Tight junction stabilization prevents HepaRG cell death in drug-induced intrahepatic cholestasis.
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ABSTRACT:
SUBMITTER: Sonoi R
PROVIDER: S-EPMC8272035 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs.
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Project description:Tricellular tight junctions (tTJs) are specialized structures that occur where the corners of three cells meet to seal adjacent intercellular space. The molecular components of tTJs include tricellulin (TRIC) and lipolysis-stimulated lipoprotein receptor (LSR) which recruits TRIC, are required for normal hearing. Although loss of TRIC causes hearing loss with degeneration of cochlear cells, the detailed mechanisms remains unclear. In the present study, by using temperature-sensitive mouse cochlear cells, US/VOT-E36 cell line, we investigated the changes of TRIC and LSR during cochlear cell differentiation and the effects of histone deacetylase (HDAC) inhibitors against cell degeneration induced by loss of TRIC and LSR. During cell differentiation induced by the temperature change, expression of TRIC and LSR were clearly induced. Treatment with metformin enhanced expression TRIC and LSR via AMPK during cell differentiation. Loss of TRIC and LSR by the siRNAs induced cell death in differentiated cells. Treatment with HDAC inhibitors trichostatin A and HDAC6 inhibitor prevented the cell death induced by loss of TRIC and LSR. Collectively, these findings suggest that both tTJ proteins TRIC and LSR have crucial roles for the differentiated cochlear cell survival, and that HDAC inhibitors may be potential therapeutic agents to prevent hearing loss.
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Project description:β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro.ConclusionWe report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).
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Project description:Recent progress in understanding the molecular mechanisms of bile formation and cholestasis have led to new insights into the pathogenesis of drug-induced cholestasis. This review summarizes their variable clinical presentations, examines the role of transport proteins in hepatic drug clearance and toxicity, and addresses the increasing importance of genetic determinants, as well as practical aspects of diagnosis and management.
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Project description:Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.
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Project description:Intrahepatic cholestasis of pregnancy (ICP) is an idiopathic liver disease while the biochemical characteristic is the elevated level of total bile acid (TBA). The present study investigated whether miR-148a mediates the induced effect of estrogen on the development of ICP and the proper mechanism: PXR/MRP3 signal pathway. mRNA expression was detected by qPCR, protein expression was detected by western blotting, the concentration of estrogen and TBA were detected by reagent kit respectively. In the cinical research, it was found that miR-148a expression was positive related with the concentration of TBA in the serum of ICP patients. In in vitro research, estradiol (500 nmol/L, 12 h) significantly upregulated miR-148a expression and LV-148a-siRNA inhibited the function of estradiol (500 nmol/L, 48 h) on TBA secretion. In addition, gene silence of miR-148a upregulated PXR expression which was inhibited by estradiol in LO2 cells. Pretreatment of rifampin (10 μmol/L), the agonist of PXR alleviated the TBA secretion induced by estradiol (500 nmol/L, 48 h). miR-148a-siRNA and PXR had a synergistic action on TBA secretion of LO2. Both of miR-148a-siRNA and rifampin (10 μmol/L) inhibited the upregulated effect of estradiol on MRP3 expression. This research has demonstrated that miR-148a may be involved in the induction of estrogen on ICP via PXR signal pathway, and MRP3 may be involved.
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Project description:Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen-induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety.
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Project description:MicroRNA-132/212 has been supposed as a critical gene related to the blood-brain barrier (BBB) protection after stroke, but its regulation pathway including the upstream regulator and downstream targets is still unclear. Herein, we demonstrated the cAMP response element-binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) to be the upstream regulator of miRNA-132/212 using CRTC1 knockout and wild-type mice. CRTC1 deletion led to the reduction of miRNA-132/212 expression in mice brain after ischemic stroke, significantly increased infarct volume, and aggravated BBB permeability with worsening neurological deficits. Furthermore, we identified that miRNA-132 repressed Claudin-1, tight junction-associated protein-1 (TJAP-1), and RNA-binding Fox-1 (RBFox-1) by directly binding to their respective 3'-untranslated regions, which alleviated the ischemic damage by enhancing neuronal survival and BBB integrity. Moreover, the co-culture of endothelial cells with CRTC1-deficient neurons aggravated the cell vulnerability to hypoxia, also supporting the idea that miRNA-132/212 cluster is regulated by CRTC1 and acts as a crucial role in the mitigation of ischemic damage. This work is a step forward for understanding the role of miRNA-132/212 in neurovascular interaction and may be helpful for potential gene therapy of ischemic stroke.
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Project description:Bile acids and bile salts have essential functions in the liver and in the small intestine. Their synthesis in the liver provides a metabolic pathway for the catabolism of cholesterol and their detergent properties promote the solubilisation of essential nutrients and vitamins in the small intestine. Inherited conditions that prevent the synthesis of bile acids or their excretion cause cholestasis, or impaired bile flow. These disorders generally lead to severe human liver disease, underscoring the essential role of bile acids in metabolism. Recent advances in the elucidation of gene defects underlying familial cholestasis syndromes has greatly increased knowledge about the process of bile flow. The expression of key proteins involved in bile flow is tightly regulated by transcription factors of the nuclear hormone receptor family, which function as sensors of bile acids and cholesterol. Here we review the genetics of familial cholestasis disorders, the functions of the affected genes in bile flow, and their regulation by bile acids and cholesterol.
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Project description:Sepsis induces both intestinal hyperpermeability and epithelial apoptosis. While each has been implicated in mediating sepsis mortality, the relationship between these two processes is unclear. We hypothesized that preventing intestinal apoptosis would prevent gut barrier dysfunction. To test this hypothesis, transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in the gut epithelium (Fabpl-Bcl-2 mice) and wild-type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture and orally gavaged with fluorescein isothiocyanate conjugated-dextran (FD-4) 5 h before sacrifice. Serum FD-4 concentration was assayed to measure intestinal permeability, and jejunal tight junctions were assayed for mRNA and protein expression. Baseline FD-4 concentration was similar between WT and Fabpl-Bcl-2 mice. Intestinal permeability increased 6, 12, 24, and 48 h following sepsis in WT mice; however, FD-4 concentration was significantly lower at each timepoint in Fabpl-Bcl-2 mice. In addition, there were no statistically significant changes in permeability between septic and sham transgenic mice. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice, while claudin 4 mRNA levels were higher in Fabpl-Bcl-2 mice. In contrast, no differences were detected in claudins 2, 7, 15, JAM-A, or ZO-1. Protein levels followed the same trend for all tight junction mediators different between WT and Fabpl-Bcl-2 mice except occludin was significantly higher in transgenic mice. Together these results demonstrate that decreasing intestinal epithelial apoptosis prevents hyperpermeability following sepsis via tight junction alterations which may be at least partially responsible for improved survival conferred by Bcl-2 overexpression.
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