Hypoxia-inducible Factor1 Stabilization in Nonhypoxic Conditions: Role of Oxidation and Intracellular Ascorbate Depletion

Centre de recherche de L'Hôtel-Dieu de Québec, Department of Medicine, Université Laval, Québec, QC, G1R 2J6, Canada.
Molecular biology of the cell (Impact Factor: 4.47). 02/2008; 19(1):86-94. DOI: 10.1091/mbc.E07-06-0612
Source: PubMed


Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1alpha, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1alpha accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein-coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1alpha protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1alpha and allowing HIF-1alpha protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1alpha stability is mediated through the Ang II-mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1alpha stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II-related cell responses.

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Available from: Darren E. Richard, Jan 24, 2014
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    • "The report of HIF-1α accumulation by exogenous addition of H2O2 though supports the role of ROS in HIF-1 activation during normoxic condition. Interestingly, involvement of ROS in HIF-1 activation by several other stimuli like exposures to Ang II [16], [24], [30], thrombin [11], [31] or transition metals [15] has also been reported. In most of these cases ROS was found to affect the PHD activity to stabilize HIF-1α but direct addition of H2O2 or thrombin induced ROS generation was found to activate NF-kB for HIF-1α transcription [11], [26], [31]. "
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    ABSTRACT: Oxygen sensing transcription factor HIF-1 is activated due to accumulation of regulatory subunit HIF-1α by posttranslational stability mechanism during hypoxia or by several other stimuli even in normoxia. HIF-1α is also regulated by NF-kB mediated transcription mechanism. Reactive oxygen species (ROS) act as an important regulator of HIF-1 either by affecting prolyl hydroxylase activity, the critical determinant of HIF-1α stabilization or by activating NF-kB to promote HIF-1α transcription. Insulin is known to activate HIF-1 by a ROS dependent mechanism but the molecular mechanism of HIF-1α regulation is not known so far. Here we show that insulin regulates HIF-1α by a novel transcriptional mechanism by a ROS-sensitive activation of Sp1 in 3T3-L1 preadipocyte. Insulin shows little effect on HIF-1α protein stability, but increases HIF-1α promoter activity. Mutation analyses, electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirm the role of Sp1 in HIF-1α transcription. We further demonstrate that insulin-induced ROS generation initiates signaling pathway involving phosphatidylinositol 3-kinase and protein kinase C for Sp1 mediated HIF-1α transcription. In summary, we reveal that insulin regulates HIF-1α by a novel transcriptional mechanism involving Sp1.
    PLoS ONE 04/2013; 8(4):e62128. DOI:10.1371/journal.pone.0062128 · 3.23 Impact Factor
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    • "On the one hand, ROS mediate transcriptional activation via NF-κB [88] and translational activation via PI3K/AKt/4E-BP1 pathway [89], increasing HIF production. On the other hand, ROS deplete cellular ascorbate, a cofactor for PHD activity, and inhibit HIFα hydroxylation and VHL binding [81, 90, 91], suppressing HIF degradation (see Figure 3). Increased HIF activity promotes angiogenesis. "
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    ABSTRACT: Reactive oxygen species (ROS) are derived from the metabolism of oxygen and are traditionally viewed as toxic byproducts that cause damage to biomolecules. It is now becoming widely acknowledged that ROS are key modulators in a variety of biological processes and pathological states. ROS mediate key signaling transduction pathways by reversible oxidation of certain signaling components and are involved in the signaling of growth factors, G-protein-coupled receptors, Notch, and Wnt and its downstream cascades including MAPK, JAK-STAT, NF-κB, and PI3K/AKT. Vascular formation and development is one of the most important events during embryogenesis and is vital for postnasal tissue repair. In this paper, we will discuss how ROS regulate different steps in vascular development, including smooth muscle cell differentiation, angiogenesis, endothelial progenitor cells recruitment, and vascular cell migration.
    Oxidative Medicine and Cellular Longevity 01/2013; 2013(9):374963. DOI:10.1155/2013/374963 · 3.36 Impact Factor
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    • "Investigators have reported normoxic expression of HIF-1α in mesenchymal stem cells [23], cartilage [24], mouse brain and kidney [25], rat kidney [26], and pulmonary and vascular smooth muscle cells [27,28] among other tissues. One goal of the present study was to determine if rat testicular HIF-1 from normoxic and hypoxic testes is capable of binding DNA. "
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    ABSTRACT: Background Spermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’) identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1. Methods The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis. Results The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter. Conclusions The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.
    Reproductive Biology and Endocrinology 12/2012; 10(1):104. DOI:10.1186/1477-7827-10-104 · 2.23 Impact Factor
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