RESUMO
UNLABELLED: Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP-12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl(4) ) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP-12 elastin degradation was defined mechanistically using CD11b-DTR and MMP-12 knockout mice. In a CCl(4) model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP-12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP-12 was bound in greater quantities to its inhibitor TIMP-1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP-12. Exposure of CCl(4) in MMP-12(-/-) mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP-12(-/-) mice compared with WT. CONCLUSION: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage-derived MMP-12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies.
Assuntos
Elastina/metabolismo , Cirrose Hepática Experimental/metabolismo , Macrófagos/enzimologia , Metaloproteinase 12 da Matriz/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Fígado/metabolismo , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Tropoelastina/biossínteseRESUMO
Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover, these findings, together with our previous reports and the emerging data from in vivo studies of TIMP inhibition, provide strong evidence that TIMP-1 is mechanistically central to liver fibrosis and an important potential therapeutic target.
Assuntos
Proliferação de Células , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/genética , RNA Interferente Pequeno/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Células Cultivadas , Inativação Gênica , Células Estreladas do Fígado/citologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
UNLABELLED: Hepatic myofibroblast apoptosis is critical to resolution of liver fibrosis. We show that human hepatic myofibroblasts co-express p75(NTR) (p75 neurotrophin receptor) and sortilin, thus facilitating differential responses to mature and pro nerve growth factor (proNGF). Although mature NGF is proapoptotic, proNGF protects human hepatic myofibroblasts from apoptosis. Moreover, in recovery from experimental liver fibrosis, the decrease in proNGF parallels loss of hepatic myofibroblasts by apoptosis. Macrophage-derived matrix metalloproteinase 7 (MMP7) cleaves proNGF in a concentration-dependent manner, and its expression in the liver coincides with falling proNGF levels. To define the dominant effect of p75(NTR)-mediated events in experimental liver fibrosis, we have used a mouse lacking the p75(NTR) ligand-binding domain but expressing the intracellular domain. We show that absence of p75(NTR) ligand-mediated signals leads to significantly retarded architectural resolution and reduced hepatic myofibroblast loss by apoptosis. Lack of the ligand-competent p75(NTR) limits hepatocyte and oval cell proliferative capacity in vivo without preventing hepatic stellate cell transdifferentiation. CONCLUSION: NGF species have a differential effect on hepatic myofibroblast survival. Our data suggest that cleavage of proNGF by MMP7 during the early phase of recovery from liver fibrosis alters the pro/mature NGF balance to facilitate hepatic myofibroblast loss. Whereas fibrosis develops in the absence of p75(NTR) signaling, the dominant effects of loss of p75(NTR) ligand-mediated events are the retardation of liver fibrosis resolution via regulation of hepatic myofibroblast proliferation and apoptosis, and the reduction of hepatocyte and oval cell proliferation.
Assuntos
Apoptose/fisiologia , Proliferação de Células , Fibroblastos/metabolismo , Cirrose Hepática/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Recuperação de Função Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacologia , Receptor de Fator de Crescimento Neural/genéticaRESUMO
BACKGROUND AND AIMS: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix-degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP-1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N-cadherin at the cell surface. RESULTS: N-cadherin expression was upregulated in human HSC during activation in culture. Addition of function-blocking antibodies or a peptide targeting the extracellular domain of N-cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N-cadherin into 20-100 kDa fragments. MMP-2 became activated early during HSC apoptosis and directly cleaved N-cadherin in vitro. Addition of activated MMP-2 to HSCs in culture resulted in enhanced apoptosis and loss of N-cadherin. CONCLUSIONS: Together, these studies identify a role for both N-cadherin and MMP-2 in mediating HSC apoptosis, where N-cadherin works to provide a cell survival stimulus and MMP-2 promotes HSC apoptosis concomitant with N-cadherin degradation.
Assuntos
Antígenos CD/metabolismo , Apoptose , Caderinas/metabolismo , Células Estreladas do Fígado/enzimologia , Cirrose Hepática Experimental/enzimologia , Fígado/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono , Caspase 3/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Ativação Enzimática , Gliotoxina/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
PURPOSE: The treatment of metastatic colorectal carcinoma represents a major clinical challenge. We investigated the hypothesis that the desmoplastic reaction within the liver elicited by metastatic adenocarcinoma, characterized by collagen I deposition and altered collagen IV distribution, promotes the growth and survival of hepatic colorectal carcinoma metastases. EXPERIMENTAL DESIGN: Partial hepatectomy specimens for metastatic colorectal adenocarcinoma were examined immunohistochemically for differential integrin expression. Human colorectal adenocarcinoma cell lines HT-29, KM12SM, and KM12c were grown on wild-type collagen I or IV, or cleavage-resistant r/r collagen I, and assessed for their growth, survival, and resistance to 5-fluorouracil. The effect of alpha(v)beta(3) and alpha(v)beta(5) integrin blockade by neutralizing antibodies was examined. RESULTS: Collagen I, in contrast to collagen IV, significantly enhanced the growth, survival, and chemoresistance of colorectal carcinoma cells. Blockade of the alpha(v)beta(3) and alpha(v)beta(5) integrins significantly reduced colorectal carcinoma cell proliferation on collagen, especially in the cell line with the most metastatic potential. These in vitro findings correlated with the pattern of integrin expression identified within resected hepatic colorectal carcinoma metastases. Using matrix metalloproteinase-resistant r/r collagen I as a dominant negative ligand for alpha(v) integrins, we showed a key role for this integrin-ligand interaction in mediating the survival and proliferation of colorectal carcinoma cells. CONCLUSIONS: Desmoplasia has an important role in the development of hepatic colorectal carcinoma metastasis. The interaction between integrin and collagen I is identified as a potential therapeutic target.
Assuntos
Colágeno Tipo IV/metabolismo , Colágeno Tipo I/metabolismo , Integrina alfaV/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Fluoruracila/farmacologia , Hepatectomia , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Integrina alfaV/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Células Tumorais CultivadasRESUMO
PURPOSE: The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype. EXPERIMENTAL DESIGN: PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models. RESULTS: In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [3H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([3H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1). CONCLUSIONS: These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Colágeno Tipo I/fisiologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/metabolismo , Apoptose , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Colágeno/química , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Pâncreas/citologia , Neoplasias Pancreáticas/metabolismo , Fenótipo , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.
Assuntos
Colágeno/biossíntese , Matriz Extracelular/metabolismo , Fígado/citologia , Fígado/metabolismo , Actinas/genética , Animais , Membrana Basal/metabolismo , Adesão Celular , Divisão Celular , Células Cultivadas , Colágeno Tipo I/genética , Técnicas In Vitro , Integrinas/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Vitamina A/metabolismoAssuntos
Hepatopatias/enzimologia , Fígado/enzimologia , Metaloproteinases da Matriz/metabolismo , Traumatismo por Reperfusão/enzimologia , Animais , Isquemia Fria/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Fígado/efeitos dos fármacos , Hepatopatias/prevenção & controle , Inibidores de Metaloproteinases de Matriz , Traumatismo por Reperfusão/prevenção & controle , Isquemia Quente/efeitos adversosRESUMO
Apoptosis of hepatic stellate cells (HSC) has previously been shown to occur during spontaneous resolution of experimental liver fibrosis. TIMP-1 has also been shown to have a key role because of its ability to inhibit apoptosis of HSC via matrix metalloproteinase (MMP) inhibition. This has led to further study of novel substrates for MMPs that might impact on HSC survival. N-Cadherin is known to mediate cell-cell contacts in fibroblasts. In this study we demonstrate that N-Cadherin is expressed by activated rat HSC. Furthermore, during apoptosis of HSC, the N-Cadherin is cleaved into smaller fragments. Apoptosis of HSC may be inhibited by TIMP-1. This is associated with reduced fragmentation of N-Cadherin. N-Cadherin may have an important role in supporting HSC survival while N-Cadherin cleavage may play a part in promoting HSC apoptosis in recovery from liver fibrosis.
RESUMO
Transforming growth factor beta-1 (TGF-beta 1) plays a pivotal role in tissue fibrogenesis. Understanding the factors that control resolution of fibrosis is critical to devising means to combat clinical fibrosis. Future challenges would include designing ways to block the fibrosis-specific actions of TGF-beta. Blockade of transforming growth factor beta (TGF-beta) activity in vivo in animal models has proven to be an effective means of inhibiting the fibrotic response to injury in various organs. Similarly, transgenic animals in which TGF-beta 1 expression is artificially enhanced show marked spontaneous fibrosis or increased fibrotic response to injury. TGF-beta is known to effect fibroplasias, not only by its well known action of increasing extracellular matrix synthesis but also by coordinately regulating key proteins which mediate connective tissue homeostasis. This includes down-regulation of interstitial collagenase and other matrix metalloproteinases and up-regulation of antiproteases such as tissue inhibitor of metalloproteinase I and plasminogen activator inhibitor. Whilst inhibition of TGF-beta activity appears to be well tolerated in rodents over several weeks, the ultimately lethal phenotype of TGF-beta 1 knockout mice warns us that this pluripotent cytokine is essential for normal health. Therefore, downstream pathways activated by TGF-beta, which might be specific for its fibrotic effects, might be more useful targets for human fibrotic disease therapy. For example, the TGF-beta response protein connective tissue growth factor may be a good target for antifibrotics but definitive evidence awaits development of suitable genetically modified animal models and specific inhibitors.
Assuntos
Cirrose Hepática Experimental/tratamento farmacológico , Fator de Crescimento Transformador beta/fisiologia , Animais , Matriz Extracelular/metabolismo , Humanos , Cirrose Hepática Experimental/etiologia , Cirrose Hepática Experimental/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Ratos , Tioacetamida , Fator de Crescimento Transformador beta1RESUMO
Both the identity and source of the rodent collagenase(s) that mediates matrix remodeling in liver fibrosis remain elusive. We have recently demonstrated an unequivocal role for scar-associated macrophages (SAMs) in the spontaneous resolution of liver fibrosis and sought to determine whether SAMs are the source of matrix metalloproteinase (MMP) 13 (collagenase 3), considered to be the primary interstitial collagenase in rodents. In this study, we demonstrate an association between MMP13 expression and the presence of SAMs in the regression of experimental liver fibrosis. mmp13 gene expression was restricted to regions of fibrosis that were rich in SAMs. Both MMP13 mRNA and protein colocalized to large phagocytes within and directly apposed to hepatic scars. Using the CD11b-DTR-transgenic mouse to deplete SAMs in a model of chronic CCl(4) injury, we found that SAM depletion resulted in a 5-fold reduction in mmp13 message (p = 0.005). Furthermore, resolution of CCl(4)-induced fibrosis was retarded in MMP13-deficient mice. Thus, SAMs selectively, during resolution of fibrosis induce and use the major collagenase MMP13 to mediate the resorption of interstitial matrix and successfully remodel the fibrotic liver.
Assuntos
Cicatriz/enzimologia , Cirrose Hepática Experimental/fisiopatologia , Fígado/enzimologia , Macrófagos/enzimologia , Metaloproteinase 13 da Matriz/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/patologia , Macrófagos/fisiologia , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.
Assuntos
Proliferação de Células , Colágeno Tipo I/metabolismo , Inibidores do Crescimento/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fígado/metabolismo , Fígado/patologia , Peptídeo Hidrolases/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Colágeno Tipo I/genética , Reagentes de Ligações Cruzadas , Inibidores do Crescimento/genética , Humanos , Hidrólise , Fígado/enzimologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Ratos , Ratos Sprague-Dawley , Transglutaminases/metabolismoRESUMO
BACKGROUND/AIMS: Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation. METHODS: Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. RESULTS: Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells. CONCLUSIONS: This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.
Assuntos
Colágeno/biossíntese , Hepatócitos/citologia , Hepatócitos/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Células 3T3 , Animais , Divisão Celular/fisiologia , Expressão Gênica/fisiologia , Hemostáticos/farmacologia , Cirrose Hepática/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Mastócitos/metabolismo , Camundongos , Mitógenos/farmacologia , RNA Mensageiro/análise , Ratos , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/agonistas , Serina Endopeptidases/farmacologia , Pele/citologia , Trombina/farmacologia , TriptasesRESUMO
There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9-12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis-Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues.
Assuntos
Pulmão/enzimologia , Mastócitos/enzimologia , Serina Endopeptidases/genética , Pele/enzimologia , Western Blotting , Eletroforese em Gel Bidimensional , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Isoformas de Proteínas , Especificidade por Substrato , TriptasesRESUMO
Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.
Assuntos
Apoptose , Integrina alfaVbeta3/metabolismo , Fígado/metabolismo , Laranja de Acridina/farmacologia , Actinas/metabolismo , Animais , Western Blotting , Caspase 3 , Caspases/metabolismo , Divisão Celular , Núcleo Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , Fragmentação do DNA , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fibrose , Corantes Fluorescentes/farmacologia , Inativação Gênica , Humanos , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Ligantes , Fígado/citologia , Metaloproteinase 9 da Matriz/metabolismo , Microscopia Confocal , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/metabolismo , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Vitronectina/metabolismoRESUMO
BACKGROUND/AIMS: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. METHODS: MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by (14)C gelatin degradation. RESULTS: In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl(4) treatment in rats. Between 3 and 7 days of resolution from CCl(4) liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. CONCLUSIONS: Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.
Assuntos
Fibrose/patologia , Cirrose Hepática Experimental/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Adulto , Animais , Modelos Animais de Doenças , Fibrose/metabolismo , Humanos , Cirrose Hepática Experimental/patologia , Masculino , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/genética , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.
Assuntos
Apoptose/fisiologia , Cirrose Hepática/patologia , Fator de Crescimento Neural/biossíntese , Comunicação Parácrina , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Caspase 3 , Caspases/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Hepatócitos/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Fator de Crescimento Neural/farmacologia , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND & AIMS: Liver fibrosis and cirrhosis result from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs). Previously considered irreversible, we have studied a model of cirrhosis to determine the mechanisms mediating and limiting spontaneous recovery. METHODS: A micronodular cirrhosis was induced in rats after 12 weeks of CCl(4) intoxication. Livers were analyzed for evidence of matrix degradation, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression, stellate cell apoptosis, tissue transglutaminase (tTg) expression, and matrix cross-linking during spontaneous recovery of up to 366 days. RESULTS: Over 366 days of recovery, micronodular cirrhosis underwent significant remodeling to a macronodular cirrhosis. Expression of collagen-1 and TIMP messenger RNA (mRNA) decreased significantly and active MMPs were shown in livers during remodeling of fibrosis. Resolution also was characterized by apoptosis of HSCs, predominantly at the margins of fibrotic septa. Residual septa, not remodeled at 366 days, were characterized by tTg-mediated cross-linking and relative hypocellularity. CONCLUSION: Recovery from comparatively advanced cirrhosis is possible and results in remodeling from a micronodular cirrhosis to a macronodular cirrhosis. We suggest resolution is limited by tTg-mediated matrix cross-linking and a failure of HSC apoptosis.