dylan; Bridges, Esther; Wigfield, Simon; Li, Ji-Liang; Russell, Angela; Swietach, Pawel; Harris, Adrian L

    2016-07-01

    Tumor hypoxia is associated clinically with therapeutic resistance and poor patient outcomes. One feature of tumor hypoxia is activated expression of carbonic anhydrase IX (CA9), a regulator of pH and tumor growth. In this study, we investigated the hypothesis that impeding the reuptake of bicarbonate produced extracellularly by CA9 could exacerbate the intracellular acidity produced by hypoxic conditions, perhaps compromising cell growth and viability as a result. In 8 of 10 cancer cell lines, we found that hypoxia induced the expression of at least one bicarbonate transporter. The most robust and frequent inductions were of the sodium-driven bicarbonate transporters SLC4A4 and SLC4A9, which rely upon both HIF1α and HIF2α activity for their expression. In cancer cell spheroids, SLC4A4 or SLC4A9 disruption by either genetic or pharmaceutical approaches acidified intracellular pH and reduced cell growth. Furthermore, treatment of spheroids with S0859, a small-molecule inhibitor of sodium-driven bicarbonate transporters, increased apoptosis in the cell lines tested. Finally, RNAi-mediated attenuation of SLC4A9 increased apoptosis in MDA-MB-231 breast cancer spheroids and dramatically reduced growth of MDA-MB-231 breast tumors or U87 gliomas in murine xenografts. Our findings suggest that disrupting pH homeostasis by blocking bicarbonate import might broadly relieve the common resistance of hypoxic tumors to anticancer therapy. Cancer Res; 76(13); 3744-55. ©2016 AACR.

  13. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis

    PubMed Central

    Gupta, Ram; Chaudhary, Anita R; Shah, Binita N; Jadhav, Avinash V; Zambad, Shitalkumar P; Gupta, Ramesh Chandra; Deshpande, Shailesh; Chauthaiwale, Vijay; Dutt, Chaitanya

    2014-01-01

    Background and aim Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn’s disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD. PMID:24493931

  14. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

    PubMed Central

    Gardner, Lauren H.; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C.

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells. PMID:27148974

  15. Coiled-coil Coactivators Play a Structural Role Mediating Interactions in Hypoxia-inducible Factor Heterodimerization*

    PubMed Central

    Guo, Yirui; Scheuermann, Thomas H.; Partch, Carrie L.; Tomchick, Diana R.; Gardner, Kevin H.

    2015-01-01

    The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. These findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated. PMID:25627682

  16. Natural Product-Derived Small Molecule Activators of Hypoxia-Inducible Factor-1 (HIF-1)

    PubMed Central

    Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1α and a constitutively expressed HIF-1β subunit. In general, the availability and activity of the HIF-1α subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders. The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through pharmacological evaluation of specifically selected individual compounds. The combination of natural products chemistry with appropriate high-throughput screening bioassays could provide an alternative approach to discover novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke. PMID:16842166

  17. Hypoxia Inducible Factor 1α Promotes Endogenous Adaptive Response in Rat Model of Chronic Cerebral Hypoperfusion

    PubMed Central

    Yang, Ying; Ju, Jieyang; Deng, Min; Wang, Jing; Liu, Hui; Xiong, Li; Zhang, Junjian

    2017-01-01

    Hypoxia inducible factor 1α (HIF-1α), a pivotal regulator of gene expression in response to hypoxia and ischemia, is now considered to regulate both pro-survival and pro-death responses depending on the duration and severity of the stress. We previously showed that chronic global cerebral hypoperfusion (CCH) triggered long-lasting accumulation of HIF-1α protein in the hippocampus of rats. However, the role of the stabilized HIF-1α in CCH is obscure. Here, we knock down endogenous HIF-1α to determine whether and how HIF-1α affects the disease processes and phenotypes of CCH. Lentivirus expressing HIF-1α small hairpin RNA was injected into the bilateral hippocampus and bilateral ventricles to knock down HIF-1α gene expression in the hippocampus and other brain areas. Permanent bilateral common carotid artery occlusions, known as 2-vessel occlusions (2VOs), were used to induce CCH in rats. Angiogenesis, oxidative stress, histopathological changes of the brain, and cognitive function were tested. Knockdown of HIF-1α prior to 2VO significantly exacerbates the impairment of learning and memory after four weeks of CCH. Mechanically, reduced cerebral angiogenesis, increased oxidative damage, and increased density of astrocytes and microglia in the cortex and some subregions of hippocampus are also shown after four weeks of CCH. Furthermore, HIF-1α knockdown also disrupts upregulation of regulated downstream genes. Our findings suggest that HIF-1α-protects the brain from oxidative stress and inflammation response in the disease process of CCH. Accumulated HIF-1α during CCH mediates endogenous adaptive processes to defend against more severe hypoperfusion injury of the brain, which may provide a therapeutic benefit. PMID:28106731

  18. Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling

    PubMed Central

    Zhu, Pengcheng; Huang, Lei; Ge, Xiaona; Yan, Fei; Wu, Renliang; Ao, Qilin

    2006-01-01

    Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle α-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin. PMID:17222214

  19. Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6β4-dependent Akt, GSK-3β, and HSF-1 in mesenchymal stem cells.

    PubMed

    Son, T W; Yun, S P; Yong, M S; Seo, B N; Ryu, J M; Youn, H Y; Oh, Y M; Han, H J

    2013-03-28

    Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [(3)H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6β4, except uncoordinated family member (UNC) 5A-C, and neogenin. Among them, IN α6β4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3β (GSK-3β) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3β were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and formation

  20. Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6β4-dependent Akt, GSK-3β, and HSF-1 in mesenchymal stem cells

    PubMed Central

    Son, T W; Yun, S P; Yong, M S; Seo, B N; Ryu, J M; Youn, H Y; Oh, Y M; Han, H J

    2013-01-01

    Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [3H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6β4, except uncoordinated family member (UNC) 5A–C, and neogenin. Among them, IN α6β4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3β (GSK-3β) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3β were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and

  1. HIF Prolyl-Hydoxylase-2 Senses High Salt Intake to Increase Hypoxia Inducible Factor-1α Levels in the Renal Medulla

    PubMed Central

    Wang, Zhengchao; Zhu, Qing; Xia, Min; Li, Pin-Lan; Hinton, Shante J.; Li, Ningjun

    2010-01-01

    High salt induces the expression of transcription factor hypoxia-inducible factor (HIF)-1α and its target genes in the renal medulla, which is an important renal adaptive mechanism to high salt intake. HIF prolyl hydroxylase domain-containing proteins (PHDs) have been identified as major enzymes to promote the degradation of HIF-1α. PHD2 is the predominant isoform of PHDs in the kidney and primarily expressed in the renal medulla. The present study tested the hypothesis that PHD2 responds to high salt and mediates high salt-induced increase in HIF-1α levels in the renal medulla. In normotensive rats, high salt intake (4% NaCl, 10 days) significantly inhibited PHD2 expressions and enzyme activities in the renal medulla. Renal medullary overexpression of PHD2 transgene significantly decreased HIF-1α levels. PHD2 transgene also blocked high salt-induced activation of HIF-1α target genes heme oxygenase-1 and nitric oxide synthase-2 in the renal medulla. In Dahl salt-sensitive hypertensive rats, however, high salt intake did not inhibit the expression and activities of PHD2 in the renal medulla. Correspondingly, renal medullary HIF-1α levels were not up-regulated by high salt intake in these rats. After transfection of PHD2 shRNA, HIF-1α and its target genes were significantly up-regulated by high salt intake in Dahl S rats. Overexpression of PHD2 transgene in the renal medulla impaired renal sodium excretion after salt loading. These data suggest that high salt intake inhibits PHD2 in the renal medulla, thereby upregulating the HIF-1α expression. The lack of PHD-mediated response to high salt may represent a pathogenic mechanism producing salt sensitive hypertension. PMID:20308610

  2. Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells.

    PubMed

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  3. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  4. Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction

    PubMed Central

    Pistilli, Emidio E.; Bogdanovich, Sasha; Mosqueira, Matias; Lachey, Jennifer; Seehra, Jasbir

    2010-01-01

    Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-β (TGFβ) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings. PMID:19864340

  5. Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction.

    PubMed

    Pistilli, Emidio E; Bogdanovich, Sasha; Mosqueira, Matias; Lachey, Jennifer; Seehra, Jasbir; Khurana, Tejvir S

    2010-01-01

    Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-beta (TGFbeta) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings.

  6. Hypoxia Induces a HIF-1-Dependent Transition from Collective-to-Amoeboid Dissemination in Epithelial Cancer Cells.

    PubMed

    Lehmann, Steffi; Te Boekhorst, Veronika; Odenthal, Julia; Bianchi, Roberta; van Helvert, Sjoerd; Ikenberg, Kristian; Ilina, Olga; Stoma, Szymon; Xandry, Jael; Jiang, Liying; Grenman, Reidar; Rudin, Markus; Friedl, Peter

    2017-02-06

    Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent (∼30%) or independent (∼70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs.

  7. Hypoxia imaging predicts success of hypoxia-induced cytosine deaminase/5-fluorocytosine gene therapy in a murine lung tumor model.

    PubMed

    Lee, B-F; Lee, C-H; Chiu, N-T; Hsia, C-C; Shen, L-H; Shiau, A-L

    2012-04-01

    Tc-99m-HL91 is a hypoxia imaging biomarker. The aim of this study was to investigate the value of Tc-99m-HL91 imaging for hypoxia-induced cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy in a murine lung tumor model. C57BL/6 mice were implanted with Lewis lung carcinoma cells transduced with the hypoxia-inducible promoter-driven CD gene (LL2/CD) or luciferase gene (LL2/Luc) serving as the control. When tumor volumes reached 100 mm(3), pretreatment images were acquired after injection of Tc-99m-HL91. The mice were divided into low and high hypoxic groups based on the tumor-to-non-tumor ratio of Tc-99m-HL91. They were injected daily with 5-FC (500 mg kg(-1)) or the vehicle for 1 week. When tumor volumes reached 1000 mm(3), autoradiography and histological examinations were performed. Treatment with 5-FC delayed tumor growth and enhanced the survival of mice bearing high hypoxic LL2/CD tumors. The therapeutic effect of hypoxia-induced CD/5-FC gene therapy was more pronounced in high hypoxic tumors than in low hypoxic tumors. This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of hypoxia-regulated CD/5-FC gene therapy in animal tumor models. Our results suggest that hypoxia imaging using Tc-99m-HL91 has the predictive value for the success of hypoxia-directed treatment regimens.

  8. High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

    PubMed

    Nijmeh, Hala; Balasubramaniam, Vivek; Burns, Nana; Ahmad, Aftab; Stenmark, Kurt R; Gerasimovskaya, Evgenia V

    2014-04-01

    Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

  9. Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype.

    PubMed

    Watson, Chris J; Collier, Patrick; Tea, Isaac; Neary, Roisin; Watson, Jenny A; Robinson, Claire; Phelan, Dermot; Ledwidge, Mark T; McDonald, Kenneth M; McCann, Amanda; Sharaf, Osama; Baugh, John A

    2014-04-15

    Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2'-deoxycytidine suppressed the pro-fibrotic effects of TGFβ. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.

  10. The design of dextran-based hypoxia-inducible hydrogels via in situ oxygen-consuming reaction.

    PubMed

    Park, Kyung Min; Blatchley, Michael R; Gerecht, Sharon

    2014-11-01

    Hypoxia plays a critical role in the development and wound healing process, as well as a number of pathological conditions. Here, dextran-based hypoxia-inducible (Dex-HI) hydrogels formed with in situ oxygen consumption via a laccase-medicated reaction are reported. Oxygen levels and gradients were accurately predicted by mathematical simulation. It is demonstrated that Dex-HI hydrogels provide prolonged hypoxic conditions up to 12 h. The Dex-HI hydrogel offers an innovative approach to delineate not only the mechanism by which hypoxia regulates cellular responses, but may facilitate the discovery of new pathways involved in the generation of hypoxic and oxygen gradient environments.

  11. Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation.

    PubMed

    Naranjo-Suárez, Salvador; Castellanos, María Carmen; Alvarez-Tejado, Miguel; Vara, Alicia; Landázuri, Manuel O; del Peso, Luis

    2003-08-22

    Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxia-inducible factors (HIFs). In the presence of oxygen, specific HIF residues become hydroxylated by the action of a recently described group of dioxygenases. These post-translational modifications target HIF for proteosomal degradation and prevent its transcriptional activity. Despite these detailed studies, little is known about the regulation of HIF by stimuli other than hypoxia. Here we report that, in rat pheochromocytoma PC12 cells, nerve growth factor (NGF) stimulation results in a decrease of both basal and hypoxia-induced levels of HIF-2 alpha protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2 alpha protein level upon stimulation was observed even in the presence of HIF-hydroxylase inhibitors such as deferoxamine or dimethyloxoglutarate. Thus, in contrast to the response to hypoxia, the effect of NGF on HIF-2 alpha protein levels is not mediated by the HIF hydroxilases. Quantitative real time (RT)-PCR showed that NGF stimulation results in a decrease of the HIF-2 alpha mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2 alpha mRNA because it did not affect HIF-1 alpha mRNA levels. NGF treatment reduced HIF-2 alpha mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2 alpha correlates with reduction of both basal and hypoxia-induced vascular endothelial growth factor mRNA levels. Reporter assays suggest that the reduced expression of hypoxia-inducible genes upon NGF treatment is related, at least in part, to the reduction of HIF-2 alpha protein. Hence, in PC12 cells the level of HIF-2 alpha protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.

  12. Plasma from human volunteers subjected to remote ischemic preconditioning protects human endothelial cells from hypoxia-induced cell damage.

    PubMed

    Weber, Nina C; Riedemann, Isabelle; Smit, Kirsten F; Zitta, Karina; van de Vondervoort, Djai; Zuurbier, Coert J; Hollmann, Markus W; Preckel, Benedikt; Albrecht, Martin

    2015-03-01

    Short repeated cycles of peripheral ischemia/reperfusion (I/R) can protect distant organs from subsequent prolonged I/R injury; a phenomenon known as remote ischemic preconditioning (RIPC). A RIPC-mediated release of humoral factors might play a key role in this protection and vascular endothelial cells are potential targets for these secreted factors. In the present study, RIPC-plasma obtained from healthy male volunteers was tested for its ability to protect human umbilical endothelial cells (HUVEC) from hypoxia-induced cell damage. 10 healthy male volunteers were subjected to a RIPC-protocol consisting of 4 × 5 min inflation/deflation of a blood pressure cuff located at the upper arm. Plasma was collected before (T0; control), directly after (T1) and 1 h after (T2) the RIPC procedure. HUVEC were subjected to 24 h hypoxia damage and simultaneously incubated with 5% of the respective RIPC-plasma. Cell damage was evaluated by lactate dehydrogenase (LDH)-measurements. Western blot experiments of hypoxia inducible factor 1 alpha (HIF1alpha), phosphorylated signal transducer and activator of transcription 5 (STAT5), protein kinase B (AKT) and extracellular signal-related kinase 1/2 (ERK-1/2) were performed. Furthermore, the concentrations of hVEGF were evaluated in the RIPC-plasma by sandwich ELISA. Hypoxia-induced cell damage was significantly reduced by plasma T1 (p = 0.02 vs T0). The protective effect of plasma T1 was accompanied by an augmentation of the intracellular HIF1alpha (p = 0.01 vs T0) and increased phosphorylation of ERK-1/2 (p = 0.03 vs T0). Phosphorylation of AKT and STAT5 remained unchanged. Analysis of the protective RIPC-plasma T1 showed significantly reduced levels of hVEGF (p = 0.01 vs T0). RIPC plasma protects endothelial cells from hypoxia-induced cell damage and humoral mediators as well as intracellular HIF1alpha may be involved.

  13. Chronic Intermittent Hypoxia Induces Chronic Low-Grade Neuroinflammation in the Dorsal Hippocampus of Mice

    PubMed Central

    Sapin, Emilie; Peyron, Christelle; Roche, Frédéric; Gay, Nadine; Carcenac, Carole; Savasta, Marc; Levy, Patrick; Dematteis, Maurice

    2015-01-01

    : Sapin E, Peyron C, Roche F, Gay N, Carcenac C, Savasta M, Levy P, Dematteis M. Chronic intermittent hypoxia induces chronic low-grade neuroinflammation in the dorsal hippocampus of mice. SLEEP 2015;38(10):1537–1546. PMID:26085297

  14. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  15. Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish

    PubMed Central

    Köblitz, Louise; Fiechtner, Birgit; Baus, Katharina; Lussnig, Rebecca; Pelster, Bernd

    2015-01-01

    The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for

  16. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

    SciTech Connect

    Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.; Akita, Geoffrey Y.; Cheng, Seng H.; Gregory, Richard J.; Jiang Canwen

    2007-12-21

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.

  17. Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1alpha in human colon cancer cells

    SciTech Connect

    Riganti, Chiara

    2009-11-01

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na{sup +}/K{sup +}-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca{sup ++}]{sub i}). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca{sup ++}]{sub i} and this event was dependent on the calcium influx via the Na{sup +}/Ca{sup ++} exchanger. The increased [Ca{sup ++}]{sub i} enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na{sup +}/Ca{sup ++} exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

  18. MiR-142 modulates human pancreatic cancer proliferation and invasion by targeting hypoxia-inducible factor 1 (HIF-1α) in the tumor microenvironments

    PubMed Central

    Lu, Yebin; Ji, Niandong; Sun, Weijia; Gong, Xuejun; Wang, Xitao

    2017-01-01

    ABSTRACT MicroRNAs regulate most protein-coding genes, including genes important in cancer and other diseases. In this study, we demonstrated that the expression of miR-142 could be significantly suppressed in pancreatic cancer specimens and cell lines compared to their adjacent tissues and normal pancreatic cells. Growth and invasion of PANC-1 and SW1990 cells were attenuated by overexpression of miR-142 in vitro. With the help of bioinformatics analysis, hypoxia-inducible factor 1 (HIF-1α) was identified to be a direct target of miR-142, and a luciferase reporter experiment confirmed this discovery. Overexpression of miR-142 decreases protein expression of HIF-1α. In the hypoxic microenvironment, HIF-1α was up-regulated while miR-142 was down-regulated. The invaded cells significantly increased in the hypoxic microenvironment compared to the normoxic microenvironment. The hypoxia treatment induced cells’ proliferation, and invasion could be inhibited by miR-142 overexpression or HIF-1α inhibition. Moreover, expression of epithelial-mesenchymal transition (EMT) markers, Vimentin, VEGF-C and E-cad, was altered under hypoxia conditions and regulated by miR-142/HIF-1α. Above all, these findings provided insights on the functional mechanism of miR-142, suggesting that the miR-142/HIF-1α axis may interfere with the proliferative and invasive properties of pancreatic cancer cells, and indicated that miR-142 could be a potential therapeutic target for pancreatic cancer. PMID:28069592

  19. Cooperation between HMGA1 and HIF-1 Contributes to Hypoxia-Induced VEGF and Visfatin Gene Expression in 3T3-L1 Adipocytes

    PubMed Central

    Messineo, Sebastiano; Laria, Anna Elisa; Arcidiacono, Biagio; Chiefari, Eusebio; Luque Huertas, Raúl M.; Foti, Daniela P.; Brunetti, Antonio

    2016-01-01

    The architectural transcription factor high-mobility group AT-hook 1 (HMGA1) is a chromatin regulator with implications in several biological processes, including tumorigenesis, inflammation, and metabolism. Previous studies have indicated a role for this factor in promoting the early stages of adipogenesis, while inhibiting adipocyte terminal differentiation, and decreasing fat mass. It has been demonstrated that hypoxia – through the hypoxia-inducible factor 1 (HIF-1) – plays a major role in triggering changes in the adipose tissue of the obese, leading to inhibition of adipocyte differentiation, adipose cell dysfunction, inflammation, insulin resistance, and type 2 diabetes. To examine the possible cooperation between HMGA1 and HIF-1, herein, we investigated the role of HMGA1 in the regulation of Visfatin and VEGF, two genes normally expressed in adipose cells, which are both responsive to hypoxia. We demonstrated that HMGA1 enhanced Visfatin and VEGF gene expression in human embryonic kidney (HEK) 293 cells in hypoxic conditions, whereas HMGA1 knockdown in differentiated 3T3-L1 adipocytes reduced these effects. Reporter gene analysis showed that Visfatin and VEGF transcriptional activity was increased by the addition of either HMGA1 or HIF-1 and even further by the combination of both factors. As demonstrated by chromatin immunoprecipitation in intact cells, HMGA1 directly interacted with the VEGF gene, and this interaction was enhanced in hypoxic conditions. Furthermore, as indicated by co-immunoprecipitation studies, HMGA1 and HIF-1 physically interacted with each other, supporting the notion that this association may corroborate a functional link between these factors. Therefore, our findings provide evidence for molecular cross-talk between HMGA1 and HIF-1, and this may be important for elucidating protein and gene networks relevant to obesity. PMID:27445976

  20. Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells

    PubMed Central

    Sheng, Lingling; Mao, Xiyuan; Yu, Qingxiong; Yu, Dong

    2017-01-01

    Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor. PMID:28123468

  1. Metal allergens induce nitric oxide production by mouse dermal fibroblasts via the hypoxia-inducible factor-2α-dependent pathway.

    PubMed

    Kuroishi, Toshinobu; Bando, Kanan; Endo, Yasuo; Sugawara, Shunji

    2013-09-01

    Nickel (Ni) has been shown to be one of the most frequent metal allergens. We have already reported a murine metal allergy model with pathogen-associated molecular patterns (PAMPs) as adjuvants. Interleukin (IL)-1β plays a critical role in our mouse model. Because nonimmune cells, including fibroblasts, play important roles in local allergic inflammation, we investigated whether Ni induces inflammatory responses in mouse dermal fibroblasts (MDF). We also analyzed the synergistic effects between Ni, PAMPs, and IL-1β. MDF stimulated with Ni produced a significantly higher amount of nitric oxide (NO) in a dose-dependent manner. NO production was augmented by costimulation with IL-1β but not with PAMPs. On the other hand, IL-1β or PAMPs induced a significantly higher amount of IL-6 production by MDF, but no augmentation was detected in the presence of Ni. A specific inhibitor for inducible nitric oxide synthase (iNOS) inhibited Ni-induced NO production. iNOS mRNA expression was significantly higher in MDF stimulated with Ni, IL-1β, or both. A specific inhibitor for hypoxia-inducible factor (HIF)-2α, but not HIF-1α, inhibited NO production. Another frequent metal allergen, cobalt, also induced iNOS expression and NO production by MDF via the HIF-2α-dependent pathway. The inhibitor for iNOS augmen