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1.
BMC Med Genet ; 17: 21, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26969407

RESUMO

BACKGROUND: Plasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder. METHODS & RESULTS: Active plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes. CONCLUSIONS: The functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.


Assuntos
Fator XIIa/genética , Calicreínas/genética , Polimorfismo de Nucleotídeo Único , Sistema Renina-Angiotensina/genética , Renina/sangue , Adolescente , Adulto , Idoso , Alelos , Angiotensina I/sangue , Angiotensinogênio/sangue , Animais , Pressão Sanguínea , Proteínas de Ciclo Celular , Linhagem Celular , Regulação da Expressão Gênica , Loci Gênicos , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Hipertensão/genética , Sistema Justaglomerular/citologia , Calicreínas/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pré-Calicreína/metabolismo , Renina/genética , Serina Endopeptidases/metabolismo , Transferases , Adulto Jovem
2.
PLoS One ; 9(1): e84132, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465394

RESUMO

RATIONALE: The chromogranin A-derived peptide pancreastatin (PST) is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced. METHODS AND RESULTS: We used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273-301-amide) as "bait" and mouse liver homogenate as "prey", and identified GRP78 (a.k.a. "78 kDa Glucose Regulated Protein", HSPA5, BIP) as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones), involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the "adaptive UPR") in CHGA(-/-) mice compared to wild-type (+/+). By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(-/-) mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM). PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin) in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis). In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP) increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP. CONCLUSIONS: Our results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion), but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic response to UPR activation. PST decreases insulin-stimulated cellular glucose uptake, and PST as well as other chaperone ATPase activity inhibitors augment expression of G6Pase; GRP78 over-expression antagonizes this PST action. Analysis of the novel PST/GRP78 interaction may provide a new avenue of investigation into cellular glycemic control as well as dysglycemia.


Assuntos
Cromogranina A/química , Cromogranina A/metabolismo , Proteínas de Choque Térmico/metabolismo , Fígado/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Cromogranina A/genética , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Ligação Proteica , Espectrometria de Massas em Tandem
3.
Hum Mol Genet ; 22(18): 3624-40, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23674521

RESUMO

Hypertension is a common hereditary syndrome with unclear pathogenesis. Chromogranin A (Chga), which catalyzes formation and cargo storage of regulated secretory granules in neuroendocrine cells, contributes to blood pressure homeostasis centrally and peripherally. Elevated Chga occurs in spontaneously hypertensive rat (SHR) adrenal glands and plasma, but central expression is unexplored. In this report, we measured SHR and Wistar-Kyoto rat (control) Chga expression in central and peripheral nervous systems, and found Chga protein to be decreased in the SHR brainstem, yet increased in the adrenal and the plasma. By re-sequencing, we systematically identified five promoter, two coding and one 3'-untranslated region (3'-UTR) polymorphism at the SHR (versus WKY or BN) Chga locus. Using HXB/BXH recombinant inbred (RI) strain linkage and correlations, we demonstrated genetic determination of Chga expression in SHR, including a cis-quantitative trait loci (QTLs) (i.e. at the Chga locus), and such expression influenced biochemical determinants of blood pressure, including a cascade of catecholamine biosynthetic enzymes, catecholamines themselves and steroids. Luciferase reporter assays demonstrated that the 3'-UTR polymorphism (which disrupts a microRNA miR-22 motif) and promoter polymorphisms altered gene expression consistent with the decline in SHR central Chga expression. Coding region polymorphisms did not account for changes in Chga expression or function. Thus, we hypothesized that the 3'-UTR and promoter mutations lead to dysregulation (diminution) of Chga in brainstem cardiovascular control nuclei, ultimately contributing to the pathogenesis of hypertension in SHR. Accordingly, we demonstrated that in vivo administration of miR-22 antagomir to SHR causes substantial (∼18 mmHg) reductions in blood pressure, opening a novel therapeutic avenue for hypertension.


Assuntos
Cromogranina A/genética , Cromogranina A/metabolismo , Hipertensão/genética , MicroRNAs/genética , Regiões Promotoras Genéticas , Regiões 3' não Traduzidas , Glândulas Suprarrenais/metabolismo , Animais , Pressão Sanguínea/genética , Tronco Encefálico/metabolismo , Linhagem Celular Tumoral , Cromogranina A/sangue , Cromogranina A/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Ligação Genética , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , MicroRNAs/metabolismo , Células PC12 , Polimorfismo Genético , Estrutura Secundária de Proteína , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Alinhamento de Sequência , Transcrição Gênica
4.
J Hypertens ; 31(1): 123-33, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23149563

RESUMO

OBJECTIVES: The neuropeptide Y(2) G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10,000 human participants indicate its involvement in control of obesity and BMI. Here we searched for genetic variation across the human NPY2R locus and probed its functional effects, especially in the proximal promoter. METHODS AND RESULTS: Twin pair studies indicated substantial heritability for multiple cardiometabolic traits, including BMI, SBP, DBP, and PYY, an endogenous agonist at NPY2R. Systematic polymorphism discovery by resequencing across NPY2R uncovered 21 genetic variants, 10 of which were common [minor allele frequency (MAF) >5%], creating one to two linkage disequilibrium blocks in multiple biogeographic ancestries. In vivo, NPY2R haplotypes were associated with both BMI (P = 3.75E-04) and PYY (P = 4.01E-06). Computational approaches revealed that proximal promoter variants G-1606A, C-599T, and A-224G disrupt predicted IRF1 (A>G), FOXI1 (T>C), and SNAI1 (A>G) response elements. In neuroendocrine cells transfected with NPY2R promoter/luciferase reporter plasmids, all three variants and their resulting haplotypes influenced transcription (G-1606A, P < 2.97E-06; C-599T, P < 1.17E-06; A-224G, P < 2.04E-06), and transcription was differentially augmented or impaired by coexpression of either the cognate full-length transcription factors or their specific siRNAs at each site. Endogenous expression of transcripts for NPY2R, IRF1, and SNAI1 was documented in neuroendocrine cells, and the NPY2R mRNA was differentially expressed in two neuroendocrine tissues (adrenal gland, brainstem) of a rodent model of hypertension and the metabolic syndrome, the spontaneously hypertensive rat. CONCLUSION: We conclude that common genetic variation in the proximal NPY2R promoter influences transcription factor binding so as to alter gene expression in neuroendocrine cells, and consequently cardiometabolic traits in humans. These results unveil a novel control point, whereby cis-acting genetic variation contributes to control of complex cardiometabolic traits, and point to new transcriptional strategies for intervention into neuropeptide actions and their cardiometabolic consequences.


Assuntos
Predisposição Genética para Doença/genética , Variação Genética/genética , Síndrome Metabólica/genética , Obesidade/genética , Regiões Promotoras Genéticas/genética , Receptores de Neuropeptídeo Y/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Polimorfismo Genético , Ratos , Ratos Endogâmicos WKY , Fatores de Risco
5.
J Hypertens ; 30(10): 1961-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22871890

RESUMO

BACKGROUND: Cathepsin L (CTSL1) catalyzes the formation of peptides that influence blood pressure (BP). Naturally occurring genetic variation or targeted ablation of the Ctsl1 locus in mice yield cardiovascular pathology. Here, we searched for genetic variation across the human CTSL1 locus and probed its functional effects, especially in the proximal promoter. METHODS AND RESULTS: Systematic polymorphism discovery by re-sequencing across CTSL1 in 81 patients uncovered 38 genetic variants, five of which were relatively common (MAF >5%), creating a single linkage disequilibrium block in multiple biogeographic ancestries. One of these five common variants lay in a functional domain of the gene: promoter C-171A (rs3118869), which disrupts a predicted xenobiotic response element (XRE; match C>A). In transfected CTSL1 promoter/luciferase reporter plasmids, C-171A allele influenced transcription (C>A, P = 3.36E-6), and transcription was also augmented by co-exposure to the aryl hydrocarbon receptor (AHR) complex (AHR:ARNT) in the presence of their ligand dioxin (P = 6.81E-8); allele (C vs. A) and AHR:ARNT/dioxin stimulus interacted to control gene expression (interaction P = 0.033). Endogenous Ctsl1, Ahr, and Arnt transcripts were present in chromaffin cells. Promoter functional C-171A genotype also predicted hypertension (P = 1.0E-3), SBP (P = 4.0E-4), and DBP (P = 3.0E-3), in an additive pattern for diploid genotypes (A/A > C/A > C/C) in 868 patients, and the results were extended by validation analysis into an independent population sample of 986 patients. CONCLUSION: We conclude that common genetic variation in the proximal CTSL1 promoter, especially at position C-171A, is functional in cells, and alters transcription so as to explain the association of CTSL1 with BP in vivo. At the XRE, endogenous genetic variation plus exogenous aryl hydrocarbon stimulation interact to control CTSL1 gene expression. These results unveil a novel control point whereby heredity and environment can intersect to control a complex trait, and point to new transcriptional strategies for intervention into transmitter biosynthesis and its cardiovascular consequences.


Assuntos
Pressão Sanguínea/genética , Catepsina L/genética , Interação Gene-Ambiente , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Transcrição Gênica , Xenobióticos/farmacologia , Humanos
6.
Circ Cardiovasc Genet ; 5(4): 430-40, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22670052

RESUMO

BACKGROUND: Essential hypertension, a common complex disease, displays substantial genetic influence. Contemporary methods to dissect the genetic basis of complex diseases such as the genomewide association study are powerful, yet a large gap exists between the fraction of population trait variance explained by such associations and total disease heritability. METHODS AND RESULTS: We developed a novel, integrative method (combining animal models, transcriptomics, bioinformatics, molecular biology, and trait-extreme phenotypes) to identify candidate genes for essential hypertension and the metabolic syndrome. We first undertook transcriptome profiling on adrenal glands from blood pressure extreme mouse strains: the hypertensive BPH (blood pressure high) and hypotensive BPL (blood pressure low). Microarray data clustering revealed a striking pattern of global underexpression of intermediary metabolism transcripts in BPH. The MITRA algorithm identified a conserved motif in the transcriptional regulatory regions of the underexpressed metabolic genes, and we then hypothesized that regulation through this motif contributed to the global underexpression. Luciferase reporter assays demonstrated transcriptional activity of the motif through transcription factors HOXA3, SRY, and YY1. We finally hypothesized that genetic variation at HOXA3, SRY, and YY1 might predict blood pressure and other metabolic syndrome traits in humans. Tagging variants for each locus were associated with blood pressure in a human population blood pressure extreme sample with the most extensive associations for YY1 tagging single nucleotide polymorphism rs11625658 on systolic blood pressure, diastolic blood pressure, body mass index, and fasting glucose. Meta-analysis extended the YY1 results into 2 additional large population samples with significant effects preserved on diastolic blood pressure, body mass index, and fasting glucose. CONCLUSIONS: The results outline an innovative, systematic approach to the genetic pathogenesis of complex cardiovascular disease traits and point to transcription factor YY1 as a potential candidate gene involved in essential hypertension and the cardiometabolic syndrome.


Assuntos
Biologia Computacional/métodos , Hipertensão/genética , Síndrome Metabólica/genética , Miocárdio/metabolismo , Sistemas Neurossecretores/metabolismo , Transcriptoma/genética , Glândulas Suprarrenais/metabolismo , Animais , Sequência de Bases , Pressão Sanguínea/genética , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença , Humanos , Luciferases/metabolismo , Masculino , Metanálise como Assunto , Camundongos , Dados de Sequência Molecular , Miocárdio/patologia , Motivos de Nucleotídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional/genética
7.
Blood Press ; 20(4): 222-31, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21428728

RESUMO

Genetic strategies such as linkage analysis and quantitative trait locus (QTL) mapping have identified a multitude of loci implicated in the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR). While several candidate genetic regions have been identified in the SHR and its control, the Wistar-Kyoto rat (WKY), systematic follow-up of candidate identification with polymorphism discovery has not been widespread. In the current report, we develop a data-mining strategy to identify candidate genes for hypertension in the SHR, and then sequence each gene in the SHR and WKY strains. We integrate blood pressure QTL data, microarray data and data-mining methods. First, we determined the set of genes differentially expressed in SHR and WKY adrenal glands. Next, the chromosomal position of all differentially expressed genes was compared with peak marker position of all reported SHR blood pressure QTLs. We also identified the set of differentially expressed genes with the most extreme fold-change. Finally, the QTL positional candidates and the genes with extreme differential expression were proposed as candidate genes if they had biologically plausible roles in hypertensive pathology. We identified seven candidate genes that merit resequencing (catechol-O-methyltransferase [Comt], chromogranin A [Chga], dopamine beta-hydroxylase [Dbh], electron transferring flavoprotein dehydrogenase [Etfdh], endothelin receptor type B [Ednrb], neuropeptide Y [Npy] and phenylethanolamine-N-methyltransferase [Pnmt]), and then discovered polymorphism in four of these seven candidate genes. Chga is proposed as the strongest candidate for additional functional investigation. Our method for candidate gene identification is portable and can be applied to microarray data from any tissue, in any disease model with a QTL database.


Assuntos
Hipertensão/genética , Ratos Endogâmicos SHR/genética , Animais , Mapeamento Cromossômico , Mineração de Dados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hipertensão/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo Genético , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos WKY
8.
Curr Hypertens Rep ; 13(1): 36-45, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21104344

RESUMO

Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or "granins"), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca(2+). Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension.


Assuntos
Catecolaminas/metabolismo , Cromogranina A/genética , Cromogranina B/genética , Hipertensão/genética , Polimorfismo Genético/genética , Secretogranina II/genética , Análise de Variância , Catecolaminas/genética , Distribuição de Qui-Quadrado , Cromograninas/genética , Cromograninas/metabolismo , Intervalos de Confiança , Progressão da Doença , Feminino , Variação Genética , Genótipo , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Razão de Chances , Fatores de Risco
9.
Cell Mol Neurobiol ; 30(8): 1395-400, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21061160

RESUMO

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.


Assuntos
Cromogranina A/genética , Predisposição Genética para Doença , Variação Genética , Hipertensão Renal/genética , Humanos , Hipertensão Renal/fisiopatologia , Rim/fisiopatologia , Testes de Função Renal , Nefroesclerose/genética , Nefroesclerose/fisiopatologia , Fenótipo , Caracteres Sexuais
10.
Endocrinology ; 151(10): 4820-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20668031

RESUMO

Exaggerated adrenergic activity is associated with human hypertension. The peptide urocortin 2 (Ucn 2) inhibits catecholamine synthesis and secretion from adrenal chromaffin cells in vitro and administration to mammals lowers blood pressure (BP). The chromogranin A-null mouse (Chga-/-) manifests systemic hypertension because of excessive catecholamine secretion from the adrenal and decreased catecholamine storage. In the present study, we investigated whether systemic administration of Ucn 2 could reduce BP and adrenal and plasma levels of catecholamines in vivo. Ucn 2 peptide was administered to freely moving, conscious Chga-/- and wild-type control mice. Telemetry and HPLC measured changes in BP and catecholamine levels, respectively. In both groups of mice, Ucn 2 dose-dependently decreased BP, and this effect was mediated by corticotropin factor-receptor type 2. However, in Chga-/- mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in wild-type mice (P=0.04). In Chga-/- mice only, Ucn 2 decreased adrenal and plasma levels of catecholamines as well as adrenal levels of tyrosine hydroxylase protein and phosphorylation. In vitro mechanistic studies demonstrated that Ucn 2 reduces both catecholamine secretion and tyrosine hydroxylase promoter activity, suggesting that the exaggerated action of Ucn 2 to reduce BP in the Chga-/- mouse is mediated through inhibition of both catecholamine synthesis and secretion. The data suggest that Ucn 2 may be therapeutically useful in regulating the exaggerated sympathoadrenal function of hyperadrenergic hypertension.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Catecolaminas/sangue , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Urocortinas/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Fibras Adrenérgicas/metabolismo , Fibras Adrenérgicas/patologia , Animais , Doenças do Sistema Nervoso Autônomo/genética , Doenças do Sistema Nervoso Autônomo/metabolismo , Doenças do Sistema Nervoso Autônomo/patologia , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Catecolaminas/metabolismo , Cromogranina A/genética , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/genética , Hipertensão/genética , Hipertensão/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Urocortinas/administração & dosagem
11.
Mamm Genome ; 21(3-4): 195-204, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20204374

RESUMO

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.


Assuntos
Biologia Computacional/métodos , Sequência Conservada , Variação Genética , Feniletanolamina N-Metiltransferase/genética , Regiões Promotoras Genéticas/genética , Alelos , Substituição de Aminoácidos/genética , Animais , Especificidade de Anticorpos , Sequência de Bases , Cromatina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Ensaio de Desvio de Mobilidade Eletroforética , Haplótipos/genética , Humanos , Dados de Sequência Molecular , Células PC12 , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Ratos , Fatores de Transcrição SOXF/genética , Fator de Transcrição Sp1/genética , Transcrição Gênica , Transfecção
12.
Physiol Genomics ; 40(3): 195-207, 2010 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-19952279

RESUMO

Chromogranin A (CHGA) has a crucial role in formation of regulated secretory granules in neuroendocrine tissues and is also a prohormone that is proteolytically processed into peptides with diverse and complex actions. CHGA and several of its peptide products, including catestatin and pancreastatin, are implicated in pathogenesis of essential hypertension, insulin resistance, and the metabolic syndrome. The Chga knockout mouse (Chga KO) displays severe hypertension coupled with reduction in size, number, and density of regulated secretory granules. We performed genome-wide transcriptome profiling in Chga KO adrenal gland and liver for insight into biochemical and physiological systems altered in this monogenic mouse model of hypertension. Adrenal gene expression pathway prediction of enhanced insulin sensitivity (P = 0.03) in Chga KO was confirmed with glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) measurements: blood glucose was normal in Chga KO, blood insulin was reduced 4.5-fold (P < 0.0001), and HOMA-IR was decreased 3.8-fold (P < 0.002). Remarkably, such observations conclusively dissociate fundamental features of the metabolic syndrome in this monogenic hypertension model. Exogenous pancreastatin treatment restored insulin sensitivity in the Chga KO to near-normal levels. Gene expression predictions of decreased adrenal cholesterol biosynthesis (P < 0.001) and increased hepatic cholesterol biosynthesis (P < 0.001) were verified with tissue total cholesterol assays: Chga KO adrenal cholesterol decreased 1.8-fold (P = 0.039) and hepatic cholesterol increased 1.8-fold (P = 0.018). Transcriptional regulatory network prediction identified sets of transcription factors that may provide insight into the unclear mechanistic links among CHGA, cholesterol, insulin sensitivity, and the metabolic syndrome. These experiments demonstrate, for the first time, that genetic variation at the CHGA locus impacts insulin sensitivity and tissue cholesterol levels in an intact, living organism. The Chga KO may constitute a unique model for studying the relationship between the CHGA locus and disease phenotypes of the metabolic syndrome.


Assuntos
Cromogranina A/genética , Perfilação da Expressão Gênica , Hipertensão/genética , Animais , Glicemia/metabolismo , Cromogranina A/metabolismo , Hipertensão/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais
13.
Clin Exp Hypertens ; 31(7): 521-33, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19886850

RESUMO

The contribution of inflammation to hypertension and target organ damage is under investigation. The matrix metalloproteinase (MMP) enzymes are inflammatory mediators that may contribute to hypertension and its target organ consequences. Here we probe MMPs as inflammatory mediators in hypertension, by studying all three MMP classes in uncomplicated hypertension as well hypertension with profound renal damage, such as hypertensive end-stage renal disease (ESRD). We assayed plasma levels of five MMPs: one collagenase (MMP-1), two gelatinases (MMP-2, MMP-9), and two stromelysins (MMP-3, MMP-10). In hypertension, MMP-9 was elevated versus normotensive controls. Systolic blood pressure (SBP) in all three subject groups positively correlated with MMP-9. In hypertensive-ESRD, MMP-2 and MMP-10 were elevated compared to both hypertensive and normotensive subjects. Several correlations occurred across MMPs, suggesting coordinate biosynthetic control. Our results suggest discrete patterns of MMP overexpression in hypertension, with MMP-9 elevated early, and MMP-2 and MMP-10 linked to target organ damage.


Assuntos
Hipertensão Renal/enzimologia , Hipertensão/enzimologia , Falência Renal Crônica/enzimologia , Metaloproteinases da Matriz/sangue , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Hipertensão/sangue , Hipertensão/etiologia , Hipertensão Renal/sangue , Hipertensão Renal/etiologia , Mediadores da Inflamação/sangue , Falência Renal Crônica/sangue , Falência Renal Crônica/etiologia , Masculino , Metaloproteinase 1 da Matriz/sangue , Metaloproteinase 10 da Matriz/sangue , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 3 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Pessoa de Meia-Idade
14.
Diabetes Obes Metab ; 8(6): 621-33, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17026486

RESUMO

Chromogranins or secretogranins (granins), present in secretory granules of virtually all neuroendocrine cells and neurones, are structurally related proteins encoded by different genetic loci: chromogranins A and B, and secretogranins II through VI. Compelling evidence supports both intracellular and extracellular functions for this protein family. Within the cells of origin, a granulogenic or sorting role in the regulated pathway of hormone or neurotransmitter secretion has been documented, especially for chromogranin A (CHGA). Granins also function as pro-hormones, giving rise by proteolytic processing to an array of peptide fragments for which diverse autocrine, paracrine, and endocrine activities have been demonstrated. CHGA measurements yield insight into the pathogenesis of such human diseases as essential hypertension, in which deficiency of the catecholamine release-inhibitory CHGA fragment catestatin may trigger sympathoadrenal overactivity as an aetiologic culprit in the syndrome. The CHGA dysglycaemic fragment pancreastatin is functional in humans in vivo, affecting both carbohydrate (glucose) and lipid (fatty acid) metabolism. Pancreastatin is cleaved from CHGA in hormone storage granules in vivo, and its plasma concentration varies in human disease. The pancreastatin region of CHGA gives rise to three naturally occurring human variants, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. These observations establish a role for pancreastatin in human intermediary metabolism and disease, and suggest that qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose disposition.


Assuntos
Catecolaminas/metabolismo , Síndrome Metabólica/metabolismo , Hormônios Pancreáticos/fisiologia , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Glicemia/metabolismo , Bovinos , Cromogranina A/fisiologia , Diabetes Mellitus Tipo 2/sangue , Humanos , Camundongos , Dados de Sequência Molecular , Hormônios Pancreáticos/genética , Ratos , Alinhamento de Sequência
15.
Am J Hypertens ; 18(5 Pt 1): 633-52, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15882546

RESUMO

Genetic studies of essential hypertension, a complex, polygenic, and age-dependent disorder, have not been able to completely elucidate the genes responsible for development of the trait. We used a novel strategy to compare gene expression in the adrenal gland of two independent rodent models of human essential hypertension (the spontaneously hypertensive rat, SHR, and the blood pressure high mouse, BPH), with the goal of uncovering shared, common genetic mechanisms of hypertension across mammalian species that might, therefore, be pertinent to human hypertension. We deliberately studied young, 4- to 5-week-old, "prehypertensive" SHR and BPH that had not yet developed complete elevations in blood pressure (BP), so that we could minimize the impact of chronic, sustained BP elevation, age, and other confounding factors on gene expression, therefore increasing the likelihood that differential expression reflects relatively early pathogenic mechanisms in hypertension, rather than later responses to, or compensations for BP elevation. We compared transcript expression patterns of genes orthologous between the rat and the mouse, and presented candidate genes for hypertension that are differentially expressed in the same direction in SHR and BPH (ie, overexpressed in both SHR and BPH, or underexpressed in both SHR and BPH). Then we used a systems biology approach to analyze expression patterns in biochemical pathways and networks to isolate systems involved in hypertension pathology in both SHR and BPH. We found transcript pattern evidence for involvement of several systems in the pathology of hypertension in SHR and BPH: adrenal catecholamines and sympathetic function; steroid hormone synthesis, catabolism, and its contribution to enhanced glucocorticoid sensitivity in SHR; oxidative stress and its role as a common mechanism of vascular and end-organ injury; and intermediary metabolism with global but mechanistically different perturbations in SHR and BPH. Approximately 10% of the differentially expressed orthologous genes we studied shared a common direction of expression in the two hypertensive rodent strains, suggesting fundamental transcriptional mechanisms in common whereby mammals can elevate BP or respond to such elevation; even these shared orthologs spanned a diverse set of biological processes, reinforcing the multifactorial and complex nature of hypertension.


Assuntos
Glândulas Suprarrenais/metabolismo , Perfilação da Expressão Gênica , Hipertensão/genética , Animais , Catecolaminas/biossíntese , Modelos Animais de Doenças , Humanos , Hipertensão/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroides/biossíntese , Sistema Nervoso Simpático/fisiologia
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