RESUMO
Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm-/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm-/- mice with aging, but the resulting â¼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm-/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm-/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Nefropatias/genética , Rim/patologia , Degeneração Macular/genética , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Epitélio Pigmentado da Retina/patologia , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Eritropoetina/sangue , Eritropoetina/metabolismo , Humanos , Rim/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Pulmão/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Distribuição TecidualRESUMO
OBJECTIVE: Small-molecule hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) inhibitors are being explored in clinical studies for the treatment of anemia. HIF-P4H-2 (also known as PHD2 or EglN1) inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. We studied here whether HIF-P4H-2 inhibition could also protect against atherosclerosis. APPROACH AND RESULTS: Atherosclerosis development was studied in low-density lipoprotein (LDL) receptor-deficient mice treated with an oral HIF-P4H inhibitor, FG-4497, and in HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice, all mice being fed a high-fat diet. FG-4497 administration to LDL receptor-deficient mice reduced the area of atherosclerotic plaques by ≈50% when compared with vehicle-treated controls and also reduced their weight gain, insulin resistance, liver and white adipose tissue (WAT) weights, adipocyte size, number of inflammation-associated WAT macrophage aggregates and the high-fat diet-induced increases in serum cholesterol levels. The levels of atherosclerosis-protecting circulating autoantibodies against copper-oxidized LDL were increased. The decrease in atherosclerotic plaque areas correlated with the reductions in weight, serum cholesterol levels, and WAT macrophage aggregates and the autoantibody increase. FG-4497 treatment stabilized HIF-1α and HIF-2α and altered the expression of glucose and lipid metabolism and inflammation-associated genes in liver and WAT. The HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice likewise had a ≈50% reduction in atherosclerotic plaque areas, reduced WAT macrophage aggregate numbers, and increased autoantibodies against oxidized LDL, but did not have reduced serum cholesterol levels. CONCLUSIONS: HIF-P4H-2 inhibition may be a novel strategy for protecting against the development of atherosclerosis. The mechanisms involve beneficial modulation of the serum lipid profile and innate immune system and reduced inflammation.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Inibidores Enzimáticos/farmacologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/enzimologia , Adiposidade/efeitos dos fármacos , Animais , Aorta/enzimologia , Aorta/imunologia , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/enzimologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Autoanticorpos/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Colesterol/sangue , Modelos Animais de Doenças , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/sangue , Resistência à Insulina , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica , Estabilidade Proteica , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Aumento de Peso/efeitos dos fármacosRESUMO
Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2ß2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.
Assuntos
Condrócitos/enzimologia , Matriz Extracelular/metabolismo , Osteocondrodisplasias/enzimologia , Pró-Colágeno-Prolina Dioxigenase/deficiência , Animais , Apoptose , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Osteocondrodisplasias/embriologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/fisiopatologia , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.
Assuntos
Eritropoese/fisiologia , Eritropoetina/sangue , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Western Blotting , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hematócrito , Hemoglobinas/metabolismo , Hepcidinas , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The hypoxia-inducible transcription factor (HIF) controls (in an oxygen-dependent manner) the expression of a large number of genes whose products are involved in the response of cells to hypoxia. HIF is an αß dimer that binds to hypoxia response elements (HREs) in its target genes. Human HIF-α has three isoforms, HIF-1α, HIF-2α and HIF-3α, of which the roles of HIF-3α are largely unknown, although it is usually regarded as a negative regulator of HIF-1α and HIF-2α. The human HIF-3α locus is subject to extensive alternative splicing, leading to at least seven variants. We analyzed here the effects of the long variants and the short variant HIF-3α4 on the hypoxia response. All these variants were found to interact with HIF-ß, HIF-1α and HIF-2α. The long HIF-3α variants were localized in the nucleus in hypoxia, while HIF-3α4 was cytoplasmic. Interaction of the HIF-3α variants with HIF-1α inhibited the nuclear translocation of both. None of the long HIF-3α variants was capable of efficient induction of an HRE reporter in overexpression experiments, but instead inhibited the transcriptional activation of the reporter by HIF-1 and HIF-2. Unexpectedly, siRNA knock-down of the endogenous HIF-3α variants led to downregulation of certain HIF target genes, while overexpression of individual long HIF-3α variants upregulated certain HIF target genes in a variant and target gene-specific manner under conditions in which HIF-ß was not a limiting factor. These data indicate that the HIF-3α variants may have more versatile and specific roles in the regulation of the hypoxia response than previously anticipated.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Variação Genética/genética , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Hipóxia Celular/genética , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras , Células Tumorais CultivadasRESUMO
Prolyl 4-hydroxylases (P4Hs) catalyze the hydroxylation of collagens and hypoxia-inducible factor (HIF)-α subunits. We studied the zebrafish homologue of the recently characterized human transmembrane P4H (P4H-TM) that can hydroxylate HIF-α, but not collagens, in vitro and influence HIF-α levels in cellulo. The zebrafish P4H-TM mRNA had its highest expression in the eye and brain and lower levels in other tissues, including the kidney. Morpholino knockdown of P4H-TM in embryos resulted in a reduction in the size of the eye and head and morphological alterations in the head from 2 days postfertilization onward. In addition, pericardial edema, regarded as a sign of kidney dysfunction, developed from 3 days postfertilization onward. The phenotype was dependent on the P4H-TM catalytic activity because similar results were obtained with morpholinos targeting either translation initiation or catalytic residues of the enzyme. Structural and functional analyses of the morphant pronephric kidneys revealed fragmented glomerular basement membranes (BMs), disorganized podocyte foot processes, and severely compromised pronephric kidney function leading to proteinuria. The opacity of the eye lens was increased due to the presence of extra nuclei and deposits, and the structure of the lens capsule BM was altered. Our data suggest that P4H-TM catalytic activity is required for the proper development of the glomerular and lens capsule BMs. Many HIF target genes were induced in the P4H-TM-deficient morphants, but the observed phenotype is not likely to be mediated at least solely via the HIF pathway, and thus P4H-TM probably has additional, as yet unknown, substrates.
Assuntos
Membrana Basal/metabolismo , Regulação Enzimológica da Expressão Gênica , Rim/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Catálise , Coração/fisiologia , Hipóxia , Hibridização In Situ , Microscopia Eletrônica de Transmissão/métodos , Modelos Biológicos , Podócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Peixe-ZebraRESUMO
Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34-47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.
Assuntos
Dioxigenases/metabolismo , Glucose/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Doença Aguda , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Circulação Coronária/genética , Dioxigenases/genética , Glucose/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glicólise/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Especificidade de Órgãos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Promoter hypermethylation is one of the common mechanisms leading to gene silencing in various human cancers. Using a combination of pharmacologic unmasking and microarray techniques, we identified 59 candidate hypermethylated genes, including LOXL1, a lysyl oxidase-like gene, in human bladder cancer cells. We further showed that LOXL1 and LOXL4 are commonly silenced genes in human bladder cancer cells, and this silence is predominantly related to promoter methylation. We also found LOXL1 and LOXL4 gene methylation and loss of expression in primary bladder tumors. In addition, somatic mutations were identified in LOXL4, but not in LOXL1 in bladder cancer. Moreover, reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells leads to a decrease of colony formation ability. Further studies indicated that the overexpression of LOXL1 and LOXL4 could antagonize Ras in activating the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, our current study suggests for the first time that lysyl oxidase-like genes can act as tumor suppressor genes and exert their functions through the inhibition of the Ras/ERK signaling pathway in human bladder cancer.
Assuntos
Aminoácido Oxirredutases/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Neoplasias da Bexiga Urinária/genética , Proteínas ras/antagonistas & inibidores , Aminoácido Oxirredutases/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Citoplasma/enzimologia , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Mutação , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Proteína-Lisina 6-Oxidase , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteínas ras/metabolismoRESUMO
Collagens and proteins with collagen-like domains form large superfamilies in various species, and the numbers of known family members are increasing constantly. Vertebrates have at least 27 collagen types with 42 distinct polypeptide chains, >20 additional proteins with collagen-like domains and approximately 20 isoenzymes of various collagen-modifying enzymes. Caenorhabditis elegans has approximately 175 cuticle collagen polypeptides and two basement membrane collagens. Drosophila melanogaster has far fewer collagens than many other species but has approximately 20 polypeptides similar to the catalytic subunits of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. More than 1300 mutations have so far been characterized in 23 of the 42 human collagen genes in various diseases, and many mouse models and C. elegans mutants are also available to analyse the collagen gene family and their modifying enzymes.
Assuntos
Colágeno/genética , Colágeno/metabolismo , Mutação , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno/química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genes de Helmintos , Genes de Insetos , Humanos , Camundongos , Família Multigênica , Estrutura Terciária de ProteínaRESUMO
Hypoxia-inducible transcription factor (HIF) is regulated by two oxygen-dependent events that are catalyzed by the HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF-P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF-P4Hs were at least 40-50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2-oxoglutarate in the absence of any peptide substrate. The purified HIF-P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF-P4Hs in vitro, especially of HIF-P4H-2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF-P4H-2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF-P4Hs during storage at 4 degrees C. The well-known stabilization of HIF-alpha by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF-alpha even in concentrations that produce no stabilization of HIF-alpha.
Assuntos
Desferroxamina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metais/farmacologia , Oxigênio/farmacologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cobalto/farmacologia , Humanos , Ferro/farmacologia , Dados de Sequência Molecular , Níquel/farmacologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/isolamento & purificação , Soroalbumina Bovina/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Zinco/farmacologiaRESUMO
We show here that mice hypomorphic for hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2) (Hif-p4h-2 (gt/gt)), the main regulator of the stability of the HIFα subunits, have normoxic stabilization of HIF-1α and HIF-2α in their skeletal muscles. The size of the capillaries, but not their number, was increased in the skeletal muscles of the Hif-p4h-2 (gt/gt) mice, whereas the amount of glycogen was reduced. The expression levels of genes for glycolytic enzymes, glycogen branching enzyme 1 and monocarboxylate transporter 4, were increased in the Hif-p4h-2 (gt/gt) skeletal muscles, whereas no significant increases were detected in the levels of any vasculature-influencing factor studied. Serum lactate levels of the Hif-p4h-2 (gt/gt) mice recovered faster than those of the wild type following exercise. The Hif-p4h-2 (gt/gt) mice had elevated hepatic phosphoenolpyruvate carboxykinase activity, which may have contributed to the faster clearance of lactate. The Hif-p4h-2 (gt/gt) mice had smaller infarct size following limb ischemia-reperfusion injury. The increased capillary size correlated with the reduced infarct size. Following ischemia-reperfusion, glycogen content and ATP/ADP and CrP/Cr levels of the skeletal muscle of the Hif-p4h-2 (gt/gt) mice were higher than in the wild type. The higher glycogen content correlated with increased expression of phosphofructokinase messenger RNA (mRNA) and the increased ATP/ADP and CrP/Cr levels with reduced apoptosis, suggesting that HIF-P4H-2 deficiency supported energy metabolism during ischemia-reperfusion and protection against injury. Key messages: HIF-P4H-2 deficiency protects skeletal muscle from ischemia-reperfusion injury. The mechanisms involved are mediated via normoxic HIF-1α and HIF-2α stabilization. HIF-P4H-2 deficiency increases capillary size but not number. HIF-P4H-2 deficiency maintains energy metabolism during ischemia-reperfusion.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Traumatismo por Reperfusão/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Glicogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Fatores de Proteção , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND: The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. METHODS AND RESULTS: Murine Lox gene was disrupted by routine methods. Lox(-/-) mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox(-/-) aortas were observed, and electron microscopy of the aortic walls of the Lox(-/-) fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox(-/-) fetuses. The wall of the aorta in the Lox(-/-) fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox(+/+) aortas. In Lox(-/-) fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. CONCLUSIONS: Lox has an essential role in the development and function of the cardiovascular system. Inactivation of the Lox gene causes structural alterations in the arterial walls, leading to abnormalities in the cardiovascular functions. Alterations in LOX activity may also play a critical role in certain human cardiovascular diseases.
Assuntos
Aneurisma Aórtico/genética , Anormalidades Cardiovasculares/genética , Doenças Cardiovasculares/genética , Proteína-Lisina 6-Oxidase/deficiência , Proteína-Lisina 6-Oxidase/genética , Animais , Animais Recém-Nascidos , Aorta/diagnóstico por imagem , Aorta/embriologia , Aorta/patologia , Aneurisma Aórtico/congênito , Aneurisma Aórtico/patologia , Velocidade do Fluxo Sanguíneo , Anormalidades Cardiovasculares/mortalidade , Anormalidades Cardiovasculares/patologia , Doenças Cardiovasculares/congênito , Doenças Cardiovasculares/patologia , Doenças Fetais/embriologia , Doenças Fetais/genética , Doenças Fetais/patologia , Viabilidade Fetal/genética , Marcação de Genes , Genes Letais , Coração/embriologia , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Miocárdio/patologia , Fenótipo , Ultrassonografia DopplerRESUMO
Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.
Assuntos
Mutação , Isomerases de Dissulfetos de Proteínas/química , Ribonuclease Pancreático/química , Animais , Bovinos , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2-deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Glucose/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Metabolismo dos Lipídeos/fisiologia , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Adipócitos/metabolismo , Animais , Dieta , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Inflamação/genética , Inflamação/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Obesidade/genéticaRESUMO
Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Insuficiência Cardíaca/complicações , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/imunologia , Constrição Patológica/fisiopatologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Pressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Disfunção Ventricular Esquerda/etiologia , Suporte de Carga/fisiologiaRESUMO
Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2(gt/gt) mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2(gt/gt) hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2(gt/gt) hearts than in wild-type hearts. Hif-p4h-2(gt/gt) mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2(gt/gt) hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/patologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miocárdio/patologia , Pró-Colágeno-Prolina Dioxigenase/genética , Animais , Apoptose , Hipóxia Celular , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Vasos Coronários/ultraestrutura , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Coração/fisiopatologia , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , RNA Mensageiro/genética , Receptor TIE-2/metabolismo , Transdução de SinaisRESUMO
The hypoxia-inducible transcription factors (HIFs) play a central role in the response of cells to hypoxia. HIFs are alphabeta dimers, the human alpha subunit having three isoforms. HIF-3alpha is unique among the HIF-alpha isoforms in that its gene is subject to extensive alternative splicing. Database analyses have predicted the generation of six HIF-3alpha splice variants that utilize three alternative transcription initiation sites. None of these variants is likely to act as an efficient transcription factor, but some of them have been reported to inhibit HIF-1 and HIF-2 functions. We analyzed here for the first time in detail whether these six variants are indeed generated in various human tissues and cell lines. We identified four novel variants, named here HIF-3alpha7 to HIF-3alpha10, whereas we obtained no evidence for the predicted HIF-3alpha3 and HIF-3alpha5. Distinct differences in the expression patterns of the variants were found between human tissues, the levels being particularly low in many cancer cell lines. Hypoxia upregulated transcription from all three alternative HIF-3alpha promoters. siRNA experiments showed that this induction is mediated specifically by HIF-1 and not by HIF-2. The tissue-specific differences in the expression patterns and levels of the HIF-3alpha variants can be expected to modulate the hypoxia response of various tissues and cell types to different extents during development and in pathological situations. A further level of regulation is brought about by the fact that the levels of the HIF-3alpha transcripts themselves are regulated by hypoxia and by changes in HIF-1 levels.
Assuntos
Processamento Alternativo/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/genética , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Hipóxia Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Metilação , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 A resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 A resolution. The structures reveal the double-stranded beta-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates.
Assuntos
Proteínas de Algas/química , Chlamydomonas reinhardtii/enzimologia , Pró-Colágeno-Prolina Dioxigenase/química , Proteínas de Protozoários/química , Proteínas de Algas/metabolismo , Animais , Sítios de Ligação/fisiologia , Parede Celular/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Peptídeos/química , Peptídeos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/química , Prolina/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas de Protozoários/metabolismo , Piridinas/química , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologia , Zinco/químicaRESUMO
Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.
Assuntos
Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Perda do Embrião/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Animais , Membrana Basal/patologia , Domínio Catalítico , Colágeno Tipo IV/genética , Colágeno Tipo IV/ultraestrutura , Desenvolvimento Embrionário/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Deleção de Genes , Camundongos , Microscopia Eletrônica , Gravidez , Pró-Colágeno-Prolina Dioxigenase/metabolismoRESUMO
The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by two oxygen-dependent events that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH. Fumarate and succinate were identified as in vitro inhibitors of all three HIF-P4Hs, fumarate having K(i) values of 50-80 microM and succinate 350-460 microM, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with K(i) values of 400-1000 microM and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a K(i) of 110 microM. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1alpha and increased production of vascular endothelial growth factor and erythropoietin. Similar, although much smaller, changes were found in cultured fibroblasts from a patient with fumarate hydratase (FH) deficiency and upon silencing FH using small interfering RNA. No such effects were seen upon culturing of cells with succinate diethyl or dimethyl ester. As FIH was not inhibited by fumarate, our data indicate that the transcriptional activity of HIF is quite high even when binding of the coactivator p300 is prevented. Our data also support recent suggestions that the increased fumarate and succinate levels present in the FH and succinate dehydrogenase-deficient tumors, respectively, can inhibit the HIF-P4Hs with consequent stabilization of HIF-alphas and effects on tumor pathology.