RESUMO
BACKGROUND & OBJECTIVES: Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease. DESIGN: Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings. RESULTS: Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin ß1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes. CONCLUSIONS: Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.
Assuntos
Ductos Biliares/citologia , Colestase/complicações , Hepatite Alcoólica/etiologia , Neutrófilos/fisiologia , Adulto , Animais , Ductos Biliares/patologia , Colestase/patologia , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Hepatite Alcoólica/patologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Animais , Apoptose/fisiologia , Sinalização do Cálcio/fisiologia , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/fisiologia , Células Cultivadas , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Receptores de Inositol 1,4,5-Trifosfato/genética , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Regeneração Hepática/fisiologia , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Análise de SobrevidaRESUMO
The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.
Assuntos
Proteína de Ligação a CREB/genética , AMP Cíclico/metabolismo , Hepatócitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , RNA Mensageiro/genética , Elementos de Resposta , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Dactinomicina/farmacologia , Jejum , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Mutação , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Tionucleotídeos/farmacologiaRESUMO
Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is the most prevalent isoform of the InsP3R in hepatocytes and is concentrated under the canalicular membrane, where it plays an important role in bile secretion. We hypothesized that altered calcium (Ca(2+)) signaling may be involved in metabolic dysfunction, as InsP3R-mediated Ca(2+) signals have been implicated in the regulation of hepatic glucose homeostasis. Here, we find that InsP3R-II, but not InsP3R-I, is reduced in the livers of obese mice. In our investigation of the functional consequences of InsP3R-II deficiency, we found that organic anion secretion at the canalicular membrane and Ca(2+) signals were impaired. However, mice lacking InsP3R-II showed no deficits in energy balance, glucose production, glucose tolerance, or susceptibility to hepatic steatosis. Thus, our results suggest that reduced InsP3R-II expression is not sufficient to account for any disruptions in metabolic homeostasis that are observed in mouse models of obesity. We conclude that metabolic homeostasis is maintained independently of InsP3R-II. Loss of InsP3R-II does impair secretion of bile components; therefore, we suggest that conditions of obesity would lead to a decrease in this Ca(2+)-sensitive process.
Assuntos
Homeostase/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos Obesos/metabolismo , Animais , Bile/metabolismo , Composição Corporal/fisiologia , Sinalização do Cálcio/genética , Colesterol/metabolismo , Dieta Hiperlipídica , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Teste de Tolerância a Glucose , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.
Assuntos
Sinalização do Cálcio , Hepatite Alcoólica , Hepatócitos , Neutrófilos , Elastase Pancreática , Humanos , Neutrófilos/metabolismo , Neutrófilos/patologia , Animais , Camundongos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Hepatite Alcoólica/genética , Elastase Pancreática/metabolismo , Masculino , Proliferação de Células , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , FemininoRESUMO
Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and ß-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells. Significance statement: Compression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.
RESUMO
UNLABELLED: Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short-term regulation of Bsep activity. Ca(2+) regulates targeting and insertion of transporters in many cell systems, and Ca(2+) release near the canalicular membrane is mediated by the type II inositol 1,4,5-trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido-fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen- and endotoxin-induced cholestasis. CONCLUSION: These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares/metabolismo , Sinalização do Cálcio/fisiologia , Colestase/metabolismo , Hepatócitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Canalículos Biliares/metabolismo , Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Colágeno , Modelos Animais de Doenças , Hepatócitos/citologia , Lipopolissacarídeos/farmacologia , Masculino , Processamento de Proteína Pós-Traducional/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. CONCLUSION: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bile/metabolismo , Bilirrubina/sangue , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
BACKGROUND: Hepatic stellate cells (HSC) are important mediators of liver fibrosis. HSC express purinergic receptors for extracellular ATP that induce fibrogenesis. Pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) is a highly bioavailable purinoceptor inhibitor. We sought to determine whether PPADS could prevent experimental liver fibrosis in rats. MATERIALS AND METHODS: The effect of PPADS as an inhibitor of HSC purinoceptors was compared to the effect of suramin using confocal video microscopy. Rats were treated with CCl4, dimethylnitrosamine, or common bile duct ligation in the presence or absence of PPADS. Fibrosis in liver sections was assessed using Trichrome and Sirius red stains. In HSC isolated from experimental animals, proliferation was determined by bromodeoxyuridine uptake, apoptosis was determined using Annexin V flow cytometry, and transcription of alpha(1)-procollagen and fibronectin were determined using quantitative RT-PCR. RESULTS: Both PPADS and suramin inhibited HSC purinoceptor activation, but PPADS had a more durable effect. PPADS completely blocked the development of cirrhosis due to CCl4 or dimethylnitrosamine but not due to bile duct ligation. PPADS inhibited HSC proliferation, but had no effect on HSC apoptosis. PPADS inhibited transcription of alpha(1)-procollagen and fibronectin by HSC. CONCLUSION: Blockade of purinergic receptors is a novel approach to prevention of non- biliary liver fibrosis. The primary action of PPADS is to inhibit HSC proliferation and fibrogenesis. Future design of purinergic receptor inhibitors may be an effective pharmacologic treatment to prevent liver fibrosis.
Assuntos
Cirrose Hepática/prevenção & controle , Antagonistas Purinérgicos , Fosfato de Piridoxal/análogos & derivados , Animais , Intoxicação por Tetracloreto de Carbono , Ensaio de Imunoadsorção Enzimática , Cirrose Hepática/induzido quimicamente , Masculino , Microscopia Confocal , Fosfato de Piridoxal/uso terapêutico , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves.
Assuntos
Canais de Cálcio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Neuropeptídeos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Receptores de Inositol 1,4,5-Trifosfato , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Células Tumorais CultivadasRESUMO
Portal fibroblasts (PF) are a newly isolated population of fibrogenic cells in the liver postulated to play a significant role in early biliary fibrosis. Because transforming growth factor-beta (TGF)-beta is a key growth factor in fibrosis, we characterized the response of PF to TGF-beta. We demonstrate that PF produce significant amounts of TGF-beta2 and, unlike activated hepatic stellate cells (HSC), express all three TGF-beta receptors and are growth inhibited by TGF-beta1 and TGF-beta2. Fibroblast growth factor (FGF)-2, but not platelet derived growth factor (PDGF), causes PF proliferation. These data suggest a mechanism whereby HSC eclipse PF as the dominant myofibroblast population in biliary fibrosis.
Assuntos
Comunicação Autócrina/fisiologia , Fibroblastos/citologia , Fígado/citologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Ratos , Receptores de Fatores de Crescimento Transformadores beta/análise , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta2RESUMO
INTRODUCTION: One of the major advances in liver research in the past decade was the ability to isolate distinct liver cell populations. Although there are established methods of isolating hepatocytes, cholangiocytes, and stellate cells, before this study no technique for liver fibroblast isolation had been devised. Consequently, we developed a technique to isolate primary rat liver fibroblasts. METHODS: Fibroblasts were isolated from a freshly perfused rat liver with a modification of the procedure for isolation of rat cholangiocytes. Cell markers were assessed with the use of confocal immunofluorescence. Cell morphology was assessed with transmission electron microscopy. Expression of procollagen-1 was assessed by reverse transcription polymerase chain reaction. RESULTS: The appearance of cells with fibroblast morphology was first noted at 48 hours, and almost all cells in culture had fibroblast morphology at 96 hours. Putative fibroblasts stained for vimentin, but not for smooth muscle actin, von Willebrand factor, or cytokeratins. Cell morphology was consistent with that of fibroblasts and showed no features of epithelial, endothelial, or smooth muscle cells. Liver fibroblasts expressed procollagen-1 mRNA. CONCLUSION: Primary isolated rat fibroblasts can be produced from a freshly perfused rat liver with a modification of standard cell culture methods. The role of fibroblasts in liver physiology can now be studied directly.
Assuntos
Fibroblastos/citologia , Fígado/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Separação Celular/métodos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Fígado/metabolismo , Masculino , Microscopia Confocal , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Portal fibroblasts are newly identified, potentially fibrogenic liver cells that are distinct from hepatic stellate cells. The ectonucleotidase* nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is restricted to portal fibroblasts in the normal liver. However, the fate of NTPDase2 after bile duct ligation (BDL) is unknown. AIMS: The aim of this study was to assess the effect of experimental rat and disease-mediated human biliary cirrhosis on NTPDase2 expression in the liver. METHODS: Cirrhosis was induced in experimental rats via BDL and carbon tetrachloride (CCl4) administration. Archived human liver biopsy specimens from normal liver, primary biliary cirrhosis, or hepatitis C cirrhosis were examined. Changes in expression of NTPDase2 were determined using confocal immunofluorescence, immunoblot, and real-time polymerase chain reaction. RESULTS: Confocal immunofluorescence demonstrated a decrease in NTPDase2 expression after BDL. Immunoblot and real-time polymerase chain reaction demonstrated a decrease in NTPDase2 expression by portal fibroblasts after BDL. No decrease in NTPDase2 protein was noted after CCl4 administration, and NTPDase2 messenger ribonucleic acid was markedly up-regulated after CCl4 administration. Confocal immunofluorescence demonstrated a shift of NTPDase2 expression from portal areas to central areas that colocalized with alpha-smooth muscle actin after CCl4 administration. In human biopsy specimens, NTPDase2 expression was lost in cirrhosis owing to primary biliary cirrhosis, whereas NTPDase2 expression was shifted to bridging fibrous bands in cirrhosis owing to hepatitis C. CONCLUSIONS: Loss of NTPDase2 is a common pathway in both rat and human manifestations of biliary cirrhosis. Conversely, in non-biliary cirrhosis, NTPDase2 is shifted from the portal area to bridging fibrous bands. Elucidations of the mechanisms regulating NTPDase2 expression may lead to new therapeutic approaches to fibrotic liver disease.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cirrose Hepática Biliar/enzimologia , Cirrose Hepática Biliar/genética , Animais , Regulação para Baixo , Hepatite C/enzimologia , Hepatite C/genética , Humanos , Cirrose Hepática Biliar/etiologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Adenosine is produced during cellular hypoxia and apoptosis, resulting in elevated tissue levels at sites of injury. Adenosine is also known to regulate a number of cellular responses to injury, but its role in hepatic stellate cell (HSC) biology and liver fibrosis is poorly understood. We tested the effect of adenosine on the cytosolic Ca2+ concentration, chemotaxis, and upregulation of activation markers in HSCs. We showed that adenosine did not induce an increase in the cytosolic Ca2+ concentration in LX-2 cells and, in addition, inhibited increases in the cytosolic Ca2+ concentration in response to ATP and PDGF. Using a Transwell system, we showed that adenosine strongly inhibited PDGF-induced HSC chemotaxis in a dose-dependent manner. This inhibition was mediated via the A(2a) receptor, was reversible, was reproduced by forskolin, and was blocked by the adenylate cyclase inhibitor 2,5-dideoxyadenosine. Adenosine also upregulated the production of TGF-beta and collagen I mRNA. In conclusion, adenosine reversibly inhibits Ca2+ fluxes and chemotaxis of HSCs and upregulates TGF-beta and collagen I mRNA. We propose that adenosine provides 1) a "stop" signal to HSCs when they reach sites of tissue injury with high adenosine concentrations and 2) stimulates transdifferentiation of HSCs by upregulating collagen and TGF-beta production.
Assuntos
Adenosina/farmacologia , Sinalização do Cálcio/fisiologia , Quimiotaxia/fisiologia , Fígado/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Colágeno Tipo I/genética , Citosol/efeitos dos fármacos , Citosol/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Derivado de Plaquetas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND/AIMS: A G-protein-coupled succinate receptor has recently been identified in several tissues, including the liver. The objectives of this work were to determine the hepatic cell types that express this receptor and to determine its physiological role. METHODS: Expression and distribution of the succinate receptor was determined by RT-PCR and confocal immunofluorescence. Biochemical assays were used to measure succinate and cAMP. Cytosolic Ca2+ was monitored in single cells by time-lapse imaging. Western blot was used to study the effect of succinate on activation of hepatic stellate cells. RESULTS: The succinate receptor was expressed in quiescent hepatic stellate cells, and expression decreased with activation. Ischemia induced release of succinate in isolated perfused livers. In contrast to what is observed in cell expression systems, succinate did not inhibit cAMP production or increase cytosolic Ca2+ in primary hepatic stellate cells. However, succinate accelerated stellate cell activation. CONCLUSIONS: Hepatic stellate cells express the succinate receptor. Succinate may behave as a paracrine signal by which ischemic hepatocytes trigger stellate cell activation.
Assuntos
Hepatopatias/fisiopatologia , Comunicação Parácrina , Receptores Acoplados a Proteínas G/metabolismo , Ácido Succínico/metabolismo , Animais , Imunofluorescência , Técnicas In Vitro , Infusões Intravenosas , Isquemia/metabolismo , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Perfusão , Veia Porta , Pressão , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Ácido Succínico/administração & dosagem , Ácido Succínico/farmacologia , Distribuição TecidualRESUMO
Progressive liver fibrosis (with the resultant cirrhosis) is the primary cause of chronic liver failure. Hepatic stellate cells (HSCs) are critically important mediators of liver fibrosis. In the healthy liver, HSCs are quiescent lipid-storing cells limited to the perisinusoidal endothelium. However, in the injured liver, HSCs undergo myofibroblastic transdifferentiation (activation), which is a critical step in the development of organ fibrosis. HSCs express P2Y receptors linking extracellular ATP to inositol (1,4,5)-trisphosphate-mediated cytosolic Ca(2+) signals. Here, we report that HSCs express only the type I inositol (1,4,5)-trisphosphate receptor and that the receptor shifts into the nucleus and cell extensions upon activation. These cell extensions, furthermore, express sufficient machinery to enable local application of ATP to evoke highly localized Ca(2+) signals that induce localized contractions. These autonomous units of subcellular signaling and response reveal a new level of subcellular organization, which, in turn, establishes a novel paradigm for the local control of fibrogenesis in the liver.
Assuntos
Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fígado/metabolismo , Transporte Ativo do Núcleo Celular , Trifosfato de Adenosina/metabolismo , Animais , Calreticulina/metabolismo , Núcleo Celular/metabolismo , Forma Celular , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Fígado/citologia , Cirrose Hepática/metabolismo , Masculino , Microscopia Confocal , Microscopia de Vídeo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Fatores de TempoRESUMO
Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Sequência de Bases , Canais de Cálcio/genética , Células Cultivadas , Hepatócitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Vasopressinas/fisiologiaRESUMO
Portal fibroblasts (PF) are fibrogenic liver cells distinct from hepatic stellate cells (HSC). Recent evidence suggests that PF may be important mediators of biliary fibrosis and cirrhosis. The cytokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 is upregulated in biliary fibrosis by bile duct epithelia (BDE) and induces functional responses in HSC. Thus we hypothesized that release of MCP-1 may mediate biliary fibrosis. We report that PF express functional receptors for MCP-1 that are distinct from the receptor CCR2. MCP-1 induces proliferation, increase and redistribution of alpha-smooth muscle (alpha-SMA) expression, loss of the ectonucleotidase NTPDase2, and upregulation of alpha(1)-procollagen production in PF. BDE secretions induce alpha-SMA levels in PF, and this is inhibited by MCP-1 blocking antibody. Together, these data suggest that BDE regulate PF proliferation and myofibroblastic transdifferentiation in a paracrine fashion via release of MCP-1.
Assuntos
Ductos Biliares/metabolismo , Quimiocina CCL2/administração & dosagem , Epitélio/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Fígado/metabolismo , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Bile duct epithelia are the target of a number of "cholangiopathies" characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.
Assuntos
Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/química , Ductos Biliares/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Animais , Bromodesoxiuridina/farmacologia , Proliferação de Células , Colangiocarcinoma/metabolismo , Colestase , Técnicas de Cocultura , DNA Complementar/metabolismo , Humanos , Fígado/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , TransfecçãoRESUMO
Apoptosis contributes to the regulation of cell growth and regeneration and to the development of neoplasia. Mcl-1 is an anti-apoptotic protein that is particularly important for the development of hematological and biliary malignancies, but the mechanism of action of Mcl-1 is unknown. A number of pro- and anti-apoptotic proteins exhibit their effects by modulating Ca2+ signals, so we examined the effects of Mcl-1 on components of the Ca2+ signaling pathway that are known to regulate apoptosis. Expression of Mcl-1 did not affect expression of the inositol 1,4,5-trisphosphate receptor or the size of endoplasmic reticulum Ca2+ stores. However, mitochondrial Ca2+ signals induced by either Ca2+ agonists or apoptotic stimuli were decreased in cells overexpressing Mcl-1 and increased in cells in which Mcl-1 expression was inhibited. These findings provide evidence that Mcl-1 directly inhibits Ca2+ signals within mitochondria, which may provide a novel mechanism to inhibit apoptosis and thereby promote neoplasia.