RESUMEN
BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis. METHODS: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4-/-, Abcb4-/-/Tgr5Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4-/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models. RESULTS: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4-/- livers, in Abcb4-/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4-/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfß2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4-/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4-/- livers. CONCLUSIONS: Reduced TGR5 levels in BECs from patients with PSC and Abcb4-/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells.
Asunto(s)
Sistema Biliar/efectos de los fármacos , Colangitis Esclerosante/genética , Regulación hacia Abajo/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Sistema Biliar/metabolismo , Colangitis Esclerosante/tratamiento farmacológico , Colangitis Esclerosante/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Factores de VirulenciaRESUMEN
Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.
Asunto(s)
Anfirregulina/metabolismo , Ácidos y Sales Biliares/biosíntesis , Colestasis Intrahepática/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Animales , Receptores ErbB/metabolismo , Humanos , Ratones Endogámicos C57BLRESUMEN
Bile salts represent signalling molecules with a variety of endocrine functions. Bile salt effects are mediated by different receptor molecules, comprising ligand-activated nuclear transcription factors as well as G protein-coupled membrane-bound receptors. The farnesoid X receptor (FXR) and the plasma membrane-bound G protein-coupled receptor TGR5 (Gpbar-1) are prototypic bile salt receptors of both classes and are highly expressed in the liver including the biliary tree as well as in the intestine. In liver, TGR5 is localized in different non-parenchymal cells such as sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells and small and large cholangiocytes. Through TGR5 bile salts can mediate choleretic, cell-protective as well as proliferative effects in cholangiocytes. A disturbance of these signalling mechanisms can contribute to the development of biliary diseases. In line with the important role of TGR5 for bile salt signalling, TGR5 knockout mice are more susceptible to cholestatic liver damage. Furthermore, in absence of TGR5 cholangiocyte proliferation in response to cholestasis is attenuated and intrahepatic and extrahepatic bile ducts show increased cell damage, underscoring the role of the receptor for biliary physiology. Decreased TGR5 expression may also contribute to the development or progression of cholangiopathies like primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) since reduced TGR5-dependent cell-protective mechanisms such as bicarbonate secretion renders cholangiocytes more vulnerable towards bile salt toxicity. Nevertheless, TGR5 overexpression or constant stimulation of the receptor can promote cholangiocyte proliferation leading to cyst growth in polycystic liver disease or even progression of cholangiocarcinoma. Not only the stimulation of TGR5-mediated pathways by suitable TGR5 agonists but also the inhibition of TGR5 signalling by the use of antagonists represent potential therapeutic approaches for different types of biliary diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Conductos Biliares/metabolismo , Colangitis/patología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Bicarbonatos/metabolismo , Conductos Biliares/citología , Proliferación Celular/genética , Colangitis/etiología , Quistes/etiología , Quistes/patología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Predisposición Genética a la Enfermedad , Humanos , Hígado/metabolismo , Hepatopatías/etiología , Hepatopatías/patología , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: TGR5 (G protein-coupled bile acid receptor 1, M-Bar) is a G protein-coupled cell surface receptor responsive to bile acids (BA) and different steroid hormones. TGR5 mRNA is detected almost ubiquitious in human and rodent tissues with a very high expression in gallbladder, liver and intestine. In liver, TGR5 is found in sinusoidal endothelial cells, Kupffer cells and cholangiocytes. Activation of TGR5 triggers an elevation of intracellular cyclic AMP and further downstream signalling. Key Messages: TGR5 exerts anti-inflammatory effects, protects cholangiocytes from BA-induced toxicity, promotes cholangiocyte secretion and proliferation and reduces portal perfusion pressure. Furthermore, TGR5 mediates gallbladder filling. TGR5 knockout mice have a smaller BA pool size with altered composition and develop more severe liver injury after BA feeding, common bile duct ligation or injection of lipopolysaccharide. The absence of TGR5 also reduces the proliferative and regenerative capacity after partial hepatectomy or liver damage. Stimulation of TGR5 signalling can improve steatohepatitis, portal hypertension and hepatic inflammation in rodent models of liver damage. However, TGR5 activation also promotes the proliferation of cystic and malignant-transformed cholangiocytes. CONCLUSIONS: TGR5 plays an important role in the protection of the liver from BA toxicity under cholestatic conditions. Stimulation of the receptor prevents excessive liver damage in rodent models of cholestasis, steatohepatitis, liver fibrosis and inflammation and also promotes liver regeneration. However, the activation of TGR5-dependent signalling may also trigger proliferation and apoptosis resistance of cystic cholangiocytes and malignantly transformed cholangiocytes, thus promoting cyst growth in polycystic liver disease or progression of cholangiocarcinoma. Depending on the type of liver disease stimulation as well as inhibition of TGR5, signalling may present a useful therapeutic approach.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Hepatopatías/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Modelos Biológicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
OBJECTIVE: Cholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, elevation of BA levels enhances proliferation and invasiveness of cholangiocarcinoma (CCA) cells in animal models, thus promoting tumour progression. TGR5 is a G-protein coupled BA receptor, which is highly expressed in cholangiocytes and postulated to mediate the proliferative effects of BA. DESIGN: BA-dependent cholangiocyte proliferation was examined in TGR5-knockout and wild type mice following cholic acid (CA)-feeding and CBDL. TGR5-dependent proliferation and protection from apoptosis was studied in isolated cholangiocytes and CCA cell lines following stimulation with TGR5 ligands and kinase inhibitors. TGR5 expression was analysed in human CCA tissue. RESULTS: Cholangiocyte proliferation was significantly reduced in TGR5-knockout mice in response to CA-feeding and CBDL. Taurolithocholic acid and TGR5-selective agonists induced cholangiocyte proliferation through elevation of reactive oxygen species and cSrc mediated epidermal growth factor receptor transactivation and subsequent Erk1/2 phosphorylation only in wild type but not in TGR5-knockout-derived cells. In human CCA tissue TGR5 was overexpressed and the pathway of TGR5-dependent proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase (ERK)1/2 activation also translated to CCA cell lines. Furthermore, apoptosis was inhibited by TGR5-dependent CD95 receptor serine phosphorylation. CONCLUSIONS: TGR5 is an important mediator of BA-induced cholangiocyte proliferation in vivo and in vitro. Furthermore, TGR5 protects cholangiocytes from death receptor-mediated apoptosis. These mechanisms may protect cholangiocytes from BA toxicity under cholestatic conditions, however, they may trigger proliferation and apoptosis resistance in malignantly transformed cholangiocytes, thus promoting CCA progression.
Asunto(s)
Ácidos y Sales Biliares/fisiología , Neoplasias de los Conductos Biliares/metabolismo , Proliferación Celular/fisiología , Colangiocarcinoma/metabolismo , Conducto Colédoco/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Conducto Colédoco/metabolismo , Conducto Colédoco/cirugía , Humanos , Ligadura , Masculino , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/deficienciaRESUMEN
Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.