The von Hippel Lindau/Hypoxia-inducible Factor (HIF) Pathway Regulates the Transcription of the HIF-Proline Hydroxylase Genes in Response to Low Oxygen

Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo s/n, 28029 Madrid, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2004; 278(49):48690-5. DOI: 10.1074/jbc.M308862200
Source: PubMed


Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIFalpha proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIFalpha is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalyze HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIFalpha escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIFalpha in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIFalpha resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIFalpha.

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    • "HIF1 and HIF2 are known to regulate several common transcriptional targets, but independently are also capable of transcriptionally regulating specific target genes [14]. To assess the transcriptional function of the cell lines, qRT-PCR was performed for canonical HIF targets, egl nine homolog 3, (Egln3) [30] and vascular endothelial growth factor (Vegfa) [31]. Murine embryonic stem (ES) cells expressing a construct with WT Vhl [32] that exhibit maximal HIF regulation and Vhl null ES cells, where both HIFs are endogenously stabilized, were employed as controls. "
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    ABSTRACT: Background Hypoxia Inducible Factors (HIF1α and HIF2α) are commonly stabilized and play key roles related to cell growth and metabolic programming in clear cell renal cell carcinoma. The relationship of these factors to discretely alter cell metabolic activities has largely been described in cancer cells, or in hypoxic conditions, where other confounding factors undoubtedly compete. These transcription factors and their specific roles in promoting cancer metabolic phenotypes from the earliest stages are poorly understood in pre-malignant cells. Methods We undertook an analysis of SV40-transformed primary kidney epithelial cells derived from newborn mice genetically engineered to express a stabilized HIF1α or HIF2α transgene. We examined the metabolic profile in relation to each gene. Results Although the cells proliferated similarly, the metabolic profile of each genotype of cell was markedly different and correlated with altered gene expression of factors influencing components of metabolic signaling. HIF1α promoted high levels of glycolysis as well as increased oxidative phosphorylation in complete media, but oxidative phosphorylation was suppressed when supplied with single carbon source media. HIF2α, in contrast, supported oxidative phosphorylation in complete media or single glucose carbon source, but these cells were not responsive to glutamine nutrient sources. This finding correlates to HIF2α-specific induction of Glul, effectively reducing glutamine utilization by limiting the glutamate pool, and knockdown of Glul allows these cells to perform oxidative phosphorylation in glutamine media. Conclusion HIF1α and HIF2α support highly divergent patterns of kidney epithelial cell metabolic phenotype. Expression of these factors ultimately alters the nutrient resource utilization and energy generation strategy in the setting of complete or limiting nutrients.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Cobalt chloride is thought to induce a hypoxic response by stabilizing HIF-1α through disruption of the hydroxylases responsible for HIF-1α degradation by replacing the iron in the active site of the hydroxylases or by depleting ascorbate, which oxidizes the iron and inactivates the enzyme [34], [35]. In particular, egl nine homolog 3 (Prolyl hydroxylase 3; PHD3 or EGLN3) regulates HIF-1α degradation, and is also transcriptionally up-regulated by HIF-1 α, perhaps by forming a feedback loop [36]. Indeed, del Peso and colleagues reported that HIF-1α is essential for the up-regulation of EGLN3, as it is not induced in cells lacking a functional HIF-1α [36]. "
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    ABSTRACT: Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "These results demonstrated that pVHL contributes to the microtubule network alteration-induced regulation of HIF-1α in hypoxic CMs and H9c2 cells, although the affinity of HIF-1α toward the pVHL decreased in hypoxic cells. Recently, del Peso and colleagues [41] showed that the pVHL/HIF pathway regulated the transcription of EGLNs genes in response to low oxygen, suggesting that EGLNs might be involved in the process of microtubule network alteration-induced regulation of HIF-1α. "
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    ABSTRACT: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.
    Full-text · Article · Apr 2012 · PLoS ONE
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