Differential contribution of n-and c-terminal regions of hif1α and hif2α to their target gene selectivity

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.This work was supported by grants from Ministerio de Economia y Competitividad (SAF2016-76815-R and SAF2017-90794-REDT), Ministerio de Ciencia e Innovación (PID2019-106371RB-I00) and Fundació La Marató de TV3 (534/C/2016). A.A.U is supported by the CAM “Atracción de Talento” program and Universidad Autónoma de Madrid, grant SI1/PJI/2019-0039

Hypoxia-Inducible Factor 2-Dependent Pathways Driving Von Hippel–Lindau-Deficient Renal Cancer

The most common type of the renal cancers detected in humans is clear cell renal cell carcinomas (ccRCCs). These tumors are usually initiated by biallelic gene inactivation of the Von Hippel–Lindau (VHL) factor in the renal epithelium, which deregulates the hypoxia-inducible factors (HIFs) HIF1α and HIF2α, and provokes their constitutive activation irrespective of the cellular oxygen availability. While HIF1α can act as a ccRCC tumor suppressor, HIF2α has emerged as the key HIF isoform that is essential for ccRCC tumor progression. Indeed, preclinical and clinical data have shown that pharmacological inhibitors of HIF2α can efficiently combat ccRCC growth. In this review, we discuss the molecular basis underlying the oncogenic potential of HIF2α in ccRCC by focusing on those pathways primarily controlled by HIF2α that are thought to influence the progression of these tumors

Hypoxia-inducible factor 2α drives hepatosteatosis through the fatty acid translocase CD36

Background & Aims: Molecular mechanisms by which hypoxia might contribute to hepatosteatosis, the earliest stage in non-alcoholic fatty liver disease (NAFLD) pathogenesis, remain still to be elucidated. We aimed to assess the impact of hypoxia-inducible factor 2α (HIF2α) on the fatty acid translocase CD36 expression and function in vivo and in vitro. Methods: CD36 expression and intracellular lipid content were determined in hypoxic hepatocytes, and in hypoxic CD36- or HIF2α -silenced human liver cells. Histological analysis, and HIF2α and CD36 expression were evaluated in livers from animals in which von Hippel-Lindau (Vhl) gene is inactivated (Vhl -deficient mice), or both Vhl and Hif2a are simultaneously inactivated (Vhl Hif2α -deficient mice), and from 33 biopsy-proven NAFLD patients and 18 subjects with histologically normal liver. Results: In hypoxic hepatocytes, CD36 expression and intracellular lipid content were augmented. Noteworthy, CD36 knockdown significantly reduced lipid accumulation, and HIF2A gene silencing markedly reverted both hypoxia-induced events in hypoxic liver cells. Moreover livers from Vhl -deficient mice showed histologic characteristics of non-alcoholic steatohepatitis (NASH) and increased CD36 mRNA and protein amounts, whereas both significantly decreased and NASH features markedly ameliorated in Vhl Hif2α -deficient mice. In addition, both HIF2α and CD36 were significantly overexpressed within the liver of NAFLD patients and, interestingly, a significant positive correlation between hepatic transcript levels of CD36 and erythropoietin (EPO), a HIF2α -dependent gene target, was observed in NAFLD patients. Conclusions: This study provides evidence that HIF2α drives lipid accumulation in human hepatocytes by upregulating CD36 expression and function, and could contribute to hepatosteatosis setup. f/f f/f /f f/f f/f f/fThis work was supported by PI13/01299, PI17/00535 and CIBEREHD from Instituto de Salud Carlos III (ISCIII/FEDER, Spain) to CGM; CP14/00181, PI16/00823 and PI19/00123 (ISCIII/FEDER, Spain), and Beca Eduardo Gallego 2016 (Fundación Francisco Cobos, Spain) to AGR; SAF2016-76815 (Ministerio de Economía y Competitividad/FEDER, Spain), 534/C/2016 (TV3 Marató, Spain) and CIBERCV (ISCIII/FEDER, Spain) to JA

El factor de respuesta a hipoxia HIF1a suprime la proliferación de células tumorales a través de la inhibición de la biosíntesis de aspartato

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de lectura: 26-04-2019Esta tesis tiene embargado el acceso al texto completo hasta el 26-10-2020El aspartato celular controla la proliferación de las células tumorales debido a su función en la síntesis de proteínas y de ácidos nucleicos. Sin embargo, se desconocen las rutas de señalización endógenas que gobiernan el metabolismo del aspartato y si los supresores tumorales ejecutan su acción mediante el control de sus rutas de biosíntesis. El factor inducible por hipoxia HIF1α puede funcionar como supresor tumoral reprimiendo la proliferación autónoma de célula en numerosos tipos de tumores, pero se desconoce en gran medida los mecanismos que subyacen a esta función supresora de la progresión tumoral. En esta tesis se identifica que HIF1 actúa como un represor directo de la biosíntesis de aspartato mediante la inhibición simultánea de la expresión de varias proteínas clave en la producción de aspartato, incluyendo las transaminasas glutamato-oxalacetato GOT1 y GOT2, citosólica y mitocondrial, respectivamente, así como la subunidad succinato deshidrogenasa-A (SDH-A) del complejo II mitocondrial. En consecuencia, HIF1 inhibe la producción de aspartato tanto por la vía oxidativa de la glutamina – implicando a SDH-A y GOT2 - como por la vía de carboxilación reductora de la glutamina – involucrando a GOT1. Por otro lado, la adición exógena de aspartato es capaz de recuperar la proliferación de aquellas células tumorales que expresan HIF1α, demostrando que la supresión de la síntesis de aspartato por parte de HIF1α es esencial para entender su potencial antiproliferativo. Además, la expresión de las enzimas GOT1 y GOT2 está disminuida in vivo en carcinomas renales humanos deficientes para VHL (variedad de tumor donde HIF1α actúa como supresor tumoral por antonomasia), poniendo de manifiesto el valor clínico de estos descubrimientos. En conclusión, demostramos que HIF1α reprime la biosíntesis de aspartato tanto en el citosol como en la mitocondria y que este mecanismo es parte de la base molecular subyacente a la actividad supresora de tumores llevada a cabo por HIF1α.Cellular aspartate drives cancer cell proliferation through its role in protein and nucleotide synthesis. However, signaling pathways that rewire aspartate metabolism and if tumor suppressor proteins execute their actions through control of aspartate biosynthesis pathways remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can act as a tumor suppressor by inhibiting cell autonomous growth in numerous cancer types, but mechanisms underlying its function as suppressor of tumor progression remain elusive. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins including cytosolic glutamic-oxaloacetic transaminase (GOT)-1 and mitochondrial GOT2, as well as succinate dehydrogenase subunit A (SDH-A) of mitochondrial complex II. Thus, HIF1α suppresses aspartate production through both glutamine oxidative, involving SDH-A and GOT2, and glutamine reductive pathways, involving GOT1. Strikingly, aspartate supplementation is sufficient to relieve the HIF1α-dependent repression of tumor cell proliferation, revealing that HIF1α-dependent suppression of aspartate biosynthesis is essential to explain its antiproliferative potential. Moreover, the relevance of these data is highlighted by the fact that GOT1 and GOT2 are repressed in vivo in VHL-deficient human renal carcinomas, a paradigmatic tumor type where HIF1α acts as a tumor suppressor, underscoring the clinical value of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is part of the molecular basis of the HIF1α tumor suppressor activity

Hypoxia-Inducible Factor 2-Dependent Pathways Driving Von Hippel–Lindau-Deficient Renal Cancer

The most common type of the renal cancers detected in humans is clear cell renal cell carcinomas (ccRCCs). These tumors are usually initiated by biallelic gene inactivation of the Von Hippel–Lindau (VHL) factor in the renal epithelium, which deregulates the hypoxia-inducible factors (HIFs) HIF1α and HIF2α, and provokes their constitutive activation irrespective of the cellular oxygen availability. While HIF1α can act as a ccRCC tumor suppressor, HIF2α has emerged as the key HIF isoform that is essential for ccRCC tumor progression. Indeed, preclinical and clinical data have shown that pharmacological inhibitors of HIF2α can efficiently combat ccRCC growth. In this review, we discuss the molecular basis underlying the oncogenic potential of HIF2α in ccRCC by focusing on those pathways primarily controlled by HIF2α that are thought to influence the progression of these tumors.This work was supported by grants from Ministerio de Educación y Ciencia (SAF2013-46058-R; SAF2016-76815), TV3 Marató (534/C/2016), and CIBERCV. OR has a contract for accessing the Spanish System of Science, Technology and Innovation (SECTI) funded by the University of Castilla La Mancha (UCLM).Peer reviewe

Role of Mitochondrial Complex IV in Age-Dependent Obesity

Aging is associated with progressive white adipose tissue (WAT) enlargement initiated early in life, but the molecular mechanisms involved remain unknown. Here we show that mitochondrial complex IV (CIV) activity and assembly are already repressed in white adipocytes of middle-aged mice and involve a HIF1A-dependent decline of essential CIV components such as COX5B. At the molecular level, HIF1A binds to the Cox5b proximal promoter and represses its expression. Silencing of Cox5b decreased fatty acid oxidation and promoted intracellular lipid accumulation. Moreover, local in vivo Cox5b silencing in WAT of young mice increased the size of adipocytes, whereas restoration of COX5B expression in aging mice counteracted adipocyte enlargement. An age-dependent reduction in COX5B gene expression was also found in human visceral adipose tissue. Collectively, our findings establish a pivotal role for CIV dysfunction in progressive white adipocyte enlargement during aging, which can be restored to alleviate age-dependent WAT expansion.publisher: Elsevier articletitle: Role of Mitochondrial Complex IV in Age-Dependent Obesity journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2016.08.041 content_type: article copyright: © 2016 The Authors.status: publishe

Role of Mitochondrial Complex IV in Age-Dependent Obesity.

Aging is associated with progressive white adipose tissue (WAT) enlargement initiated early in life, but the molecular mechanisms involved remain unknown. Here we show that mitochondrial complex IV (CIV) activity and assembly are already repressed in white adipocytes of middle-aged mice and involve a HIF1A-dependent decline of essential CIV components such as COX5B. At the molecular level, HIF1A binds to the Cox5b proximal promoter and represses its expression. Silencing of Cox5b decreased fatty acid oxidation and promoted intracellular lipid accumulation. Moreover, local in vivo Cox5b silencing in WAT of young mice increased the size of adipocytes, whereas restoration of COX5B expression in aging mice counteracted adipocyte enlargement. An age-dependent reduction in COX5B gene expression was also found in human visceral adipose tissue. Collectively, our findings establish a pivotal role for CIV dysfunction in progressive white adipocyte enlargement during aging, which can be restored to alleviate age-dependent WAT expansion