RESUMEN
DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.
Asunto(s)
Epigénesis Genética/genética , Músculo Liso Vascular/fisiología , Neointima/genética , Proproteína Convertasas/genética , Proteína Desglicasa DJ-1/genética , Receptores de Lisoesfingolípidos/genética , Serina Endopeptidasas/genética , Acetilación/efectos de los fármacos , Animales , Aterosclerosis/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Epigénesis Genética/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H2O2, and exogenous H2O2 elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H2O2-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.
Asunto(s)
Angiotensina II/toxicidad , Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Neointima , Receptores de Lisoesfingolípidos/metabolismo , Acetilación , Animales , Sitios de Unión , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epigénesis Genética/efectos de los fármacos , Histonas/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Oxidación-Reducción , Regiones Promotoras Genéticas , Ratas Sprague-Dawley , Receptores de Lisoesfingolípidos/genética , Transducción de Señal/efectos de los fármacos , Receptores de Esfingosina-1-Fosfato , Factores de TiempoRESUMEN
BACKGROUND: DJ-1 protein plays multifunctional roles including transcriptional regulation and scavenging oxidative stress; thus, it may be associated with the development of renal disorders. We investigated whether DJ-1 protein regulates the expression of (pro)renin receptor (PRR), a newly identified member of renin-angiotensin system. METHODS: The levels of mRNA and protein were determined by real-time PCR and western blot, respectively. H2O2 production was tested by using fluorescence probe. Histone modification was determined by chromatin immunoprecipitation. RESULTS: The expression of PRR was significantly higher in the kidney from DJ-1 knockout mice (DJ-1-/-) compared with wild-type mice (DJ-1+/+). Histone deacetylase 1 recruitment at the PRR promoter was lower, and histone H3 acetylation and RNA polymerase II recruitment were higher in DJ-1-/- than in DJ-1+/+. Knockdown or inhibition of histone deacetylase 1 restored PRR expression in mesangial cells from DJ-1+/+. H2O2 production was greater in DJ-1-/- cells compared with DJ-1+/+ cells. These changes in PRR expression and epigenetic modification in DJ-1-/- cells were induced by H2O2 treatment and reversed completely by addition of an antioxidant reagent. Prorenin-stimulated ERK1/2 phosphorylation was greater in DJ-1-/- than in DJ-1+/+ cells and this was inhibited by a PRR-inhibitory peptide, and by AT1 and AT2 receptor inhibitors. The expression of renal fibrotic genes was higher in DJ-1-/- than in DJ-1+/+ cells and decreased in PRR-knockdown DJ-1-/- cells. CONCLUSIONS: We conclude that DJ-1 protein regulates the expression of renal PRR through H2O2-mediated epigenetic modification. GENERAL SIGNIFICANCE: We suggest that renal DJ-1 protein may be an important molecule in the acceleration of renal pathogenesis through PRR regulation.
Asunto(s)
Epigénesis Genética , Peróxido de Hidrógeno/metabolismo , Riñón/metabolismo , Proteínas Oncogénicas/metabolismo , Regiones Promotoras Genéticas , Receptores de Superficie Celular/biosíntesis , Acetilación/efectos de los fármacos , Animales , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Histonas/metabolismo , Peróxido de Hidrógeno/farmacología , Riñón/patología , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Oncogénicas/genética , Oxidantes/metabolismo , Oxidantes/farmacología , Peroxirredoxinas , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Desglicasa DJ-1 , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/genética , Receptor de Angiotensina Tipo 2/metabolismo , Receptores de Superficie Celular/genética , Receptor de ProreninaRESUMEN
RATIONALE: Inhibition of histone deacetylases (HDACs) results in attenuated development of hypertension in deoxycorticosterone acetate-induced hypertensive rats and spontaneously hypertensive rats. However, the molecular mechanism remains elusive. OBJECTIVE: We hypothesized that HDAC inhibition attenuates transcriptional activity of mineralocorticoid receptor (MR) through its acetylation and prevents development of hypertension in deoxycorticosterone acetate-induced hypertensive rats. METHODS AND RESULTS: Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II on promoters of target genes was analyzed by chromatin immunoprecipitation assay. Live cell imaging was performed for visualization of nuclear translocation of MR. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Transcriptional activity of MR was determined by luciferase assay. For establishment of a hyperaldosteronism animal, Sprague-Dawley rats underwent uninephrectomy and received subcutaneous injection of 40 mg/kg per week of deoxycorticosterone acetate and drinking water containing 1% NaCl. Treatment with a HDAC class I inhibitor resulted in reduced expression of MR target genes in accordance with reduced recruitment of MR and RNA polymerase II on promoters of target genes. HDAC inhibition promoted MR acetylation, leading to decreased transcriptional activity of MR. Knockdown or inhibition of HDAC3 resulted in reduced expression of MR target genes induced by mineralocorticoids. CONCLUSIONS: These results indicate that HDAC inhibition attenuates transcriptional activity of MR through its acetylation and prevents development of hypertension in deoxycorticosterone acetate-induced hypertensive rats.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Hipertensión Renal/prevención & control , Receptores de Mineralocorticoides/genética , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Aldosterona/farmacología , Animales , ADN Polimerasa II/metabolismo , Desoxicorticosterona/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Hipertensión Renal/inducido químicamente , Masculino , Mineralocorticoides/farmacología , Nefrectomía , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Azole antifungals such as ketoconazole are generally known to induce a variety of heart function side effects, e.g., long-QT syndrome and ventricular arrhythmias. However, a clear mechanism for the action of ketoconazole in heart cells has not been reported. In the present study, we assessed the correlation between ketoconazole-induced apoptosis and the alteration of genes in response to ketoconazole in rat cardiomyocytes. Cardiomyocyte viability was significantly inhibited by treatment with ketoconazole. Ketoconazole also stimulated H2O2 generation and TUNEL-positive apoptosis in a dose-dependent manner. DNA microarray technology revealed that 10,571 genes were differentially expressed by more than threefold in ketoconazole-exposed cardiomyocytes compared with untreated controls. Among these genes, parkin, which encodes a component of the multiprotein E3 ubiquitin ligase complex, was predominantly overexpressed among those classified as apoptosis- and reactive oxygen species (ROS)-related genes. The expression of parkin was also elevated in cardiomyocytes treated with exogenous H2O2. Moreover, cell viability and apoptosis in response to ketoconazole were inhibited in cardiomyocytes treated with ROS inhibitors and transfected with parkin siRNA. From the present findings, we concluded that ketoconazole may increase the expression of parkin via the ROS-mediated pathway, which consequently results in the apoptosis and decreased viability of cardiomyocytes.
Asunto(s)
Apoptosis/efectos de los fármacos , Cetoconazol/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genética , Animales , Antifúngicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Etiquetado Corte-Fin in Situ , Miocitos Cardíacos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose-response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Hipertensión/genética , Simportadores de Cloruro de Sodio-Potasio/genética , Agonistas alfa-Adrenérgicos/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Secuencia de Bases , Bumetanida/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Datos de Secuencia Molecular , Fenilefrina/farmacología , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12 , Regulación hacia ArribaRESUMEN
OBJECTIVE: Carvacrol (2-methyl-5-(1-methylethyl) phenol), a cyclic monoterpene, exerts protective activities in a variety of pathological states including tumor growth, inflammation, and oxidative stress. However, it is unknown whether carvacrol affects events in vascular cells during the development of atherosclerotic neointima. We investigated the effects of carvacrol on the migration and proliferation of rat aortic smooth muscle cells (RASMCs) and on vascular neointima formation. METHODS AND RESULTS: Carvacrol significantly inhibited platelet-derived growth factor (PDGF)-BB-stimulated RASMC migration and proliferation in a concentration-dependent manner. Cell viability was not affected by treatment with carvacrol. Carvacrol attenuated the expression of NADPH oxidase (NOX) 1 and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 in response to PDGF-BB. Moreover, carvacrol suppressed the PDGF-BB-stimulated generation of H2O2 and inhibited the activity of NOX in RASMCs. Treatment with carvacrol inhibited PDGF-BB-induced aortic sprout outgrowth, balloon injury-evoked vascular neointima formation, and expression of proliferating cell nuclear antigen in the neointima. CONCLUSION: These findings indicate that carvacrol inhibits migration and proliferation of RASMCs by suppressing the reactive oxygen species-mediated MAPK signaling pathway in these cells, thereby attenuating vascular neointimal formation. Carvacrol may be a promising agent for preventing vascular restenosis or atherosclerosis.
Asunto(s)
Antioxidantes/farmacología , Aterosclerosis/prevención & control , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Monoterpenos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Neointima , Especies Reactivas de Oxígeno/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Becaplermina , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cimenos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-sis/farmacología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIMS: DJ-1/park7, a multifunctional protein, may play essential roles in the vascular system. However, the function of DJ-1/park7 in vascular contractility has remained unclear. The present study was designed to investigate whether the DJ-1/park7 is involved in the regulation of vascular contractility and systolic blood pressure (SBP). METHODS AND RESULTS: Norepinephrine (NE) elevated contraction in endothelium-intact vessels in a dose-dependent manner, to a greater extent in DJ-1/park7 knockout (DJ-1/park7(-/-)) mice than in wild-type (DJ-1/park7(+/+)) mice. Acetylcholine inhibited NE-evoked contraction in endothelium-intact vessels, and this was markedly impaired in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+). Nitric oxide (NO) production (82.1 ± 2.8% of control) and endothelial NO synthase (eNOS) expression (61.7 ± 8.9%) were lower, but H2O2 production (126.4 ± 8.6%) was higher, in endothelial cells from DJ-1/park7(-/-) mice than in those from DJ-1/park7(+/+) controls; these effects were reversed by DJ-1/park7-overexpressing endothelial cells from DJ-1/park7(-/-) mice. Histone deacetylase (HDAC)-1 recruitment and H3 histone acetylation at the eNOS promoter were elevated and diminished, respectively, in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+) controls. Moreover, SBP was significantly elevated in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+) controls, but this elevation was inhibited in mice treated with valproic acid, an inhibitor of Class I HDACs including HDAC-1. CONCLUSION: These results demonstrate that DJ-1/park7 protein may be implicated in the regulation of vascular contractility and blood pressure, probably by the impairment of NO production through H2O2-mediated epigenetic inhibition of eNOS expression.
Asunto(s)
Endotelio Vascular/metabolismo , Epigénesis Genética/fisiología , Óxido Nítrico Sintasa de Tipo III/genética , Proteínas Oncogénicas/metabolismo , Vasodilatación , Animales , Presión Sanguínea , Epigénesis Genética/genética , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Oncogénicas/genética , Peroxirredoxinas , Proteína Desglicasa DJ-1 , Vasodilatación/genética , Vasodilatación/fisiologíaRESUMEN
OBJECTIVE: DJ-1-a multifunctional protein responding to oxidative stress-is a possible regulator of the inflammatory response that plays an important role in atherosclerosis. Stromal cell-derived factor (SDF)-1 and its receptor, chemokine receptor type 4 (CXCR4), have been implicated in the recruitment of inflammatory cells during atherosclerosis. Here we investigated the hypothesis that DJ-1 protein might participate in CD3+ T cell functions in response to SDF-1 and contribute to the pathogenesis of atherosclerosis. METHODS AND RESULTS: SDF-1 stimulated migration in mouse CD3+ T cells in a dose-dependent manner. SDF-1 also elevated the phosphorylation level of extracellular-regulated kinase (ERK) 1/2 in CD3+ T cells. These SDF-1-induced responses were greater in CD3+ T cells from DJ-1 gene knockout (DJ-1(-/-)) mice than in those from wild type (DJ-1(+/+)) mice and were abolished by treatment with WZ811 and PD98059, inhibitors of CXCR4 and ERK1/2, respectively. Flow cytometry revealed that expression of the CXCR4 receptor was greater in CD3+ T cells from DJ-1(-/-) mice than in those from the controls. Moreover, expression of the CD3 protein was observed in the neointimal plaque from carotid artery-ligated mice and was stronger in DJ-1(-/-) mice compared with controls. The CD3+ T cell subsets, Th1 and Th17, showed increased production of interferon-γ and interleukin-17 in DJ-1(-/-) compared with DJ-1(+/+) mice. CONCLUSION: DJ-1 protein is involved in the SDF-1-induced CD3+ T cell migration via overexpression of the CXCR4 receptor, and that DJ-1 acts as an inhibitory regulator in vascular remodeling such as neointima formation.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proteínas Oncogénicas/fisiología , Peroxirredoxinas/fisiología , Receptores CXCR4/biosíntesis , Linfocitos T/fisiología , Animales , Complejo CD3/biosíntesis , Quimiocina CXCL12 , Masculino , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neointima , Proteínas Oncogénicas/genética , Peroxirredoxinas/genética , Fosforilación , Proteína Desglicasa DJ-1 , Linfocitos T/efectos de los fármacosRESUMEN
OBJECTIVES: Promoter hypomethylation leads to upregulation of Naâº-Kâº-2Clâ» cotransporter 1 (NKCC1) in the spontaneously hypertensive rat (SHR). We hypothesized that recruitment of Specificity Protein 1 (Sp1) by CpG hypomethylation would result in upregulation of Naâº-Kâº-2Clâ» cotransporter 1 in hypertensive rats. METHODS: Sham-operated Wistar-Kyoto (WKY) rats (sham) and angiotensin II (Ang II)-infused WKY rats, as well as SHRs, were used in this study. We performed real-time PCR and western blot for determination of the expression levels of Nkcc1 mRNA and protein, respectively, and bisulphite sequencing for determination of the methylation status of the proximal promoter; an assay kit was used for assessment of the activity of DNA methyltransferase (DNMT), and the electrophoretic mobility shift assay (EMSA) was used for assessment of binding of Sp1 to cis-element, and promoter function was assessed using the luciferase assay. RESULTS: Both Ang II-infused WKY rats and SHRs showed higher expression of Nkcc1 mRNA and protein and less DNA methylation, compared with sham. CpG methylation at Sp1 response elements interfered with binding of Sp1, resulting in disabled promoter activity. Both types of hypertensive rats showed hypomethylation of CpG dinucleotides in Sp1 response elements in accordance with the decrease of DNMT activity. DNMT3b and MeCP2 were highly recruited to the more methylated promoter of normotensive rats, whereas the CXXC finger protein 1 (Cfp1), Sp1 and RNA polymerase II were highly recruited to the less methylated promoter of hypertensive rats. CONCLUSION: Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Naâº-Kâº-2Clâ» cotransporter 1 in hypertensive rats.
Asunto(s)
Islas de CpG , Metilación de ADN , Miembro 2 de la Familia de Transportadores de Soluto 12/fisiología , Factor de Transcripción Sp1/fisiología , Regulación hacia Arriba , Animales , Presión Sanguínea , Células HEK293 , Humanos , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Compound K (CK), an intestinal metabolite of ginsenosides, has pharmacological properties such as anti-angiogenesis, anti-inflammation, anti-platelet and anti-cancer activities. In the present study, we investigated the inhibitory effect of CK on vascular smooth muscle cell (VSMC) proliferation and migration in vitro and neointima formation in a rat carotid artery injury model. RESULTS: CK significantly inhibited both the proliferation and migration of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. In accordance with these findings, CK blocked the PDGF-BB-induced progression of synchronized cells through the G0/G1 phase of the cell cycle. CK also decreased the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, and proliferative cell nuclear antigen (PCNA) in response to PDGF. However, CK did not affect early signal transduction through PDGF-Rß, Akt, ERK1/2 and PLC-γ1 phosphorylation. CK attenuated PDGF-BB-induced VSMC migration by inhibiting MMP-2 and MMP-9 expression. Furthermore, the CK-treated groups showed a significant reduction in neointima formation vs. the control group. Immunohistochemical staining demonstrated decreased expression of PCNA in the neointima of the CK-treated group. CONCLUSION: Our findings demonstrated that CK was capable of suppressing the abnormal VSMC proliferation and migration. It suggested that CK can be a therapeutic agent to control pathologic cardiovascular conditions such as restenosis and atherosclerosis.
Asunto(s)
Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Ginsenósidos/farmacocinética , Músculo Liso Vascular/citología , Neointima/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-sis/antagonistas & inhibidores , Angioplastia de Balón/efectos adversos , Animales , Becaplermina , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Ginsenósidos/química , Ginsenósidos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neointima/metabolismo , Neointima/patología , Fosfolipasa C gamma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
AIMS: DJ-1/park7 is a ubiquitously expressed multifunctional protein that plays essential roles in a variety of cells. However, its function in the vascular system has not been determined. We investigated the protective roles of DJ-1/park7 in vascular disorders, especially in neointimal hyperplasia. METHODS AND RESULTS: DJ-1/park7 was strongly expressed in the neointimal layer, in which its oxidized form was predominant. Treatment of vascular smooth muscle cells (VSMCs) from the mouse aorta with H(2)O(2) increased the oxidation of DJ-1/park7 visualized on two-dimensional electrophoresis gels. The growth of VSMCs in FBS-containing media and the release of H(2)O(2) were significantly increased in DJ-1/park7(-/-) knockout mice compared with DJ-1/park7(+/+) wild-type mice. The expression of cyclin D1 and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were greater in VSMCs from the DJ-1/park7(-/-) aorta than from the DJ-1/park7(+/+) aorta. Both of these measures were inhibited by treatment with an ERK1/2 inhibitor or antioxidants and in DJ-1/park7-overexpressing cells. VSMC proliferation, cyclin D1 expression, and ERK1/2 phosphorylation in response to platelet-derived growth factor-BB were upregulated in DJ-1/park7(-/-) compared with DJ-1/park7(+/+) mice. VSMCs of DJ-1/park7(-/-) mice exhibited higher levels of sprout outgrowth of aortic strips and neointimal plaque formation elicited by carotid artery ligation compared with those of DJ-1/park7(+/+) mice. CONCLUSION: These results indicate that DJ-1/park7 is involved in the growth of VSMCs, thereby inhibiting neointimal hyperplasia, and suggest that it might play protective roles in vascular remodelling.
Asunto(s)
Proliferación Celular , Músculo Liso Vascular/citología , Neointima/prevención & control , Neointima/fisiopatología , Neovascularización Patológica/fisiopatología , Proteínas Oncogénicas/fisiología , Animales , Aorta/citología , Aorta/fisiopatología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/efectos de los fármacos , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/fisiopatología , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Peroxirredoxinas , Fosforilación/efectos de los fármacos , Proteína Desglicasa DJ-1 , Proteínas Proto-Oncogénicas c-sis/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: The (pro)renin receptor [(P)RR] non-proteolytically, through conformational change, activates prorenin which can convert angiotensinogen to angiotensin I in addition to the classic conversion of angiotensinogen to angiotensin I by circulating renin. Since renal (P)RR is upregulated in hypertension and implicated in the pathogenesis of malignant hypertension, we hypothesized that (pro)renin receptor promoter is enriched with activating histone codes in the kidney of spontaneously hypertensive rats (SHR). METHODS: The mRNA and protein expression levels were measured by real-time polymerase chain reaction (PCR) and western blot, respectively. The DNA methylation status of (P)RR promoter region was analyzed by bisulfite sequencing. The histone modifications were determined by chromatin immunoprecipitation followed by real-time PCR. RESULTS: The (P)RR mRNA expression in the kidney was about six times greater in SHR than in Wistar-Kyoto (WKY) rats. The (P)RR promoter was little methylated in the kidneys of both WKY and SHR. Acetylated histone H3 (H3Ac) and di-methylated histone H3 at lysine 4 (H3K4me2), activating histone codes, were about 25 and three times higher in SHR than in WKY, respectively. On the other hand, di-methylated histone H3 at lysine 9 (H3K9me2), a suppressive histone code, was 50 times lower in SHR than in WKY. CONCLUSION: These results suggest that the (P)RR promoter is enriched with activating histone codes in the kidneys of SHR.
Asunto(s)
Código de Histonas/genética , Riñón/metabolismo , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Presión Sanguínea/genética , Epigénesis Genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Riñón/enzimología , Riñón/patología , Masculino , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Renina/genética , Renina/metabolismo , Receptor de ProreninaRESUMEN
The renin-angiotensin system has been implicated in the development of hypertension and damages several organs. The expressions of the components of a local renin-angiotensin system (RAS) in the hypertensive rats differ from those of the normotensive rats. We hypothesized that local tissue-specific upregulation of angiotensin-converting enzyme 1 (ACE1) in hypertension is caused by epigenetic changes. Adrenal gland, aorta, heart, kidney, liver, and lung tissues were excised from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Ace1 mRNA and protein expressions were measured by real-time PCR and Western blot, respectively. Promoter methylation was revealed by bisulfite sequencing. Histone modifications, such as histone 3 acetylation (H3Ac), fourth lysine trimethylation (H3K4me3), and ninth lysine dimethylation (H3K9me2), were quantified by chromatin immunoprecipitation (ChIP), followed by real-time PCR. The expressions and associations of chromatin remodeling genes were analyzed by real-time PCR and ChIP, respectively. Local tissues from SHRs showed higher expressions of Ace1 mRNA and protein than those from the WKY rats. Ace1 promoter was mostly unmethylated in all of the tissues from both strains. The Ace1 promoter regions of SHR tissues were more enriched with H3Ac and H3K4me3, except in the lungs. The adrenal glands, hearts, and kidneys of SHRs showed less enrichment with H3K9me2. Valsartan treatment in SHRs decreased local Ace1 mRNA and protein expressions, which were accompanied by higher H3K9me2, as well as less H3Ac and H3K4me3. In conclusion, ACE1 is upregulated in local tissues of SHRs via histone code modifications.
Asunto(s)
Presión Sanguínea , Hipertensión/genética , Peptidil-Dipeptidasa A/genética , ARN Mensajero/genética , Sistema Renina-Angiotensina/genética , Regulación hacia Arriba , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/patología , Animales , Modelos Animales de Enfermedad , Código de Histonas , Hipertensión/metabolismo , Hipertensión/fisiopatología , Riñón/metabolismo , Riñón/patología , Miocardio/metabolismo , Miocardio/patología , Peptidil-Dipeptidasa A/biosíntesis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in diverse models of hypertension. We hypothesized that NKCC1 is upregulated via histone modification in the aortas of angiotensin II (Ang II)-induced hypertensive rats. An osmotic mini-pump containing Ang II was implanted in the subcutaneous tissues of the backs of Sprague-Dawley (SD) rats for 7 days. The systolic blood pressure was recorded every day by the tail-cuff method. On days 3 and 7, the mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The levels of Nkcc1 mRNA and protein in the aortas were measured using real-time PCR and Western blotting, respectively. The histone modifications and recruited proteins at the Nkcc1 promoter were determined by chromatin immunoprecipitation. The inhibition of concentration-response curves to phenylephrine by bumetanide, an inhibitor of NKCCs, was greater in Ang II-infused rats than in sham-operated (sham) rats . The levels of Nkcc1 mRNA and protein in the aortas increased gradually as Ang II was infused into the rats. Acetylated histone H3 (H3Ac), an activating histone code, was increased but trimethylated histone H3 at lysine 27 (H3K27me3), a repressive histone code, was greatly decreased in Ang II-infused rats compared with sham. RNA polymerase II was recruited to the Nkcc1 promoter with increased KDM6b. We conclude that the NKCC1 is upregulated via histone modification in the aortas of Ang II-induced hypertensive rats. Thus, we suggest that this ion transporter is epigenetically upregulated by histone modification or DNA demethylation upon the development of hypertension.
Asunto(s)
Angiotensina II , Histonas/metabolismo , Hipertensión/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Vasoconstrictores , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Presión Sanguínea , Western Blotting , Bumetanida/farmacología , Inmunoprecipitación de Cromatina , Diuréticos/farmacología , Histona Demetilasas/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Masculino , Contracción Muscular , Músculo Liso Vascular/fisiopatología , Fenilefrina/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Miembro 2 de la Familia de Transportadores de Soluto 12 , Regulación hacia Arriba , Vasoconstrictores/farmacologíaRESUMEN
Changes in the expression profiles of specific proteins leads to serious human diseases, including colitis. The proteomic changes related to colitis and the differential expression between tuberculous (TC) and ulcerative colitis (UC) in colon tissue from colitis patients has not been defined. We therefore performed a proteomic analysis of human TC and UC mucosal tissue. Total protein was obtained from the colon mucosal tissue of normal, TC, and UC patients, and resolved by 2-dimensional electrophoresis (2-DE). The results were analyzed with PDQuest using silver staining. We used matrix-assisted laser desorption ionization time-of-flight/time-of-flight spectrometry (MALDI TOF/TOF) to identify proteins differentially expressed in TC and UC. Of the over 1,000 proteins isolated, three in TC tissue and two in UC tissue displayed altered expression when compared to normal tissue. Moreover, two proteins were differentially expressed in a comparative analysis between TC and UC. These were identified as mutant ß-actin, α-enolase and Charcot-Leyden crystal protein. In particular, the expression of α-enolase was significantly greater in TC compared with normal tissue, but decreased in comparison to UC, implying that α-enolase may represent a biomarker for differential diagnosis of TC and UC. This study therefore provides a valuable resource for the molecular and diagnostic analysis of human colitis.