RESUMO
Acute respiratory distress syndrome (ARDS) is a devastating disease process characterized by severe acute lung injury, inflammatory cell recruitment to the lung, upregulation of pro-inflammatory cytokines and increased oxidative stress. Epithelial cell injury, diffuse alveolar damage and surfactant dysfunction ensue leading to refractory hypoxemic respiratory failure. There are no specific effective therapies for ARDS and novel therapeutic approaches are desperately needed. In this study we assessed the role of the cytoprotective and anti-inflammatory enzyme heme oxygenase (HO)-1 in a model of nebulized endotoxin-induced acute lung injury. HO-1 null (HO-1(-/-)) mice exhibited severe physiologic lung dysfunction following lipopolysaccharide (LPS) nebulization, but had similar inflammatory responses as wild-type (WT) mice. However, a dramatic reduction in surfactant protein-B (SP-B) expression was observed in the lungs of LPS-treated HO-1(-/-) mice compared with similarly treated WT mice. Using reciprocal bone marrow transplantation (BMT) to generate HO-1-chimeric mice, we found that absence of HO-1 in the lung parenchyma, not in bone marrow-derived inflammatory cells, was responsible for enhanced SP-B downregulation and severe physiologic lung dysfunction. These findings have implications for our understanding of the pathophysiology of ARDS and may guide future therapeutic strategies.
Assuntos
Endotoxinas/farmacologia , Heme Oxigenase-1/deficiência , Pulmão/enzimologia , Proteína B Associada a Surfactante Pulmonar/análise , Síndrome do Desconforto Respiratório/etiologia , Animais , Transplante de Medula Óssea , Camundongos , Camundongos Knockout , Surfactantes Pulmonares/química , Síndrome do Desconforto Respiratório/induzido quimicamenteRESUMO
In high-risk patients, the ideal cardiovascular gene therapy requires a strategy that provides long-term protection of myocardium against episodes of ischemic/reperfusion injury. We report the development of an efficient, long-lasting pre-emptive gene therapy strategy in a rat model of ischemic-reperfusion (I/R) injury of heart. At 6 weeks prior to myocardial injury, the human extracellular superoxide dismutase (Ec-SOD) gene was delivered by direct intramyocardial injections, using a recombinant adeno-associated virus vector. Significant myocardial protection was documented by the decrease in infarct size at 24 h post I/R, improved left ventricular function at 7 weeks postinjury, and enhanced long-term survival in the SOD treated group. This concept of preinjury delivery and 'pre-emptive' gene therapy via the expression of a secreted protein that renders paracrine therapeutic action can be an effective strategy for organ protection against future injury.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Superóxido Dismutase/genética , Transdução Genética/métodos , Animais , Líquido Extracelular/enzimologia , Masculino , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/mortalidade , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Ratos , Ratos Sprague-Dawley , Taxa de Sobrevida , Fatores de Tempo , Função VentricularRESUMO
Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.
Assuntos
Arteriosclerose/patologia , Proteínas de Transporte/fisiologia , Hipercolesterolemia/complicações , Macrófagos/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/sangue , Arteriosclerose/complicações , Glicemia/metabolismo , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Transporte/genética , Colesterol/sangue , Citocinas/sangue , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Insulina/sangue , Camundongos , Camundongos Knockout , Óxido Nítrico/sangueAssuntos
Arteriosclerose/prevenção & controle , Proteínas de Transporte/fisiologia , Hipercolesterolemia/complicações , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Proteínas de Transporte/genética , Colesterol/sangue , Colágeno/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Metabolismo dos Lipídeos , Macrófagos/patologia , Camundongos , Camundongos Knockout , Triglicerídeos/sangueRESUMO
Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.
Assuntos
Músculos Abdominais/embriologia , Proteínas/genética , Proteínas/fisiologia , Cicatrização , Animais , Desenvolvimento Ósseo , Carboxipeptidases , Adesão Celular , Agregação Celular , Divisão Celular , Células Cultivadas , Clonagem Molecular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Músculo Liso/citologia , Mutagênese Sítio-Dirigida , Fenótipo , Estrutura Terciária de Proteína , Recombinação Genética , Proteínas Repressoras , Pele/metabolismo , Pele/patologia , Frações Subcelulares , Fatores de TempoRESUMO
Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-alpha signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.
Assuntos
Heme Oxigenase (Desciclizante)/fisiologia , Hipertensão Pulmonar/imunologia , Hipóxia/imunologia , Pulmão/imunologia , Artéria Pulmonar/imunologia , Animais , Expressão Gênica , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/prevenção & controle , Pulmão/irrigação sanguínea , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Fatores de Tempo , TransgenesRESUMO
Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.
Assuntos
Injúria Renal Aguda/patologia , Heme Oxigenase (Desciclizante)/deficiência , Hipertensão Renovascular/genética , Injúria Renal Aguda/sangue , Injúria Renal Aguda/complicações , Animais , Pressão Sanguínea/genética , Cardiomegalia/etiologia , Cardiomegalia/patologia , Doença Crônica , Creatinina/sangue , Modelos Animais de Doenças , Antagonistas dos Receptores de Endotelina , Endotelina-1/genética , Endotelina-1/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Heterozigoto , Homozigoto , Hipertensão Renovascular/sangue , Hipertensão Renovascular/complicações , Imuno-Histoquímica , Rim/patologia , Proteínas de Membrana , Camundongos , Camundongos Knockout , Nefrectomia , Tamanho do Órgão , RNA Mensageiro/metabolismo , Receptor de Endotelina A , Obstrução da Artéria Renal/complicações , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Peixe-Zebra , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína Axina , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas Desgrenhadas , Ativação Enzimática , Genes Reporter , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Camundongos , Células PC12 , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas Wnt , beta CateninaRESUMO
Inflammation is a hallmark of several vascular diseases. The nuclear factor kappaB (NF-kappaB) transcription factors are dimeric proteins involved in the activation of a large number of genes in response to inflammatory stimuli. We report the involvement of a novel member of the ETS transcription factor, ESE-1, in mediating vascular inflammation. ESE-1 is induced in response to inflammatory cytokines and lipopolysaccharide in vascular smooth muscle cells, endothelial cells, and cells of the monocyte-macrophage lineage. This induction occurs within hours of stimulation and is mediated by NF-kappaB transactivation of the ESE-1 promoter. We have identified the inducible form of nitric-oxide synthase (NOS2) as a putative target for ESE-1. ESE-1 can bind to the p50 subunit of NF-kappaB, and cotransfection of ESE-1 with the p50 and p65 subunits of NF-kappaB synergistically enhances transactivation of the NOS2 promoter by ESE-1. An ESE-1-binding site within the NOS2 promoter has been identified, the site-directed mutagenesis of which completely abolishes the ability of ESE-1 to transactivate the NOS2 promoter. Finally, in a mouse model of endotoxemia, associated with acute vascular inflammation, ESE-1 is strongly expressed in vascular endothelium and smooth muscle cells. In summary, ESE-1 represents a novel mediator of vascular inflammation.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Proto-Oncogênicas , Transativadores/fisiologia , Fatores de Transcrição , Sítios de Ligação , Células Cultivadas , Citocinas/farmacologia , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Glicoproteínas de Membrana/metabolismo , Mutação , NF-kappa B/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase Tipo II , Regiões Promotoras Genéticas/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets , Sinaptotagmina I , Sinaptotagminas , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Doenças Vasculares/metabolismoRESUMO
The inducible isoform of nitric-oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), which participates in the pathophysiology of systemic inflammatory diseases such as sepsis. NOS2 is transcriptionally up-regulated by endotoxin and inflammatory cytokines, and down-regulated by transforming growth factor (TGF)-beta1. Recently we have shown that high mobility group (HMG)-I(Y) protein, an architectural transcription factor, contributes to NOS2 gene transactivation by inflammatory mediators. The aim of the present study was to determine whether regulation of HMG-I(Y) by TGF-beta1 contributes to the TGF-beta1-mediated suppression of NOS2. By Northern blot analysis, we show that TGF-beta1 decreased cytokine-induced HMG-I(Y) mRNA levels in vascular smooth muscle cells and macrophages in vitro and in vivo. Western analysis confirmed the down-regulation of HMG-I(Y) protein by TGF-beta1. To determine whether the down-regulation of HMG-I(Y) contributed to a decrease in NOS2 gene transactivation by TGF-beta1, we performed cotransfection experiments. Overexpression of HMG-I(Y) was able to restore cytokine inducibility of the NOS2 promoter that was suppressed by TGF-beta1. The effect of TGF-beta1 on NOS2 gene transactivation was not related to a decrease in binding of HMG-I(Y) to the promoter of the NOS2 gene, but due to a decrease in endogenous HMG-I(Y) protein. These data provide the first evidence that cytokine-induced HMG-I(Y) can be down-regulated by TGF-beta1. This down-regulation of HMG-I(Y) contributes to the TGF-beta1-mediated decrease in NOS2 gene transactivation by proinflammatory stimuli.
Assuntos
Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Aorta , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteína HMGA1a , Proteínas de Grupo de Alta Mobilidade/metabolismo , Interferon gama/farmacologia , Macrófagos/enzimologia , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II , Fosfoproteínas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
Heme oxygenase (HO)-1 is a stress response protein that is regulated by oxidative stress. HO-1 catalyzes the generation of biliverdin, carbon monoxide, and iron from heme. Lipopolysaccharide (LPS) and interleukin (IL)-1beta induce HO-1 through the binding of nuclear proteins to AP-1 motifs in enhancer regions upstream from the transcription start site. The DNA binding activity of AP-1 proteins depends on the reduction of cysteines in their DNA-binding domains. We found that agents that disrupt free sulfhydryl groups abolish AP-1 binding activity in nuclear proteins obtained from rat aortic smooth muscle cells and macrophages stimulated with IL-1beta or LPS. Thioredoxin (TRX) may regulate the redox status of nuclear transcription factors in response to oxidative stimuli, thus we determined the role of TRX in the physiologic regulation of HO-1. TRX underwent nuclear translocation in cells stimulated with IL-1beta and LPS. We transfected macrophages with a heterologous promoter construct containing two AP-1 sites from an upstream enhancer region in the HO-1 promoter. Recombinant TRX induced promoter activity to a level analogous to that induced by LPS, and this TRX response was abolished by mutation of the AP-1 sites. An inhibitor of TRX reductase, used to prevent TRX translocation in the reduced state, decreased HO-1 induction by IL-1beta and LPS. These data provide the first evidence that TRX contributes to the induction of HO-1 by inflammatory mediators.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Heme Oxigenase (Desciclizante)/genética , Interleucina-1/farmacologia , Macrófagos/enzimologia , Músculo Liso Vascular/enzimologia , Tiorredoxinas/metabolismo , Animais , Aorta/citologia , Aorta/enzimologia , Carbono-Oxigênio Liases/genética , Linhagem Celular , Células Cultivadas , Elementos Facilitadores Genéticos , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase-1 , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Proteínas de Membrana , Músculo Liso Vascular/citologia , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
Matrix metalloproteinases (MMP) have been identified in vulnerable areas of atherosclerotic plaques and may contribute to plaque instability through extracellular matrix degradation. Human metalloelastase (MMP-12) is a macrophage-specific MMP with broad substrate specificity and is capable of degrading proteins found in the extracellular matrix of atheromas. Despite its potential importance, little is known about the regulation of MMP-12 expression in the context of atherosclerosis. In this study, we report that in human peripheral blood-derived macrophages, MMP-12 mRNA was markedly up-regulated by several pro-atherosclerotic cytokines and growth factors including interleukin-1beta, tumor necrosis factor-alpha, macrophage colony-stimulating factor, vascular endothelial growth factor, and platelet-derived growth factor-BB. In contrast, the pleiotropic anti-inflammatory growth factor transforming growth factor-beta1 (TGF-beta1) inhibited cytokine-mediated induction of MMP-12 mRNA, protein, and enzymatic activity. Analyses of MMP-12 promoter through transient transfections and electrophoretic mobility shift assays indicated that both its induction by cytokines and its inhibition by TGF-beta1 depended on signaling through an AP-1 site at -81 base pairs. Moreover, the inhibitory effect of TGF-beta1 on MMP-12 was dependent on Smad3. Taken together, MMP-12 is induced by several factors implicated in atherosclerosis. The inhibition of MMP-12 expression by TGF-beta1 suggests that TGF-beta1, acting via Smad3, may promote plaque stability.
Assuntos
Citocinas/metabolismo , Macrófagos/enzimologia , Metaloendopeptidases/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Arteriosclerose/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Genes Dominantes , Genes Reporter , Humanos , Metaloproteinase 12 da Matriz , Metaloendopeptidases/sangue , Metaloendopeptidases/genética , Camundongos , Monócitos/enzimologia , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad3 , Fatores de Tempo , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transfecção , Regulação para CimaRESUMO
CD44 is a multifunctional cell adhesion molecule that participates in pathological states such as inflammation and tumorigenesis. CD44 is induced on vascular smooth muscle cells after arterial wall injury and may mediate their proliferation and migration into the neointima during arteriosclerosis. We have demonstrated elsewhere that the proinflammatory cytokine interleukin (IL)-1beta up-regulates CD44 mRNA and protein expression in cultured rat aortic smooth muscle cells (RASMC) by increasing gene transcription. By transient transfection of 5'-deletion constructs into RASMC, we show in the present study that a conserved AP-1 site 110 base pairs from the transcription start site of the mouse CD44 promoter is important for basal activity. Mutation of the AP-1 site significantly reduced induction of promoter activity by IL-1beta, and electrophoretic mobility shift assays demonstrated that Fos and c-Jun were present in the CD44 AP-1 binding complex after IL-1beta stimulation. In addition, cotransfection of the architectural transcription factor high mobility group (HMG)-I(Y) protein with c-Fos and c-Jun markedly increased trans-activation of the CD44 promoter. Taken together, our studies demonstrate that AP-1 proteins are a central regulatory component used by IL-1beta to modulate expression of CD44 during an inflammatory response in vascular smooth muscle cells and that transcription of CD44 by AP-1 proteins is enhanced by HMG-I(Y). -Foster, L. C., Wiesel, P., Huggins, G. S, Pañares, R., Chin, M. T., Pellacani, A., Perrella, M. A. Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.
Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Receptores de Hialuronatos/genética , Interleucina-1/farmacologia , Músculo Liso Vascular/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Proteína HMGA1a , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Sprague-Dawley , Deleção de Sequência , Transcrição GênicaRESUMO
Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that is preferentially expressed in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and, like HIF-1alpha, has been shown to bind to the HIF-1-binding site and to activate its downstream genes such as vascular endothelial growth factor (VEGF) and erythropoietin. In this report, we show that EPAS1 increased VEGF gene expression through the HIF-1-binding site. This transactivation was enhanced further by cotransfection of an aryl hydrocarbon receptor nuclear translocator expression plasmid. Deletion analysis of EPAS1 revealed a potent activation domain (amino acids 486-639) essential for EPAS1 to transactivate the VEGF promoter. We confirmed the ability of this domain to activate transcription using a Gal4 fusion protein system. Because a truncated EPAS1 protein lacking the transactivation domain at amino acids 486-639 eliminated induction of the VEGF promoter by wild-type EPAS1, the truncated protein functions as a dominant-negative mutant. Most important, infection of the cells with an adenoviral construct expressing this mutant inhibited the induction of VEGF mRNA under conditions that mimic hypoxia. Our results suggest that EPAS1 is an important regulator of VEGF gene expression. Since VEGF plays a crucial role in angiogenesis, the ability of dominant-negative EPAS1 to inhibit VEGF promoter activity raises the possibility of a novel approach to inhibiting pathological angiogenesis.
Assuntos
Mutação , Proteínas de Saccharomyces cerevisiae , Transativadores/genética , Ativação Transcricional , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Bovinos , Proteínas de Ligação a DNA/metabolismo , Desferroxamina/farmacologia , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Proteínas Fúngicas , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Linfocinas/biossíntese , Linfocinas/genética , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Recombinantes de Fusão , Deleção de Sequência , Transativadores/metabolismo , Fatores de Transcrição , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-gamma on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-gamma-treated VSMCs. IFN-gamma treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-gamma treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-gamma treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-beta1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-gamma treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-gamma inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.
Assuntos
Ciclina A/genética , Regulação da Expressão Gênica/fisiologia , Interferon gama/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Primers do DNA , Inibidores Enzimáticos/farmacologia , Masculino , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-kappaB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1beta on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1beta stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-kappaB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes.
Assuntos
Proteínas de Grupo de Alta Mobilidade/biossíntese , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Músculo Liso Vascular/metabolismo , Proteínas de Neoplasias/biossíntese , Óxido Nítrico Sintase/metabolismo , Fatores de Transcrição/biossíntese , Animais , Células Cultivadas , Distamicinas/farmacologia , Drosophila , Ativação Enzimática , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteína HMGA1a , Macrófagos Alveolares/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , NF-kappa B/farmacologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ativação Transcricional/efeitos dos fármacosRESUMO
The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules that participate in extracellular matrix binding, lymphocyte activation, cell migration, and tumor metastasis. In a mouse model of transplant-associated arteriosclerosis, CD44 protein was induced in the neointima of allografted vessels and colocalized with a subset of proliferating vascular smooth muscle cells (SMC). To elucidate the molecular mechanisms regulating CD44 expression in this model, we investigated the regulation of CD44 gene expression by interleukin (IL)-1beta. Treatment of rat aortic SMC with IL-1beta resulted in a 5.3-fold increase in cell surface CD44 expression. Northern analysis showed that IL-1beta promoted a dose- and time-dependent induction of CD44 mRNA which reached 6.6-fold after 48 h, and nuclear run-on analysis showed that IL-1beta increased the rate of CD44 gene transcription within 8 h of stimulation. In transient reporter gene transfection experiments in rat aortic SMC, a 1.4-kilobase fragment of the mouse CD44 5'-flanking sequence mediated this response to IL-1beta. Regulation of CD44 gene expression by the proinflammatory cytokine IL-1beta may contribute to SMC phenotypic modulation in the pathogenesis of arteriosclerosis.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Hialuronatos/genética , Interleucina-1/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Aorta/citologia , Aorta/fisiologia , Arteriosclerose/fisiopatologia , Sequência de Bases , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Genes Reporter/genética , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência de DNA , Transplante Homólogo/imunologiaRESUMO
The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas dos Microfilamentos , Proteínas Musculares/biossíntese , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Drosophila , Genes fos , Proteína HMGA1a , Cinética , Dados de Sequência Molecular , Músculo Liso/metabolismo , Oligodesoxirribonucleotídeos , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Fator de Resposta Sérica , TransfecçãoRESUMO
Although several cytokines and growth factors have been shown to regulate vascular endothelial growth factor (VEGF) production, little is known about how VEGF may regulate growth factors that have known mitogenic and chemotactic actions on mesenchymal cells (which are involved in the maturation of the angiogenic process). We investigated the effect of VEGF on heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression in human umbilical vein endothelial cells. HB-EGF mRNA was induced by 8-fold after 2 h of VEGF stimulation, and it returned to base line within 6 h. VEGF did not alter the half-life of HB-EGF mRNA (55 min). Nuclear run-on experiments showed a 4.9-fold increase in HB-EGF gene transcription within 2 h of VEGF stimulation, and Western analysis demonstrated an associated increase in cellular HB-EGF protein. We found that platelet-derived growth factor-BB (PDGF-BB) mRNA was also induced 3-fold after 5 h of VEGF stimulation, whereas neither endothelin 1 nor transforming growth factor-beta1 was regulated by VEGF. Finally, conditioned medium from VEGF-stimulated endothelial cells produced an increase in DNA synthesis in vascular smooth muscle cells, and this effect was blocked by a neutralizing antibody to PDGF. The induction of HB-EGF and PDGF-BB expression in endothelial cells may represent the mechanism by which VEGF recruits mesenchymal cells to form the medial and adventitial layers of arterioles and venules during the course of angiogenesis.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Linfocinas/metabolismo , Movimento Celular , Células Cultivadas , Meios de Cultivo Condicionados , Endotélio Vascular/citologia , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.