RESUMO
BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.
Assuntos
Canalículos Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Colestase/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colagogos e Coleréticos/uso terapêutico , Ácido Cólico , Claudina-1/metabolismo , Família 2 do Citocromo P450/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais , Feminino , Fibrose , Predisposição Genética para Doença , Hepatócitos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Oxazóis/uso terapêutico , Permeabilidade , Fatores de Proteção , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/metabolismo , Junções Íntimas/ultraestrutura , Ácido Ursodesoxicólico/uso terapêutico , Proteína da Zônula de Oclusão-2/deficiênciaRESUMO
Genetic cholestasis has been dissected through genetic investigation. The major PFIC genes are now described. ATP8B1 encodes FIC1, ABCB11 encodes BSEP, ABCB4 encodes MDR3, TJP2 encodes TJP2, NR1H4 encodes FXR, and MYO5B encodes MYO5B. The full spectra of phenotypes associated with mutations in each gene are discussed, along with our understanding of the disease mechanisms. Differences in treatment response and targets for future treatment are emerging.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/metabolismo , Metabolismo dos Lipídeos , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Colestase Intra-Hepática/diagnóstico , Homeostase , Humanos , Metabolismo dos Lipídeos/genética , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Proteína da Zônula de Oclusão-2/deficiência , Proteína da Zônula de Oclusão-2/genéticaRESUMO
The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.
