Induction of the mitochondrial NDUFA4L2 protein by HIF-1α decreases oxygen consumption by inhibiting complex i activity

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-1This work was supported by Ministerio de Ciencia e Innovación (SAF 2007-06592, SAF2010-14851), Comunidad Autónoma de Madrid (SAL 2006/ 0311), Metoxia Project-Health (F2 2009-222741), and Recava Network (RD 06/0014/0031) to M.O.L.; PS09/00101 and CP07/00143 to A.M.-R.; PI060701, PS09/00116, and CP08/00204 to S.C.; BFU2008-03407/BMC to J.A.; SAF2009-08007 to J.A.E.; and CSD2007-00020 to A.M.-R. and J.A.E. The CNIC is supported by the Instituto de Salud Carlos III-MICINN and the Pro-CNIC Foundation. We are grateful to Mike Murphy (Mitochondrial Biology Unit, MRC, Cambridge, UK) for the gift of MitoQ. We also thank Stephen Y. Chan and Joseph Loscalzo (Harvard Medical School, Boston, MA) for providing us ISCU expression vector

Hypoxia Inducible Factor 1-Alpha (HIF-1 Alpha) Is Induced during Reperfusion after Renal Ischemia and Is Critical for Proximal Tubule Cell Survival

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant

Accumulation of hypoxia-inducible factor-1α through a novel electrophilic, thiol antioxidant-sensitive mechanism

15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) is a peroxisome-activated proliferator receptor-γ (PPARγ) agonist which contains an α,β-unsaturated electrophilic ketone involved in nucleophilic addition reactions to thiols. Here we studied its effect on hypoxia-inducible factor-1α (HIF-1α) in human proximal tubular cells HK-2. 15d-PGJ2 induced stabilization of HIF-1α protein, without affecting HIF-1α mRNA levels or proteasome activity, leading to its nuclear accumulation and activation of HIF-induced transcription. Accumulation of HIF-1α was unaffected by selective PPARγ blockade nor mimicked by the PPARγ agonists ciglitazone and 9,10-dihydro-15d-PGJ2. N-acetylcysteine, reduced glutathione (GSH) or dithiothreitol (i.e. agents that act as thiol reducing agents and/or increase the GSH content), but not reactive oxygen species (ROS) scavengers, prevented 15d-PGJ2-induced HIF-1α accumulation whereas the inhibitor of GSH synthesis buthionine sulfoximine cooperated with 15d-PGJ2 to accumulate HIF-1α. Finally, HIF-1α expression was increased by the electrophilic α,β-unsaturated compounds acrolein and PGA2, but not by 9,10-dihydro-15d-PGJ2, which lacks the electrophilic cyclopentenone moiety. Taken together, these results point out to a new mechanism to increase pharmacologically the cell levels of HIF-1α through the electrophilic reaction of α,β-unsaturated ketones with thiol groups. © 2007 Elsevier Inc. All rights reserved.This work was supported by grants from the Spanish Ministry of Education and Science (SAF2005-06242-C03-01), the Comunidad de Madrid-Universidad Alcalá (CAM-UAH2005/001), the Fundación Médica Mutua Madrileña Automovilista, and the Comunidad de Madrid (S-SAL-0311/2006).Peer Reviewe

Analysis of HIF-prolyl hydroxylases binding to substrates

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. HIFα 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 HIF1α in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1α 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. © 2006 Elsevier Inc. All rights reserved.This work was supported by grants from Fondo de Investigaciones Sanitarias (FIS01/0264 to L.P.), from Ministerio de Ciencia y Tecnologı´a (SAF2002-02344 and SAF2005-00180 to L.P. and SAF2004-00824 to M.O.L.), and from Red Cardiovascular (RECAVA).Peer Reviewe

Defective interleukin 2 receptor expression is associated with the T cell disfunction subsequent to bone marrow transplantation

In the present work we have used monoclonal antibodies (mAb) as probes to attempt a dissection of the mechanisms underlying the immunodeficiency subsequent to bone marrow transplantation (BMT). To this end we have studied 19 allogeneic BMT recipients, analyzing the proliferative response of peripheral blood mononuclear cells (PBMC) after activation with either phytohemagglutinin (PHA), anti-CD3 or anti-CD 2 mAb. All patients presented normal proportions of CD2+ and CD3+ lymphocytes, as assessed by flow cytometry. Our results indicated that in most cases both CD 2 and CD 3-mediated activation pathways were inefficient to trigger normal T cell proliferation. The addition of exogenous interleukin 2 (IL 2) did not restore in most cases the proliferative response, pointing out that additional defects contribute to the hyporesponsiveness. This was more evident in the group of patients studied during the first 6 months. To further dissect the T cell defect we analyzed the effect of a phorbol ester (phorbol myristate acetate, PMA), which activates protein kinase C, on the anti-CD 3-induced response. Our data showed that PMA synergized with anti-CD 3 similarly to exogenous IL 2, and restored the proliferative response only in certain cases. The expression of IL 2 receptors (CD 25) as assessed by cytofluorimetry, after either PHA or anti-CD 3 and PMA stimulation, was shown to be depressed, and the addition of IL 2 did not restore it. Finally, we observed that the early increase of intracytoplasmic Ca2+ after anti-CD 3 stimulation was comparable to that detected in normal PBMC. Altogether these results indicate that a diminished CD 25 expression is associated with the T cell defect, and cannot apparently be attributed to an inability of the CD 3 molecule to transduce early activation signals thus suggesting that either protein kinase C itself or an as yet undefined metabolic step preceding IL 2 receptor expression is abnormal in variable proportions of T cells after BMT, and constitutes another manifestation of this complex immunodeficiency.This work was supported by grants from INSALUD (FIS 86/825) and CAICYT (0456-84):Peer reviewe

c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

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-1α subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1α 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

Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1 integrin fibrillar adhesions

8 páginas, 7 figuras -- PAGS nros. 2929-2936The 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 β1 and αv 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” αv focal contacts. As the culture advanced and cells became confluent, αv integrins partly relocated to the intercellular junctions in VHL(+) transfectants, which then developed large β1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL(−) cells were unable to assemble β1 fibrillar adhesions, and αv focal contacts remained unchanged at all stages of the culture. Exogenous activation of β1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL(−) cells to assemble β1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common β1-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 β1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL(−) renal cancer cellsThis work was supported by Grants SAF2001/0215 and FEDER-2FD971870 from the Ministerio de Educación y Cultura. Miguel A. Esteban-Barragán, Miguel Álvarez-Tejado, and M. Dolores Gutiérrez were supported by fellowships from Comunidad Autónoma de MadridPeer reviewe

Myocardial protection during ischemia-reperfusion

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