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ABSTRACT: Uncoupling protein 3 (UCP3) is a member of the mitochondrial inner membrane carrier superfamily that modulates energy efficiency by catalyzing proton conductance and thus decreasing the production of superoxide anion. However, its role during oxidative stress and the underlying regulatory and molecular mechanisms remain poorly understood. We sought to investigate how UCP3 expression is regulated by oxidative stress and to evaluate the putative antioxidant role of this protein. H2O2 treatment increased UCP3 expression and the nuclear accumulation of the transcription factor Nrf2 in C2C12 and HL-1 cells. Nrf2 siRNA prevented H2O2- induced UCP3 expression, increasing oxidative stress and cell death. ChIP assays identified an antioxidant response element (ARE) within the UCP3 promoter that bound Nrf2 following exposure to H2O2. Luciferase reporter experiments confirmed increased ARE activity in H2O2-treated HL-1 cells. Importantly, H2O2 increased the UCP3-mediated proton leak, suggesting a role for this protein in attenuating ROS-induced damage. Nrf2 nuclear accumulation and increased UCP3 protein were also detected in intact mouse heart subjected to a condition known to increase ROS generation. This is the first study to demonstrate that H2O2 augments UCP3 expression and it provides the first evidence of Nrf2 binding to the UCP3 promoter in response to oxidative challenge. These findings suggest that UCP3 functions as a member of the cellular antioxidant defence system that protects against oxidative stress in vivo. In conclusion, we have identified a novel regulatory process induced by an oxidative insult whereby the expression of the mitochondrial protein UCP3 is driven by the Nrf2 transcription factor, which decreases ROS production and prevents cell death.
Free radical biology & medicine 04/2013; · 5.42 Impact Factor
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Ainara Elorza,
Inés Soro-Arnáiz,
Florinda Meléndez-Rodríguez,
Victoria Rodríguez-Vaello,
Glenn Marsboom,
Guillermo de Cárcer,
Bárbara Acosta-Iborra,
Lucas Albacete-Albacete,
Angel Ordóñez,
Leticia Serrano-Oviedo,
Jose Miguel Giménez-Bachs,
Alicia Vara-Vega,
Antonio Salinas,
Ricardo Sánchez-Prieto,
Rafael Martín Del Río,
Francisco Sánchez-Madrid,
Marcos Malumbres, Manuel O Landázuri,
Julián Aragonés
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ABSTRACT: The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.
Molecular cell 10/2012; · 14.61 Impact Factor
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ABSTRACT: The oxidative phosphorylation system is one of the best-characterized metabolic pathways. In mammals, the protein components and X-ray structures are defined for all complexes except complex I. Here, we show that NDUFA4, formerly considered a constituent of NADH Dehydrogenase (CI), is instead a component of the cytochrome c oxidase (CIV). Deletion of NDUFA4 does not perturb CI. Rather, proteomic, genetic, evolutionary, and biochemical analyses reveal that NDUFA4 plays a role in CIV function and biogenesis. The change in the attribution of the NDUFA4 protein requires renaming of the gene and reconsideration of the structure of CIV. Furthermore, NDUFA4 should be considered a candidate gene for CIV rather than CI deficiencies in humans.
Cell metabolism 08/2012; 16(3):378-86. · 17.35 Impact Factor
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Daniel Tello,
Eduardo Balsa,
Bárbara Acosta-Iborra,
Esther Fuertes-Yebra,
Ainara Elorza,
Ángel Ordóñez,
María Corral-Escariz,
Inés Soro,
Elia López-Bernardo,
Ester Perales-Clemente,
Antonio Martínez-Ruiz,
José Antonio Enríquez,
Julián Aragonés,
Susana Cadenas, Manuel O Landázuri
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ABSTRACT: The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1.
Cell metabolism 11/2011; 14(6):768-79. · 17.35 Impact Factor
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Marta Miró-Murillo,
Ainara Elorza,
Inés Soro-Arnáiz,
Lucas Albacete-Albacete,
Angel Ordoñez,
Eduardo Balsa,
Alicia Vara-Vega,
Silvia Vázquez,
Esther Fuertes,
Carmen Fernández-Criado, Manuel O Landázuri,
Julián Aragonés
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ABSTRACT: Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2). Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed)-UBC-Cre-ER(T2) adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2) tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2) mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.
PLoS ONE 01/2011; 6(7):e22589. · 4.09 Impact Factor
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ABSTRACT: Under hypoxic conditions, mitochondria can represent a threat to the cell because of their capacity to generate toxic reactive oxygen species (ROS). However, cardiomyocytes are equipped with an oxygen-sensing pathway that involves prolyl hydroxylase oxygen sensors and hypoxia-inducible factors (HIFs), which induces a tightly regulated programme to keep ischaemic mitochondrial activity under control. The aim of this review is to provide an update on the pathways leading to mitochondrial reprogramming, which occurs in the myocardium during ischaemia, with particular emphasis on those induced by HIF activation. We start by studying the mechanisms of mitochondrial damage during ischaemia and upon reperfusion, highlighting the importance of the formation of the mitochondrial permeability transition pore during reperfusion and its consequences for cardiomyocyte survival. Next, we analyse hypoxia-induced metabolic reprogramming through HIF and its important consequences for mitochondrial bioenergetics, as well as the phenomenon known as the hibernating myocardium. Subsequently, we examine the mechanisms underlying ischaemic preconditioning, focusing, in particular, on those that involve the HIF pathway, such as adenosine signalling, sub-lethal ROS generation, and nitric oxide production. Finally, the role of the mitochondrial uncoupling proteins in ischaemia tolerance is discussed.
Cardiovascular research 11/2010; 88(2):219-28. · 5.80 Impact Factor
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ABSTRACT: Low oxygen tension areas are found in inflamed or diseased tissues where hypoxic cells induce survival pathways by regulating the hypoxia-inducible transcription factor (HIF). Macrophages are essential regulators of inflammation and, therefore, we have analyzed their response to hypoxia. Murine peritoneal elicited macrophages cultured under hypoxia produced higher levels of IFN-gamma and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-gamma increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-gamma induction was mediated through HIF-1alpha binding to its promoter on a new functional hypoxia response element. The requirement of HIF-alpha in the IFN-gamma induction was confirmed in RAW264.7 cells, where HIF-1alpha was knocked down, as well as in resident HIF-1alpha null macrophages. Moreover, Ag presentation capacity was enhanced in hypoxia through the up-regulation of costimulatory and Ag-presenting receptor expression. Hypoxic macrophages generated productive immune synapses with CD8 T cells that were more efficient for activation of TCR/CD3epsilon, CD3zeta and linker for activation of T cell phosphorylation, and T cell cytokine production. In addition, hypoxic macrophages bound opsonized particles with a higher efficiency, increasing their phagocytic uptake, through the up-regulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1alpha- and in IFN-gamma-silenced macrophages, indicating a link between HIF-1alpha and IFN-gamma in the functional responses of macrophages to hypoxia. Our data underscore an important role of hypoxia in the activation of macrophage cytokine production, Ag-presenting activity, and phagocytic activity due to an HIF-1alpha-mediated increase in IFN-gamma levels.
The Journal of Immunology 04/2009; 182(5):3155-64. · 5.79 Impact Factor
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ABSTRACT: HIFs [hypoxia-inducible (transcription) factors] are essential for the induction of an adaptive gene expression programme under low oxygen partial pressure. The activity of these transcription factors is mainly determined by the stability of the HIFalpha subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl 4-hydroxylases [EGLN1-EGLN3 (EGL nine homologues 1-3)]. HIFalpha contains two, N- and C-terminal, independent ODDs (oxygen-dependent degradation domains), namely NODD and CODD, that, upon hydroxylation by the EGLNs, target HIFalpha for proteasomal degradation. In vitro studies indicate that each EGLN shows a differential preference for ODDs, However, the sequence determinants for such specificity are unknown. In the present study we showed that whereas EGLN1 and EGLN2 acted upon any of these ODDs to regulate HIF1alpha protein levels and activity in vivo, EGLN3 only acted on the CODD. With the aim of identifying the region within EGLNs responsible for their differential substrate preference, we investigated the activity and binding pattern of different EGLN deletions and chimaeric constructs generated by domain swapping between EGLN1 and EGLN3. These studies revealed a region of 97 residues that was sufficient to confer the characteristic substrate binding observed for each EGLN. Within this region, we identified the minimal sequence (EGLN1 residues 236-252) involved in substrate discrimination. Importantly, mapping of these sequences on the EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site.
Biochemical Journal 01/2008; 408(2):231-40. · 4.90 Impact Factor
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ABSTRACT: Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presence of oxygen, target HIF for degradation. HIFalpha contains two independent oxygen degradation domains (N-ODD and C-ODD) that are substrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required for the binding of EGLN1 and 3 to HIF1alpha in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1alpha protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novel critical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical, residue preference at specific positions in their substrate sequences.
Biochemical and Biophysical Research Communications 01/2007; 351(2):313-20. · 2.48 Impact Factor
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ABSTRACT: Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.
Cancer Research 03/2006; 66(3):1553-60. · 7.86 Impact Factor
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ABSTRACT: Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.
Biochemical Journal 09/2005; 390(Pt 1):189-97. · 4.90 Impact Factor
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ABSTRACT: Hypoxia-inducible factors (HIF-1/HIF-2) govern the expression of critical genes for cellular adaptation to low oxygen tensions. We have previously reported that the intracellular level of phosphatidic acid (PA) rises in response to hypoxia (1% O(2)). In this report, we have explored whether components of the canonical HIF/von Hippel-Lindau (VHL) pathway are involved in the induction of PA. We found that hypoxia induces PA in a cell line constitutively expressing a stable version of HIF-1alpha. PA induction was also found in HIF-1alpha- and 2alpha-negative CHO Ka13 cells, as well as in HIF-beta-negative HepaC4 cells. These data indicate that HIF activity is neither sufficient nor necessary for oxygen-dependent PA accumulation. PA generation was also detected in cells deficient for the tumor suppressor VHL, indicating that the presence of VHL was not required for the induction of PA. Here we show that PA accumulation also occurs at moderate hypoxia (5% O(2)), although to a lesser extent to that seen at 1% O(2), revealing that PA is induced at the same hypoxia range required to activate HIF-1. Prolyl hydroxylases (PHD) and asparaginyl hydroxylase (FIH) belong to the iron (II) and 2-oxoglutarate-dependent dioxygenase family and have been proposed as oxygen sensors involved in the regulation of HIFs. Chemical inhibition of these activities by treatment with iron chelators or 2-oxoglutarate analogs also results in a marked PA accumulation similar to that observed in hypoxia. Together these data show that PA accumulation in response to hypoxia is both HIF-1/2- and VHL-independent and indicate a role of iron (II)-2-oxoglutarate-dependent dioxygenases in the oxygen-sensing mechanisms involved in hypoxia-driven phospholipid regulation.
Journal of Biological Chemistry 04/2004; 279(10):9504-11. · 4.77 Impact Factor
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ABSTRACT: Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor for the adaptation to lowered oxygen environments. We have previously reported that hypoxia induced phosphatidic acid (PA) accumulation through diacylglycerol kinase (DGK) activity and provided evidence that this PA production regulated HIF-1 expression. Here we report that hypoxia also produces a marked intracellular accumulation of diacylglycerol (DAG) in different cell types. The previously proposed inhibitor of phosphatidylcholine phospholipase C (PC-PLC)/sphingomyelin synthase (SMS) activities, D609, specifically abrogates both hypoxia-dependent DAG accumulation and hypoxia-induced HIF-1 expression. We show that DAG-dependent protein kinase C (PKC) isoforms do not play an essential role in the regulation of HIF-1 expression. D609 inhibits PA accumulation triggered by hypoxia, suggesting that DAG could act as substrate for its conversion into PA by DGK upon these conditions. Therefore, this work provides novel evidence for the existence of DAG/PA-dependent intracellular mechanisms involved in the regulation of HIF-1 expression.
Biochemical and Biophysical Research Communications 03/2004; 315(1):44-50. · 2.48 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 09/2003; 278(34):31895-901. · 4.77 Impact Factor
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ABSTRACT: The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL- cells and their wild-type VHL stably transfected counterparts. The levels of beta1 and alphav integrins were increased in VHL- cells when compared with VHL+ transfectants. Early after plating, both VHL+ and VHL- cells were capable of assembling classic "patch-like" alphav focal contacts. As the culture advanced and cells became confluent, alphav integrins partly relocated to the intercellular junctions in VHL+ transfectants, which then developed large beta1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL- cells were unable to assemble beta1 fibrillar adhesions, and alphav focal contacts remained unchanged at all stages of the culture. Exogenous activation of beta1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL- cells to assemble beta1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common beta1-integrin chain was delayed in VHL- cells when compared with VHL+ cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of beta1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL- renal cancer cells.
Cancer Research 06/2002; 62(10):2929-36. · 7.86 Impact Factor
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ABSTRACT: Hypoxia-inducible factors (HIF) belong to an evolutionary conserved family of transcription factors, the activity of which is tightly regulated by oxygen levels. We have recently demonstrated that hypoxia activates the phosphoinositide 3-kinase (PI3K)/Akt pathway in some cell types, and other works have suggested that this pathway is involved in the activation of HIF. In the present work we studied the role of this pathway in the induction of HIF by hypoxia. Under hypoxic conditions the PI3K/Akt pathway was activated in some (PC12 and HeLa) but not all cell types (HepG2) tested, whereas the HIF protein was induced by hypoxia in all cases. Kinetics analysis showed that, when observed, the activation of PI3K/Akt occurred after HIF induction. In addition, the chemical inhibition of PI3K had no significant effect on the induction of the HIF protein or its transcriptional activity but prevented Akt activation. Accordingly, transient overexpression of a dominant negative form of the regulatory subunit of PI3K in HEK293T cells did not interfere with the induction of the HIF-alpha protein by hypoxia or affect HIF-mediated transcription in any of the cell types tested. Moreover, forced activation of the PI3K/Akt pathway did not affect the transcriptional activity of HIF under normoxic or hypoxic conditions. Thus, our data suggest that the activation of PI3K/Akt by hypoxia is cell type-specific and, when observed, lies downstream of HIF activation or in a parallel pathway. Furthermore, the activity of the PI3K/Akt is not sufficient for the activation of HIF nor is it essential for its induction by hypoxia.
Journal of Biological Chemistry 05/2002; 277(16):13508-17. · 4.77 Impact Factor
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ABSTRACT: Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1alpha subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1alpha are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser(63) in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser(63) and Ser(73) impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis.
Molecular and Cellular Biology 02/2002; 22(1):12-22. · 5.53 Impact Factor
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ABSTRACT: Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor for the adaptation to lowered oxygen environments. We have previously reported that hypoxia induced phosphatidic acid (PA) accumulation through diacylglycerol kinase (DGK) activity and provided evidence that this PA production regulated HIF-1 expression. Here we report that hypoxia also produces a marked intracellular accumulation of diacylglycerol (DAG) in different cell types. The previously proposed inhibitor of phosphatidylcholine phospholipase C (PC-PLC)/sphingomyelin synthase (SMS) activities, D609, specifically abrogates both hypoxia-dependent DAG accumulation and hypoxia-induced HIF-1 expression. We show that DAG-dependent protein kinase C (PKC) isoforms do not play an essential role in the regulation of HIF-1 expression. D609 inhibits PA accumulation triggered by hypoxia, suggesting that DAG could act as substrate for its conversion into PA by DGK upon these conditions. Therefore, this work provides novel evidence for the existence of DAG/PA-dependent intracellular mechanisms involved in the regulation of HIF-1 expression.
Biochemical and Biophysical Research Communications.
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ABSTRACT: The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHL cancer syndrome and in most sporadic renal cancers. VHL is required for the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exoge- nous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL() cells and their wild-type VHL stably transfected counterparts. The levels of 1 and v integrins were increased in VHL() cells when compared with VHL() transfectants. Early after plating, both VHL() and VHL() cells were capable of assembling classic "patch-like" v focal contacts. As the culture advanced and cells became confluent, v integrins partly relocated to the intercellular junctions in VHL() transfectants, which then developed large 1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL() cells were unable to assemble 1 fibrillar adhesions, and v focal contacts remained un- changed at all stages of the culture. Exogenous activation of 1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL() cells to assemble 1 fibrillar adhesions and fi- bronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common 1-integrin chain was delayed in VHL() cells when compared with VHL() cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of 1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL() renal cancer cells.
