RESUMO
It has been suggested that left ventricular fibrosis in spontaneously hypertensive rats (SHR) is the result of both exaggerated collagen synthesis and insufficient collagen degradation. We have shown previously that chronic treatment with the angiotensin II type 1 receptor antagonist losartan results in diminished synthesis of collagen type I molecules and reversal of myocardial fibrosis in SHR. This study was designed to investigate whether losartan also affects the extracellular degradation of collagen type I fibers in the left ventricle of SHR. The study was performed in 30-week-old normotensive Wistar-Kyoto rats (WKY), untreated SHR, and SHR treated with orally administered losartan (20 mg/kg per day) for 14 weeks before they were killed. Ventricular collagenase activity was determined by degradation of [(14)C]collagen with tissue extracts. Ventricular expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) mRNA was analyzed by Northern blot. A histomorphometric study of the left ventricle was performed in all rats. Compared with WKY, SHR exhibited left ventricular hypertrophy, increased (P<0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and diminished (P<0.05) collagenase activity. After the treatment period, blood pressure was higher (P<0.05) in losartan-treated SHR than in WKY, and no significant differences were noted in the remaining parameters between the 2 strains of rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy, diminished (P<0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and increased (P<0.05) collagenase activity. These results suggest that the transcription of the TIMP-1 gene is upregulated in the hypertrophied and fibrotic left ventricle of adult SHR. Upregulation of TIMP-1 may account for diminished collagenase activity in the myocardium of those rats. Chronic angiotensin II type 1 receptor blockade with losartan resulted in inhibition of TIMP-1 expression and stimulation of collagenase activity in the left ventricle of SHR. It is proposed that angiotensin II may facilitate myocardial fibrosis in SHR by depressing the collagenase-mediated extracellular degradation of collagen fibers.
Assuntos
Antagonistas de Receptores de Angiotensina , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Hipertensão/metabolismo , Hipertensão/patologia , Miocárdio/patologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Colagenases/metabolismo , Fibrose , Expressão Gênica , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/patologia , Losartan/farmacologia , Masculino , Ratos , Ratos Endogâmicos SHR/genética , Ratos Endogâmicos SHR/fisiologia , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein-3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell-cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase-3 activation, Bcl-2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.
Assuntos
Apoptose/imunologia , Autoanticorpos/farmacologia , Queratinócitos/citologia , Queratinócitos/imunologia , Pênfigo/imunologia , Autoanticorpos/imunologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fragmentação do DNA , Relação Dose-Resposta Imunológica , Imunoglobulina G/metabolismo , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Microscopia de Fluorescência , Testes de Precipitina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2RESUMO
The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.
Assuntos
Colágeno Tipo I/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Piridinas/farmacologia , Ratos , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
OBJECTIVE: Previous studies have shown that as well as left ventricular hypertrophy, myocardial fibrosis develops early in rats with spontaneous hypertension (SHR). The present study was designed to investigate whether chronic treatment with the angiotensin II type 1 (AT1) receptor antagonist losartan modifies collagen type I metabolism and reverses left ventricular fibrosis in young SHR with left ventricular hypertrophy. DESIGN: The study was performed in 30-week-old normotensive Wistar-Kyoto (WKY) rats, untreated SHR and SHR treated with losartan (20 mg/mg per day, orally) for 14 weeks before they were killed. METHODS: Ventricular pro-alpha 1 (I) collagen messenger RNA was analyzed by Northern blot. Serum levels of the carboxy-terminal propeptide of procollagen type I (PIP) and the pyridoline cross-linked telopeptide domain of collagen type I (CITP) were determined by specific radioimmunoassays as markers of collagen type I synthesis and degradation, respectively. Collagen volume fraction was determined in the left ventricle by quantitative morphometry. RESULTS: Compared with WKY rats, SHR exhibited increased (P < 0.05) mean arterial pressure, pro-alpha 1 (I) collagen messenger RNA, PIP and left ventricular collagen volume fraction, and similar CITP values. After the treatment period, mean arterial pressure was higher (P < 0.05) in losartan-treated SHR than in WKY rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy and diminished (P < 0.05) values of mean arterial pressure, PIP and left ventricular collagen volume fraction. No changes in pro-alpha 1 (I) collagen messenger RNA and CITP values were observed with treatment in SHR. No significant differences in the left ventricular collagen volume fraction were observed between treated SHR with normal blood pressure and treated SHR with abnormally high blood pressure at the end of the treatment period. CONCLUSIONS: These results suggest that chronic AT1 blockade with losartan decreases the post-transcriptional synthesis of fibril-forming collagen type I molecules in young SHR. This effect may be involved in the ability of this drug to reverse left ventricular fibrosis in young rats with genetic hypertension. Apart from its antihypertensive action, other mechanisms may mediate the antifibrotic effect of losartan in this animal model.
Assuntos
Anti-Hipertensivos/farmacologia , Cardiomiopatias/tratamento farmacológico , Colágeno/metabolismo , Hipertensão/tratamento farmacológico , Losartan/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transcrição Gênica , Animais , Biomarcadores/sangue , Pressão Sanguínea/efeitos dos fármacos , Northern Blotting , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Colágeno/sangue , Colágeno/genética , Colágeno Tipo I , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Fibrose/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Masculino , Peptídeos/sangue , Pró-Colágeno/sangue , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
The Ca²( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF1. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Spinacia oleracea/enzimologia , Ditiotreitol/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Oxirredução , Proteínas/metabolismoRESUMO
Synthetic peptides with sequences present in extracellular matrix protein fibronectin have been described to stimulate human monocytes. We describe now that one of these peptides, FN6, induces apoptotic effects on monocytes and we investigate the molecular mechanisms involved in the regulation of this response. Incubation of monocytes with FN6 induces the activation of the small GTPase Rac. In turn, Rac mediates the increase of both JNK and p38 activities in a sustained fashion, as well as the phosphorylation levels of their respective substrates c-Jun and ATF-2. FN6 also stimulates caspases -9 and -3 and the delayed proteolysis of its substrates PARP and D4-GDI. In addition, initiator caspases-1 and -5 were activated by FN6 treatment of monocytes but, in contrast to that observed for caspases-9 and -3, this effect was not dependent on JNK or p38 activities. These kinases also mediated the increase of Bax levels, but only in some conditions Bcl-2 depletion caused by the peptide. Moreover, whereas initially only caspase-1 is involved in caspase-3 activation, later on caspase-9 seems also to participate. Therefore, we demonstrate that FN6 stimulation allows multiple, JNK and p38-dependent and -independent interacting signals to regulate the apoptotic response in human monocytes.
Assuntos
Apoptose/efeitos dos fármacos , Fibronectinas/química , Monócitos/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.
Assuntos
ATPases Transportadoras de Cálcio/química , Cloroplastos/enzimologia , Proteínas de Plantas/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Sítio Alostérico , Ânions/farmacologia , Sítios de Ligação , ATPases Transportadoras de Cálcio/metabolismo , Catálise , Ativação Enzimática/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/farmacologia , Cinética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tripsina/farmacologiaRESUMO
3,4-Methylenedioxymethamphetamine, MDMA ("Ecstasy"), has been previously shown to produce cell necrosis and fibrosis in the liver. Our aim was to study the effect of MDMA on the type I collagen production by a cell line of hepatic stellate cells (HSC), the cell type mainly responsible for collagen synthesis in the liver. We demonstrated that MDMA increases alpha1(I) procollagen mRNA levels and that this increase correlates with glutathione depletion and enhanced hydrogen peroxide production by HSC. Pre-treatment with either glutathione monoethyl ester or deferoxamine prevents the MDMA-induced alpha1(I) procollagen mRNA expression, indicating oxidative stress to be a mediator of this effect. Lipid peroxidation was not detected in MDMA-treated cells and therefore does not seem to be involved in the pro-fibrogenic action of MDMA on HSC.
Assuntos
Regulação da Expressão Gênica , Fígado/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Pró-Colágeno/metabolismo , Antioxidantes/farmacologia , Linhagem Celular , Desferroxamina/farmacologia , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo , Pró-Colágeno/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
BACKGROUND: In response to nonspecific inflammatory stimuli, circulating monocytes produce several cytokines that regulate the expression of liver acute phase protein genes. Patients with alcoholic liver disease have several manifestations of the acute phase response, including elevated serum levels of interleukin (IL)-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha). However, the role of the acute phase response on liver fibrogenesis has not been explored. EXPERIMENTAL DESIGN: In this communication we report experiments performed to investigate whether turpentine, an acute phase response inducer in rats has any effect on alpha 1 (I) procollagen gene expression in the liver. We also investigated which of the cytokines is responsible for the turpentine effect and whether IL-6 and TNF-alpha had an effect on alpha 1 (I) procollagen mRNA expression by liver fat-storing cells (FSC). RESULTS: We show that alpha 1 (I) procollagen mRNA is increased in livers of turpentine-treated rats, and that an antibody to IL-6 as well as colchicine inhibit this effect. We also show that rIL-6 induces the expression of alpha 1 (I) procollagen mRNA in cultured FSC but not in hepatocytes. We demonstrated that the IL-6 effect is a transcriptional event that requires "de novo" protein synthesis. In addition to its effect on collagen gene expression, rIL-6 also stimulates expression of transforming growth factor-beta and fibronectin mRNAs. TNF-alpha inhibits alpha 1 (I) procollagen expression in FSC by 24 to 48 hours. However, TNF-alpha induces a transient expression of alpha 1 (I) procollagen mRNA by 2 to 3 hours. This increase is preceded by the induction of IL-6 mRNA. CONCLUSIONS: We conclude that IL-6 produced during the acute phase response, alone or in conjunction with other cytokines, could play an important role in liver fibrogenesis by inducing the expression of collagen, fibronectin, and transforming growth factor-beta mRNAs in FSC.
Assuntos
Reação de Fase Aguda/metabolismo , Interleucina-6/biossíntese , Fígado/metabolismo , Pró-Colágeno/biossíntese , RNA Mensageiro/biossíntese , Animais , Northern Blotting , Linhagem Celular , Interleucina-6/fisiologia , Metabolismo dos Lipídeos , Fígado/citologia , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up-regulation of transforming growth factor beta (TGF-beta), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-beta induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-beta-elicited alpha1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-beta induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of alpha1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-beta-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (-370 to -344) essential for TGF-beta responsiveness. We further showed that TGF-beta induced the activation and binding of a C/EBPbeta-containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF-beta-mediated accumulation of H2O2 and the up-regulation of col1a1 gene in HSC.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Peróxido de Hidrogênio/metabolismo , Fígado/fisiologia , Proteínas Nucleares/fisiologia , Oxidantes/metabolismo , Pró-Colágeno/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Antioxidantes/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT , Catalase/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Colágeno/genética , Genes Reporter/genética , Peróxido de Hidrogênio/farmacologia , Fígado/citologia , Fígado/metabolismo , Camundongos/genética , Oxidantes/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Elementos de Resposta/genética , Tiocarbamatos/farmacologiaRESUMO
Tumor necrosis factor alpha (TNF-alpha) is one of the key cytokines of the acute phase response and of many inflammatory processes. This cytokine has several antifibrogenic actions and down-regulates the expression of the type I collagen genes and induces the expression of metalloproteinases. Because TNF-alpha directly antagonizes some fibrogenic actions of transforming growth factor beta(1) (TGF-beta(1)), we considered it important to map the cis-acting regulatory element of the alpha1(I) collagen (col1a1) promoter involved in TNF-alpha responsiveness in hepatic stellate cells (HSC), to investigate the transcription factors that bind to it, and to establish possible mechanisms by which TNF-alpha down-regulates its expression. In this article, we show the presence of a functional TNF-alpha-responsive element (TaRE) in the -378 to -345 region of the col1a1 promoter. This element colocalizes with a previously reported TGF-beta(1)-responsive element. We further demonstrate that TNF-alpha induces nuclear translocation and binding of transcriptional complexes containing p20C/EBPbeta, p35C/EBPbeta, and C/EBPdelta to this sequence of the promoter. Transient overexpression of C/EBPdelta or p20C/EBPbeta, the natural dominant negative form of C/EBPbeta in HSC, down-regulated activity of a CAT reporter vector driven by -412 to +110 of the col1a1 promoter. Taken together, these data suggest that the -378 to -340 region of the col1a1 promoter is the site of convergence of different stimuli that ultimately modulate col1a1 gene transcription.