ABSTRACT
While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.
Subject(s)
Erythropoietin , Response Elements , Cell Hypoxia , Erythropoietin/genetics , Gene Expression , Humans , Hypoxia/genetics , Hypoxia-Inducible Factor 1, alpha SubunitABSTRACT
Werner syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies.
Subject(s)
Exodeoxyribonucleases/metabolism , Neoplasm Proteins/metabolism , RecQ Helicases/metabolism , Werner Syndrome/genetics , Cells, Cultured , Exodeoxyribonucleases/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Mitochondria/metabolism , Mitochondrial Proteins , Reactive Oxygen Species/metabolism , RecQ Helicases/genetics , Werner Syndrome HelicaseABSTRACT
Hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR) are members of the bHLH-PAS family of transcription factors that underpin cellular responses to oxygen and to endogenous and exogenous ligands, respectively, and have central roles in the pathogenesis of renal cancer. Composed of heterodimers, they share a common HIF-1ß/ARNT subunit and similar DNA-binding motifs, raising the possibility of crosstalk between the two transcriptional pathways. Here, we identify both general and locus-specific mechanisms of interaction between HIF and AHR that act both antagonistically and cooperatively. Specifically, we observe competition for the common HIF-1ß/ARNT subunit, in cis synergy for chromatin binding, and overlap in their transcriptional targets. Recently, both HIF and AHR inhibitors have been developed for the treatment of solid tumours. However, inhibition of one pathway may promote the oncogenic effects of the other. Therefore, our work raises important questions as to whether combination therapy targeting both of these pro-tumourigenic pathways might show greater efficacy than targeting each system independently.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Cell Hypoxia/physiology , Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , Kidney/metabolismABSTRACT
Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a 'pathway tuning' model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Renal Cell/genetics , Gene Expression Regulation, Neoplastic , Kidney Neoplasms/genetics , Signal Transduction/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Datasets as Topic , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , Kidney Neoplasms/pathology , Mutation , Polymorphism, Single Nucleotide , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Hypoxia-inducible factors (HIF-1 and HIF-2) are essential mediators for the adaptive transcriptional response of cells and tissues to low-oxygen conditions. Under hypoxia or when cells are treated with various nonhypoxic stimuli, the active HIF-α subunits are mainly regulated through increased protein stabilization. For HIF-1α, it is clear that further transcriptional, translational, and posttranslational regulations are important for complete HIF-1 activity. Novel evidence links hypoxia and HIF-1 to arginine methylation, an important protein modification. These studies suggest that arginine methyltransferases may be important for hypoxic responses. Protein arginine methyltransferase 1 (PRMT1), the predominant arginine methyltransferase, can act as a transcriptional activator or repressor by modifying a diverse set of substrates. In this work, we show that PRMT1 is a repressor of both HIF-1 and HIF-2. The cellular depletion of PRMT1 by small interference RNA targeting leads to increased HIF transcriptional activity. This activation is the result of enhanced HIF-α subunit transcription, which allows increased HIF-α subunit availability. We provide evidence that PRMT1-dependent HIF-1α regulation is mediated through the activities of both specificity protein 1 (Sp1) and Sp3, two transcription factors known to control HIF-1α expression. This study therefore identifies PRMT1 as a novel regulator of HIF-1- and HIF-2-mediated responses.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Protein Processing, Post-Translational , Protein-Arginine N-Methyltransferases/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Arginine/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Hypoxia/genetics , Genes, Reporter , HEK293 Cells , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Luciferases/genetics , Luciferases/metabolism , Methylation , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Signal Transduction , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/genetics , Sp3 Transcription Factor/metabolismABSTRACT
Hypoxia-inducible transcription factor-1 (HIF-1) plays a decisive role in cell survival and adaptation to hypoxic stress by controlling the expression of genes involved in oxygen homeostasis. HIF-1 activity is fine-tuned through specific post-translational modifications of its essential HIF-1α subunit. Among these modifications, phosphorylation is important for HIF-1 transcriptional activity. Studies have shown that the mitogen-activated protein kinases, p42/p44 MAPKs, directly phosphorylate HIF-1α and increase HIF-1-mediated transcription. Pin1, a peptidyl-prolyl cis/trans isomerase, targets a number of proteins containing a phosphorylated Ser/Thr-Pro motif. Pin1 isomerization causes a change in target protein conformation which can modify their activity. Here, we identify Pin1 as an important HIF-1α partner. Immunoprecipitation and pull-down studies show that Pin1 interacts with HIF-1α. We demonstrate that the interaction between Pin1 and HIF-1α is regulated through p42/p44 MAPK pathway activation. By performing proteolysis studies, our results indicate that Pin1 catalytic activity generates a conformational change in HIF-1α. Finally, our work shows that Pin1 is required for gene-specific HIF-1 transcriptional activity. Our results indicate that the prolyl isomerase Pin1 regulates HIF-1 transcriptional activity by interacting with HIF-1α and promoting conformational changes in a p42/p44 MAPK phosphorylation-dependent manner.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Peptidylprolyl Isomerase/metabolism , Animals , Cell Line , HEK293 Cells , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/antagonists & inhibitors , Peptidylprolyl Isomerase/genetics , Phosphorylation , Protein Interaction Domains and Motifs , RNA Interference , RNA, Small Interfering/metabolism , Transcription, GeneticABSTRACT
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.