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Background & Aims: Antibody-induced bile salt export pump deficiency (AIBD) is an acquired form of intrahepatic cholestasis, which may develop following orthotopic liver transplantation (OLT) for progressive familial intrahepatic cholestasis type 2 (PFIC-2). Approximately 8-33% of patients with PFIC-2 who underwent a transplant develop bile salt export pump (BSEP) antibodies, which trans-inhibit this bile salt transporter from the extracellular, biliary side. AIBD is diagnosed by demonstration of BSEP-reactive and BSEP-inhibitory antibodies in patient serum. We developed a cell-based test directly measuring BSEP trans-inhibition by antibodies in serum samples to confirm AIBD diagnosis. Methods: Sera from healthy controls and cholestatic non-AIBD or AIBD cases were tested (1) for anticanalicular reactivity by immunofluorescence staining of human liver cryosections, (2) for anti-BSEP reactivity by immunofluorescence staining of human embryonic kidney 293 (HEK293) cells expressing BSEP-enhanced yellow fluorescent protein (EYFP) and immunodetection of BSEP-EYFP on Western blot, and (3) for BSEP trans-inhibition using HEK293 cells stably expressing Na+/taurocholate cotransporting polypeptide (NTCP)-mCherry and BSEP-EYFP. The trans-inhibition test uses [3H]-taurocholate as substrate and is divided into an uptake phase dominated by NTCP followed by BSEP-mediated export. For functional analysis, sera were bile salt depleted. Results: We found BSEP trans-inhibition by seven sera containing anti-BSEP antibodies, but not by five cholestatic or nine control sera, all lacking BSEP reactivity. Prospective screening of a patient with PFIC-2 post OLT showed seroconversion to AIBD, and the novel test method allowed monitoring of treatment response. Notably, we identified a patient with PFIC-2 post OLT with anti-BSEP antibodies yet without BSEP trans-inhibition activity, in line with asymptomatic presentation at serum sampling. Conclusions: Our cell-based assay is the first direct functional test for AIBD and allows confirmation of diagnosis as well as monitoring under therapy. We propose an updated workflow for AIBD diagnosis including this functional assay. Impact and Implications: Antibody-induced BSEP deficiency (AIBD) is a potentially serious complication that may affect patients with PFIC-2 after liver transplantation. To improve its early diagnosis and thus immediate treatment, we developed a novel functional assay to confirm AIBD diagnosis using a patient's serum and propose an updated diagnostic algorithm for AIBD.
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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.
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Sistema Biliar/efeitos dos fármacos , Colangite Esclerosante/genética , Regulação para Baixo/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Sistema Biliar/metabolismo , Colangite Esclerosante/tratamento farmacológico , Colangite Esclerosante/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Fatores de VirulênciaRESUMO
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.
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Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Colangite/patologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bicarbonatos/metabolismo , Ductos Biliares/citologia , Proliferação de Células/genética , Colangite/etiologia , Cistos/etiologia , Cistos/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Predisposição Genética para Doença , Humanos , Fígado/metabolismo , Hepatopatias/etiologia , Hepatopatias/patologia , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genéticaRESUMO
BACKGROUND & AIMS: The bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4) and the ATPase familial intrahepatic cholestasis 1 (FIC1, ATP8B1) mediate bile formation. This study aimed to determine the contribution of mutations and common variants in the FIC1, BSEP and MDR3 genes to cholestatic disorders of differing disease onset and severity. METHODS: Coding exons with flanking intron regions of ATP8B1, ABCB11, and ABCB4 were sequenced in cholestatic patients with assumed genetic cause. The effects of new variants were evaluated by bioinformatic tools and 3D protein modeling. RESULTS: In 427 patients with suspected inherited cholestasis, 149 patients carried at least one disease-causing mutation in FIC1, BSEP or MDR3, respectively. Overall, 154 different mutations were identified, of which 25 were novel. All 13 novel missense mutations were disease-causing according to bioinformatics analyses and homology modeling. Eighty-two percent of patients with at least one disease-causing mutation in either of the three genes were children. One or more common polymorphism(s) were found in FIC1 in 35.3%, BSEP in 64.3% and MDR3 in 72.6% of patients without disease-causing mutations in the respective gene. Minor allele frequencies of common polymorphisms in BSEP and MDR3 varied in our cohort compared to the general population, as described by gnomAD. However, differences in ethnic background may contribute to this effect. CONCLUSIONS: In a large cohort of patients, 154 different variants were detected in FIC1, BSEP, and MDR3, 25 of which were novel. In our cohort, frequencies for risk alleles of BSEP (p.V444A) and MDR3 (p.I237I) polymorphisms were significantly overrepresented in patients without disease-causing mutation in the respective gene, indicating that these common variants can contribute to a cholestatic phenotype. LAY SUMMARY: FIC1, BSEP, and MDR3 represent hepatobiliary transport proteins essential for bile formation. Genetic variants in these transporters underlie a broad spectrum of cholestatic liver diseases. To confirm a genetic contribution to the patients' phenotypes, gene sequencing of these three major cholestasis-related genes was performed in 427 patients and revealed 154 different variants of which 25 have not been previously reported in a database. In patients without a disease-causing mutation, common genetic variants were detected in a high number of cases, indicating that these common variants may contribute to cholestasis development.
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Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Colestase/genética , Mutação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Variação Genética , Humanos , Lactente , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
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.
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Ácidos e Sais Biliares/metabolismo , Hepatopatias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Modelos Biológicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMO
We aimed to investigate the mechanistic, functional, and therapeutic role of glycogen synthase kinase 3ß (GSK-3ß) in the regulation and activation of the proinflammatory oncogenic transcription factor nuclear factor of activated T cells (NFATc2) in pancreatic cancer. IHC, qPCR, immunoblotting, immunofluorescence microscopy, and proliferation assays were used to analyze mouse and human tissues and cell lines. Protein-protein interactions and promoter regulation were analyzed by coimmunoprecipitation, DNA pulldown, reporter, and ChIP assays. Preclinical assays were performed using a variety of pancreatic cancer cells lines, xenografts, and a genetically engineered mouse model (GEMM). GSK-3ß-dependent SP2 phosphorylation mediates NFATc2 protein stability in the nucleus of pancreatic cancer cells stimulating pancreatic cancer growth. In addition to protein stabilization, GSK-3ß also maintains NFATc2 activation through a distinct mechanism involving stabilization of NFATc2-STAT3 complexes independent of SP2 phosphorylation. For NFATc2-STAT3 complex formation, GSK-3ß-mediated phosphorylation of STAT3 at Y705 is required to stimulate euchromatin formation of NFAT target promoters, such as cyclin-dependent kinase-6, which promotes tumor growth. Finally, preclinical experiments suggest that targeting the NFATc2-STAT3-GSK-3ß module inhibits proliferation and tumor growth and interferes with inflammation-induced pancreatic cancer progression in Kras(G12D) mice. In conclusion, we describe a novel mechanism by which GSK-3ß fine-tunes NFATc2 and STAT3 transcriptional networks to integrate upstream signaling events that govern pancreatic cancer progression and growth. Furthermore, the therapeutic potential of GSK-3ß is demonstrated for the first time in a relevant Kras and inflammation-induced GEMM for pancreatic cancer.
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Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamação/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Genes ras , Humanos , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Fatores de Transcrição NFATC/genética , Motivos de Nucleotídeos , Neoplasias Pancreáticas/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Fator de Transcrição STAT3/metabolismoRESUMO
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.