RESUMO
Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression.
Assuntos
Colágeno/genética , Peróxidos Lipídicos/metabolismo , Vitamina E/farmacologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Northern Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Azul de Metileno/química , Probucol/farmacologia , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in patients with chronic liver diseases.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores do Crescimento/fisiologia , Fígado/citologia , Fígado/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Fígado/fisiologia , Masculino , Fosforilação , Ratos , Serina/biossíntese , Serina/fisiologiaRESUMO
In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized.
Assuntos
Aldeídos/metabolismo , Ferro/metabolismo , Lisina/metabolismo , Malondialdeído/metabolismo , Proteínas/metabolismo , Animais , Peso Corporal , Eletroforese em Gel Bidimensional , Fluorescência , Peróxidos Lipídicos/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Peso Molecular , Ratos , Ratos Endogâmicos , Tiobarbitúricos/metabolismoRESUMO
Although collagen is known to enhance hepatocyte differentiation and hepatocytes produce collagen in vivo, the transcriptional factors responsible for collagen type I gene expression in hepatic cells are not known. LAP (Liver Activator Protein) is a member of the C/EBP family, which in differentiated hepatocytes contributes to the high levels of liver-specific gene expression. In this study we show that LAP binds to the collagen alpha 1(I) promoter at both reverse CCAAT motifs and activates transcription. Furthermore, an upstream element, collagen element I (-370/-344), which shares homology with the LAP binding cis-element of the albumin promoter (9 of 13 bp) is described. This collagen element I stimulates transcription in both orientations and when placed in front of either a homologous or a heterologous chimeric report construct. These experiments suggest that LAP may be important in the expression of collagen in differentiated hepatocytes through both the promoter and a newly described upstream element.
Assuntos
Colágeno/genética , Proteínas de Ligação a DNA/farmacologia , Genes Reguladores , Proteínas Nucleares/farmacologia , Fatores de Transcrição/farmacologia , Ativação Transcricional , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and alpha-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGF alpha was blocked by antioxidants, such as d-alpha-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGF alpha and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity, and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGF alpha. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the alpha-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation.
Assuntos
Adipócitos/metabolismo , Colágeno/farmacologia , Fígado/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Fator de Crescimento Transformador alfa/farmacologia , Adipócitos/citologia , Animais , Sequência de Bases , Divisão Celular , Células Cultivadas , Fígado/citologia , Masculino , Dados de Sequência Molecular , NF-kappa B/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-myb , Ratos , Ratos Sprague-DawleyRESUMO
The expression of the collagen alpha 1(I) gene in activated stellate cells plays an important role during liver fibrogenesis. To identify the critical cis-elements of the collagen alpha 1(I) gene in stellate cells, we used transgenic animals bearing various collagen alpha 1(I) regulatory regions directing the expression of either a human growth hormone minigene or the bacterial beta-galactosidase gene. We found that collagen alpha 1(I)-human growth hormone transgene expression was constitutively high in tendon and skin, provided the transgene contained the -2.3 to -0.44 kb collagen regulatory region. However in the liver, expression was stimulated several-fold, as was the endogeneous gene, by the fibrogenic hepatotoxin carbon tetrachloride. This stimulation occurred whether the collagen 5' regulatory region extended -2.3, -1.6 or -0.44 kb, and in the presence or absence of much of the first intron (+292 to +1607 bp). In addition, the -0.44 kb 5' region was sufficient for high-level transgene expression in stellate cells, following their activation by culture on plastic. In contrast, in skin and tendon, high-level transcription of the collagen alpha 1(I) gene required the -2.3 to -0.44 kb 5' flanking region. Thus, two different cis-regulatory regions direct cell-specific transcription of the collagen alpha 1(I) gene in stellate cells and in skin and tendon.
Assuntos
Colágeno/genética , Fígado/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Pele/metabolismo , Tendões/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Células Cultivadas , Colágeno/biossíntese , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de ÓrgãosRESUMO
Ascorbic acid stimulates collagen gene expression in cultured fibroblasts but mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Because another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts, we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mmol/L) induced lipid peroxidation and stimulated collagen alpha 1(I) gene transcription in cultured human fibroblasts. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 mumol/L) or methylene blue (10 mumol/L) prevented the stimulation of collagen gene expression. Addition of malondialdehyde (200 mumol/L), a product of lipid peroxidation, to cultured human fibroblasts also increased two- to threefold collagen production and procollagen alpha 1(I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes, and this step may be necessary for stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.
Assuntos
Ácido Ascórbico/farmacologia , Colágeno/biossíntese , Células Cultivadas , Colágeno/genética , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Hidroxilação , Malondialdeído/farmacologia , Transcrição Gênica , Vitamina E/farmacologiaRESUMO
We have shown that lipid peroxidation stimulates collagen-alpha 1 (I) gene transcription in cultured cells. Because iron is a transitional metal known to induce lipid peroxidation, we investigated whether hepatic lipid peroxidation modulates collagen gene expression in iron-overloaded rats. In this animal model of hemochromatosis, we show colocalization with iron in the hepatic acinar zone 1 of both lipid peroxidation and increased collagen-alpha 1 (I) transcripts, using immunohistochemistry for malondialdehyde-protein adducts and in situ hybridization, respectively. Iron overload stimulated the expression of the cytokine transforming growth factor-beta (TGF-beta) in acinar zone 1, in spite of the minor degree of hepatocellular necrosis and inflammation. The formation of reactive aldehydes and TGF-beta, both inducers of collagen gene expression, may play a role in the stimulation of hepatic collagen production in iron overload. These mechanisms could be a link between iron overload and fibrosis in genetic hemochromatosis.
Assuntos
Colágeno/genética , Expressão Gênica , Hemocromatose/genética , Fígado/fisiopatologia , Fator de Crescimento Transformador beta/genética , Animais , Colágeno/classificação , Hemocromatose/metabolismo , Imuno-Histoquímica , Ferro/metabolismo , Peróxidos Lipídicos/fisiologia , Malondialdeído/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Distribuição TecidualRESUMO
The mechanisms responsible for impaired wound healing in patients with cachexia-associated infection, inflammation, and cancer are unknown. As tumor necrosis factor (TNF)-alpha is elevated in these diseases, and TNF-alpha inhibits collagen alpha1(I) gene expression in cultured fibroblasts, we analyzed whether chronically elevated serum TNF-alpha affects collagen metabolism in vivo by inoculating nude mice with Chinese hamster ovary cells secreting TNF-alpha (TNF-alpha mice) or control Chinese hamster ovary cells (control mice). Before the onset of weight loss, TNF-alpha mice had a selective decrease in collagen synthesis and collagen alpha1(I) mRNA in the skin. In addition, TNF-alpha mice displayed impaired healing of incisional and excisional skin wounds, compared with control animals, before the onset of cachexia. The expression of transforming growth factor-beta1, a potent fibrogenic factor, was inhibited by TNF-alpha in the skin. In studies with transgenic mice expressing the human growth hormone under the direction of 5' regulatory regions of the human collagen alpha1(I) gene, TNF-alpha treatment inhibited the expression of the collagen alpha1(I) human growth hormone transgene containing -2.3 kb of the 5' region, whereas transgene expression directed by -0.44 kb of the 5' region was not affected. These experiments suggest that TNF-alpha may play an important role in the impaired wound healing of chronic diseases that are characterized by a high production of this cytokine and provide insights for potential therapeutic approaches.
Assuntos
Caquexia/fisiopatologia , Colágeno/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Cicatrização/fisiologia , Animais , Caquexia/tratamento farmacológico , Caquexia/patologia , Colágeno/efeitos dos fármacos , Colágeno/genética , Cricetinae , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Pele/patologia , Pele/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/patologiaRESUMO
BACKGROUND & AIMS: Oxidative stress mediates activation and stimulates collagen production of cultured hepatic stellate (Ito) cells. The aim of this study was to assess whether oxidative stress contributes to hepatic fibrogenesis in chronic hepatitis C. METHODS: In liver biopsy specimens of patients with chronic hepatitis C, the following fibrogenesis cascade was analyzed: (1) oxidative stress, determined by the presence of malondialdehyde protein adducts; (2) activation of stellate cells as indicated by their expression of alpha-smooth muscle actin; (3) stimulation of c-myb expression in stellate cells, a critical step in the activation of these cells; and (4) induction of collagen gene expression as detected by in situ hybridization. RESULTS: Treatment with d-alpha-tocopherol (1200 IU/day for 8 weeks) in 6 of these patients, who were refractory to interferon therapy, prevented the fibrogenesis cascade observed before antioxidant treatment. In addition, d-alpha-tocopherol treatment significantly decreased the carbonyl modifications of plasma proteins, a sensitive index of oxidative stress. However, 8 weeks of d-alpha-tocopherol treatment did not significantly affect serum alanine aminotransferase levels, hepatitis C virus titers, or histological degree of hepatocellular inflammation or fibrosis. CONCLUSIONS: These data suggest that enhanced oxidative stress initiates a fibrogenesis cascade in the liver of patients with chronic hepatitis C.
Assuntos
Hepatite C/tratamento farmacológico , Hepatite C/patologia , Vitamina E/uso terapêutico , Actinas/biossíntese , Biópsia , Colágeno/biossíntese , Feminino , Hepatite C/metabolismo , Humanos , Hibridização In Situ , Fígado/metabolismo , Fígado/patologia , Masculino , Malondialdeído/análise , Pessoa de Meia-Idade , Estresse Oxidativo , Projetos Piloto , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-myb , Proto-Oncogenes , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transativadores/biossíntese , Transcrição GênicaRESUMO
Although tumor necrosis factor-alpha (TNF-alpha) inhibits collagen-alpha 1(I) gene expression in cultured hepatic stellate cells, assessment of its effects on hepatic collagen expression is complicated by the confounding variables of tissue necrosis and inflammation. Therefore, we analyzed whether chronically elevated serum TNF-alpha affects constitutive hepatic collagen metabolism in vivo by inoculating nude mice with Chinese hamster ovary (CHO) cells secreting TNF-alpha (TNF-alpha mice) or with control CHO cells (control mice). Before the onset of weight loss, collagen synthesis and collagen gene expression were inhibited in the liver of TNF-alpha mice. In transgenic mice, after 8 h, TNF-alpha (500 ng at 0 and 5 h) inhibited the liver expression of the collagen-alpha 1(I)-human growth hormone (hGH) transgene containing the first intron and -440 bp of the 5' region. Similarly, in cultured hepatic stellate cells isolated from these transgenic animals, the -440 bp collagen-alpha 1(I)-hGH transgene was responsive to TNF-alpha treatment independent of the activation of these cells. Transfection studies in stellate cells allowed further characterization of this TNF-alpha-responsive segment to -220 bp of the 5' region. Because in the skin the inhibitory effect of TNF-alpha involves a regulatory region of the collagen-alpha 1(I) gene beyond -440 bp, we herein identify a novel tissue-specific regulation of collagen-alpha 1(I) gene by TNF-alpha.
Assuntos
Colágeno/antagonistas & inibidores , Colágeno/genética , Expressão Gênica/fisiologia , Fígado/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células CHO , Cricetinae , Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento Humano/genética , Humanos , Isomerismo , Fígado/citologia , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos/genética , Valores de Referência , Fatores de Tempo , Transgenes/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We analyzed the role of oxidative stress on liver collagen gene expression in vivo. Long- and short-term supplementation with the lipophilic antioxidant D-alpha-tocopherol (40 IU/day for 8 wk or 450 IU for 48 h) to normal C57BL/6 mice selectively decreased liver collagen mRNA by approximately 70 and approximately 60%, respectively. In transgenic mice, the -0.44 kb of the promoter and the first intron of the human collagen alpha1(I) gene were sufficient to confer responsiveness to D-alpha-tocopherol. Inhibition of collagen alpha1(I) transactivation in primary cultures of quiescent stellate cells from these transgenic animals by D-alpha-tocopherol required only -0.44 kb of the 5' regulatory region. This regulation resembled that of the intact animal following D-alpha-tocopherol treatment and indicates that D-alpha-tocopherol may act directly on stellate cells. Transfection of stellate cells with collagen-LUC chimeric genes allowed localization of an "antioxidant"-responsive element to the -0.22 kb of the 5' region excluding the first intron. These findings suggest that oxidative stress, independently of confounding variables such as tissue necrosis, inflammation, cell activation, or cell proliferation, modulates in vivo collagen gene expression.
Assuntos
Colágeno/genética , Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Vitamina E/farmacologia , Animais , Células Cultivadas , Quimera/genética , Feminino , Genes Reporter/genética , Hormônio do Crescimento Humano/genética , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Valores de Referência , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacosRESUMO
Ascorbic acid stimulates collagen gene expression in cultural fibroblasts (Lyons, B. L., and Schwartz, R. L. (1984) Nucleic Acids Res. 12, 2569-2579), but the mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Since another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts (Brenner, D. A., and Chojkier, M. (1987) J. Biol. Chem 262, 17690-17696), we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mM) induced lipid peroxidation in cultured human fibroblasts judging by the production of thiobarbituric acid-reactive substances and carbonyl groups, and by the presence of malondialdehyde- and 4-hydroxynonenal-protein adducts. Ascorbic acid stimulated (2-3-fold) the net production of collagen relative to total proteins, the levels of procollagen alpha 1 (I) mRNA and the transcription of this gene. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 microM) or methylene blue (10 microM) prevented the stimulation of collagen gene expression. The addition of malondialdehyde (200 microM), a product of lipid peroxidation, to cultured human fibroblasts also increased 2-3-fold collagen production and procollagen alpha 1 (I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes and this step may be necessary for the stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.
Assuntos
Ácido Ascórbico/farmacologia , Colágeno/genética , Genes/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Feto , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Malondialdeído/metabolismo , Vitamina E/farmacologiaRESUMO
We have shown that lipid peroxidation stimulates collagen alpha 1(I) gene transcription in cultured cells. Because increased lipid peroxidation and collagen production coexist in many hepatic disorders, including experimental carbon tetrachloride intoxication, we investigated whether lipid peroxidation modulates collagen gene expression in rats treated with carbon tetrachloride. In this animal model, we show colocalization of increased collagen alpha 1(I) mRNA with lipid peroxidation by means of in situ hybridization and immunohistochemical study for malondialdehyde and 4-hydroxynonenal protein adducts, respectively. However, allyl alcohol treatment, which induced a similar degree of hepatocellular injury but without aldehyde-protein adducts, did not increase collagen alpha 1(I) gene expression, suggesting that hepatocyte necrosis is not sufficient to induce the expression of collagen type I. Furthermore, in the absence of an inflammatory response, coculture experiments of hepatocytes and Ito cells treated with carbon tetrachloride indicate that hepatocytes exert a "paracrine" stimulation of both lipid peroxidation and collagen gene expression in Ito cells. These experiments suggest that hepatocyte lipid peroxidation plays a major role in the regulation of collagen alpha 1(I) gene expression by Ito cells and that it may be a link between hepatocyte injury and hepatic fibrosis.
Assuntos
Colágeno/genética , Regulação da Expressão Gênica , Peroxidação de Lipídeos , Cirrose Hepática Experimental/etiologia , Fígado/patologia , 1-Propanol/efeitos adversos , Aldeídos/metabolismo , Animais , Intoxicação por Tetracloreto de Carbono , Células Cultivadas , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Masculino , Malondialdeído/metabolismo , Propanóis , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Expression of alpha-smooth muscle actin (alpha-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of alpha-SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal alpha-SMA E box. Therefore, we analyzed the role of c-Myb in alpha-SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the -724 and -271 alpha-SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (-223/-216 bp). Alpha-SMA/LUC minigenes with mutation (-219/-217 bp), truncation (-224 bp), or deletion (-191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a approximately 10-fold stimulation of -724 alpha-SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys(43) --> Asp) or a c-Myb antisense RNA blocked transcription from the -724 alpha-SMA/LUC or -271 alpha-SMA/LUC in activated cells. Moreover, transfection of c-myb antisense, but not sense, RNA inhibited both expression of the endogenous alpha-SMA gene and stellate cell activation, whereas transfection of c-myb stimulated alpha-SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys(43) in c-Myb is required for this effect.
Assuntos
Actinas/genética , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas Proto-Oncogênicas c-myb/fisiologia , Animais , Células Cultivadas , Imunofluorescência , Fígado/citologia , Masculino , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , RNA Antissenso , Ratos , Ratos Sprague-Dawley , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Acetaldehyde, the first product of ethanol oxidation, has been shown to stimulate collagen gene expression and to form protein-acetaldehyde adducts. Because little is known about these adducts in human liver tissue, we assessed, with an immunohistochemical procedure, the presence and location of acetaldehyde-protein adducts in liver biopsy specimens of alcoholic patients. In addition, we correlated the presence of adducts with the progression or subsequent occurrence of liver fibrosis. The group included 106 patients with high alcohol consumption (> 90 gm ethanol/day for the last 5 yr), 10 nonalcoholic patients with normal livers and 23 patients with other liver diseases. Sixty-four of the 106 alcoholic patients had a second liver biopsy, whose specimen was used to assess the progression of liver fibrosis. Polyclonal antibodies were produced against homologous low-density lipoprotein purified from rabbit serum and modified in vitro in the presence of acetaldehyde. Protein-acetaldehyde adducts could be detected by immunohistochemistry in biopsy specimens of 90 alcoholic patients (85%), in none of the 10 nonalcoholic patients with normal livers and in 65% of the patients with nonalcoholic liver disease. Acetaldehyde-modified epitopes were detected in the intracellular and extracellular compartment. Intracellular protein-acetaldehyde adducts were localized in the cytoplasm of hepatocytes with a more intense staining in zone 3. No correlation existed between the intensity of intracellular staining and the histologically assessed severity of liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acetaldeído/análise , Cirrose Hepática Alcoólica/etiologia , Fígado/química , Acetaldeído/imunologia , Adulto , Especificidade de Anticorpos , Biópsia por Agulha , Epitopos/análise , Feminino , Humanos , Imuno-Histoquímica , Fígado/patologia , Cirrose Hepática Alcoólica/patologia , Hepatopatias/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Genetic hemochromatosis (GH) is associated with excess iron deposition in hepatocytes, which results in progressive hepatic injury. The pathogenesis of hepatic injury in GH is poorly understood. In this study, we found enhanced oxidative stress in patients with GH, as evidenced by hepatic malondialdehyde (MDA)-protein adducts and by increased oxidatively modified serum proteins. MDA-lysine epitopes and oxidatively modified serum proteins, as well as immunoglobulin G autoantibodies against MDA-lysine epitopes, were increased in untreated GH patients and to a lesser extent in GH heterozygotes compared with normal individuals. These markers of ongoing oxidative stress decreased with phlebotomy treatment in GH patients. In addition, TGF-beta1 colocalized with hepatic iron and MDA protein adducts in hepatocytes and sinusoidal cells of hepatic acinar zone 1 and normalized after iron removal. Our data suggest that iron overload increases both lipid peroxidation and TGF-beta1 expression, which together could promote hepatic injury and fibrogenesis.
Assuntos
Proteínas Sanguíneas/química , Hemocromatose/genética , Hemocromatose/fisiopatologia , Ferro/metabolismo , Fígado/metabolismo , Malondialdeído/análise , Estresse Oxidativo , Adulto , Aldeídos , Autoanticorpos/análise , Biópsia , Feminino , Hemocromatose/patologia , Heterozigoto , Humanos , Imunoglobulina G/análise , Cetonas , Fígado/patologia , Masculino , Malondialdeído/imunologia , Estudos Retrospectivos , Transferrina/metabolismoRESUMO
Activated, but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and smooth muscle actin (SMA) gene expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. The mechanisms leading to stellate cell activation in vivo are unknown. The characteristic hepatic oxidative stress cascade induced in rats by CCl4 markedly stimulated stellate cell entry into S phase, nuclear factor (NF)-kappa B activity, and c-myb expression. These changes were prevented by pentoxifylline, which also decreased CCl4-induced hepatic injury. As expected, cAMP-mediated phosphorylation of CREB-Ser133 was induced in vivo in stellate cells by pentoxifylline but not by its metabolite 5, an N-1 carboxypropyl derivative, which lacks phosphodiesterase inhibitory activity. Stellate cell nuclear extracts from CCl4-treated, but not from control, animals formed a complex with the critical promoter E box of the alpha-SMA gene, which was disrupted by c-myb antibodies and competed with by c-myb cognate DNA. Treatment with pentoxifylline or metabolite 5 prevented the molecular abnormalities characteristic of stellate cell activation induced by CCl4. These results suggest that induction of c-myb plays an important role in the in vivo activation of stellate cells. Pentoxifylline blocks stellate cell activation in vivo independently of its inhibitory effects on phosphodiesterases by interfering with the oxidative stress cascade and the activation of NF-kappa B and c-myb.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pentoxifilina/farmacologia , Células 3T3 , Actinas/biossíntese , Animais , Tetracloreto de Carbono/toxicidade , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Colágeno/biossíntese , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fígado/citologia , Fígado/fisiologia , Masculino , Camundongos , NF-kappa B/biossíntese , Estresse Oxidativo , Pentoxifilina/análogos & derivados , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-myb , Ratos , Ratos Sprague-Dawley , Transativadores/biossínteseRESUMO
BACKGROUND AND AIMS: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. METHODS: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA). RESULTS: Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. CONCLUSIONS: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.