Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation.

BACKGROUND AND AIMS: Inflammatory response is crucial for bile acid (BA)-induced cholestatic liver injury, but molecular mechanisms remain to be elucidated. Solute Carrier Family 35 Member C1 (SLC35C1) can transport Guanosine diphosphate-fucose into the Golgi to facilitate protein glycosylation. Its mutation leads to the deficiency of leukocyte adhesion and enhances inflammation in humans. However, little is known about its role in liver diseases. APPROACH AND RESULTS: Hepatic SLC35C1 mRNA transcripts and protein expression were significantly increased in patients with obstructive cholestasis and mouse models of cholestasis. Immunofluorescence revealed that the upregulated SLC35C1 expression mainly occurred in hepatocytes. Liver-specific ablation of Slc35c1 ( Slc35c1 cKO ) significantly aggravated liver injury in mouse models of cholestasis induced by bile duct ligation and 1% cholic acid-feeding, evidenced by increased liver necrosis, inflammation, fibrosis, and bile ductular proliferation. The Slc35c1 cKO increased hepatic chemokine Ccl2 and Cxcl2 expression and T cell, neutrophil, and F4/80 macrophage infiltration but did not affect the levels of serum and liver BA in mouse models of cholestasis. Liquid chromatography with tandem mass spectrometry analysis revealed that hepatic Slc35c1 deficiency substantially reduced the fucosylation of cell-cell adhesion protein CEACAM1 at N153. Mechanistically, cholestatic levels of conjugated BAs stimulated SLC35C1 expression by activating the STAT3 signaling to facilitate CEACAM1 fucosylation at N153, and deficiency in the fucosylation of CEACAM1 at N135 enhanced the BA-stimulated CCL2 and CXCL2 mRNA expression in primary mouse hepatocytes and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5- ASBT cells. CONCLUSIONS: Elevated hepatic SLC35C1 expression attenuates cholestatic liver injury by enhancing CEACAM1 fucosylation to suppress CCL2 and CXCL2 expression and liver inflammation.

Organic Anion Transporting Polypeptide (OATP) 1B3 is a Significant Transporter for Hepatic Uptake of Conjugated Bile Acids in Humans.

BACKGROUND & AIMS: OATP1B3/SLCO1B3 is a human liver-specific transporter for the clearance of endogenous compounds (eg, bile acid [BA]) and xenobiotics. The functional role of OATP1B3 in humans has not been characterized, as SLCO1B3 is poorly conserved among species without mouse orthologs. METHODS: Slc10a1-knockout (Slc10a1-/-), Slc10a1hSLCO1B3 (endogenous mouse Slc10a1 promoter-driven human-SLCO1B3 expression in Slc10a1-/- mice), and human SLCO1B3 liver-specific transgenic (hSLCO1B3-LTG) mice were generated and challenged with 0.1% ursodeoxycholic-acid (UDCA), 1% cholic-acid (CA) diet, or bile duct ligation (BDL) for functional studies. Primary hepatocytes and hepatoma-PLC/RPF/5 cells were used for mechanistic studies. RESULTS: Serum BA levels in Slc10a1-/- mice were substantially increased with or without 0.1% UDCA feeding compared with wild-type (WT) mice. This increase was attenuated in Slc10a1hSLCO1B3-mice, indicating that OATP1B3 functions as a significant hepatic BA uptake transporter. In vitro assay using primary hepatocytes from WT, Slc10a1-/-, and Slc10a1hSLCO1B3-mice indicated that OATP1B3 has a similar capacity in taking up taurocholate/TCA as Ntcp. Furthermore, TCA-induced bile flow was significantly impaired in Slc10a1-/- mice but partially recovered in Slc10a1hSLC01B3-mice, indicating that OATP1B3 can partially compensate the NTCP function in vivo. Liver-specific overexpression of OATP1B3 markedly increased the level of hepatic conjugated BA and cholestatic liver injury in 1% CA-fed and BDL mice. Mechanistic studies revealed that conjugated BAs stimulated Ccl2 and Cxcl2 in hepatocytes to increase hepatic neutrophil infiltration and proinflammatory cytokine production (eg, IL-6), which activated STAT3 to repress OATP1B3 expression by binding to its promoter. CONCLUSIONS: Human OATP1B3 is a significant BA uptake transporter and can partially compensate Ntcp for conjugated BA uptake in mice. Its downregulation in cholestasis is an adaptive protective response.

Unique DUOX2+ACE2+ small cholangiocytes are pathogenic targets for primary biliary cholangitis.

Cholangiocytes play a crucial role in bile formation. Cholangiocyte injury causes cholestasis, including primary biliary cholangitis (PBC). However, the etiology of PBC remains unclear despite being characterized as an autoimmune disease. Using single-cell RNA sequencing (scRNA-seq), fluorescence-activated-cell-sorting, multiplex immunofluorescence (IF) and RNAscope analyses, we identified unique DUOX2+ACE2+ small cholangiocytes in human and mouse livers. Their selective decrease in PBC patients was associated with the severity of disease. Moreover, proteomics, scRNA-seq, and qPCR analyses indicated that polymeric immunoglobulin receptor (pIgR) was highly expressed in DUOX2+ACE2+ cholangiocytes. Serum anti-pIgR autoantibody levels were significantly increased in PBC patients, regardless of positive and negative AMA-M2. Spatial transcriptomics and multiplex IF revealed that CD27+ memory B and plasma cells accumulated in the hepatic portal tracts of PBC patients. Collectively, DUOX2+ACE2+ small cholangiocytes are pathogenic targets in PBC, and preservation of DUOX2+ACE2+ cholangiocytes and targeting anti-pIgR autoantibodies may be valuable strategies for therapeutic interventions in PBC.

Hepatic TNFRSF12A promotes bile acid-induced hepatocyte pyroptosis through NFκB/Caspase-1/GSDMD signaling in cholestasis.

Tumor necrosis factor receptor superfamily member-12A (TNFRSF12A) plays a critical role in inflammation and cell death. It is expressed in multiple tissues yet extremely low in normal liver. To date, little is known about its role in cholestasis. Therefore, we sought to delineate the role of TNFRSF12A in cholestasis and its underlying mechanisms. Human liver tissues were collected from patients with obstructive cholestasis (OC) or primary biliary cholangitis (PBC). Tnfrsf12a knockout (KO) mice were generated. Cholestasis was induced by bile-duct ligation (BDL) or 0.1% 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-feeding. Human hepatoma PLC/PRF/5-ASBT and THP1 cell lines or primary mouse hepatocytes were used for mechanistic studies. Hepatic TNFRSF12A expression was markedly increased in OC or PBC patients. Genetic ablation of Tnfrsf12a in BDL- and 0.1%DDC-induced cholestatic mice significantly attenuated cholestatic liver injury with remarkable reduction of hepatocyte pyroptosis but without changing scores of necroptosis and apoptosis. Morphological features of hepatocyte pyroptosis and increased levels of pyroptosis-related proteins, NLRP3, cleaved-Caspase-1, and cleaved-GSDMD in OC patients and BDL-mice confirmed this observation. Further mechanistic studies revealed that bile acids (BAs) induced TNFRSF12A expression by enhancing the transcription factor c-JUN binding to the TNFRSF12A promoter and subsequently initiated hepatocyte pyroptosis by the NFκB/Caspase-1/GSDMD signaling. Interestingly, TWEAK, a typical ligand of TNFRSF12A, secreted by infiltrated macrophages in cholestatic livers, enhanced TNFRSF12A-induced hepatocyte pyroptosis. Taken together, we report, for the first time, that hepatic TNFRSF12A is dramatically increased in human cholestasis. Deletion of TNFRSF12A inhibits BAs-induced hepatocyte pyroptosis through the NFκB/Caspase-1/GSDMD signaling and thereby ameliorates cholestatic liver injury. As such, targeting TNFRSF12A could be a promising approach to treating cholestasis.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

BACKGROUND AND AIMS: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. APPROACH AND RESULTS: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. CONCLUSIONS: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

SEMA7AR148W mutation promotes lipid accumulation and NAFLD progression via increased localization on the hepatocyte surface.

Genetic polymorphisms are associated with the development of nonalcoholic fatty liver disease (NAFLD). Semaphorin7a (Sema7a) deficiency in mouse peritoneal macrophages reduces fatty acid (FA) oxidation. Here, we identified 17 individuals with SEMA7A heterozygous mutations in 470 patients with biopsy-proven NAFLD. SEMA7A heterozygous mutations increased susceptibility to NAFLD, steatosis severity, and NAFLD activity scores in humans and mice. The Sema7aR145W mutation (equivalent to human SEMA7AR148W) significantly induced small lipid droplet accumulation in mouse livers compared with WT mouse livers. Mechanistically, the Sema7aR145W mutation increased N-glycosylated Sema7a and its receptor integrin ß1 proteins in the cell membranes of hepatocytes. Furthermore, Sema7aR145W mutation enhanced its protein interaction with integrin ß1 and PKC-α and increased PKC-α phosphorylation, which were both abrogated by integrin ß1 silencing. Induction of PKCα_WT, but not PKCα_dominant negative, overexpression induced transcriptional factors Srebp1, Chrebp, and Lxr expression and their downstream Acc1, Fasn, and Cd36 expression in primary mouse hepatocytes. Collectively, our findings demonstrate that the SEMA7AR148W mutation is a potentially new strong genetic determinant of NAFLD and promotes intrahepatic lipid accumulation and NAFLD in mice by enhancing PKC-α-stimulated FA and triglyceride synthesis and FA uptake. The inhibition of hepatic PKC-α signaling may lead to novel NAFLD therapies.

Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct-Ligated Mouse.

BACKGROUND & AIMS: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study. METHODS: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1-/- (Casp1-/-) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids. RESULTS: Caspase-1, NLRP1, NLRP3 and IL-1ß were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1ß (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1-/- BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1-/- BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1-/- BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1-/- BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300µM TCA), neither IL-1ß induction nor procaspase-1 cleavage were detected. CONCLUSIONS: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.

Cytoprotective Effect of Antioxidant Pentapeptides from the Protein Hydrolysate of Swim Bladders of Miiuy Croaker (Miichthys miiuy) against H2O2-Mediated Human Umbilical Vein Endothelial Cell (HUVEC) Injury.

In our previous research, ten antioxidant pentapeptides including FYKWP, FTGMD, GFEPY, YLPYA, FPPYERRQ, GFYAA, FSGLR, FPYLRH, VPDDD, and GIEWA were identified from the hydrolysate of miiuy croaker (Miichthys miiuy) swim bladder. In this work, their protective function on H2O2-induced oxidative damage to human umbilical vein endothelial cells (HUVECs) was studied. Results indicated that there was no significant difference in the HUVEC viability between the normal group and the treated groups with the 10 pentapeptides at the concentration of 100 µM for 24 h (p < 0.05). Furthermore, FPYLRH of 100 µg/mL extremely significantly (p < 0.001) increased the viability (80.58% ± 5.01%) of HUVECs with H2O2-induced oxidative damage compared with that of the model group. The protective mechanism indicated that FPYLRH could extremely significantly (p < 0.001) increase the levels of superoxide dismutase (SOD) (211.36 ± 8.29 U/mg prot) and GSH-Px (53.06 ± 2.34 U/mg prot) and decrease the contents of reactive oxygen species (ROS) (139.1 ± 11.8% of control), malondialdehyde (MDA) (13.66 ± 0.71 nM/mg), and nitric oxide (NO) (4.36 ± 0.32 µM/L) at the concentration of 100 µM in HUVECs with H2O2-induced oxidative damage compared with those of the model group. In addition, FPYLRH dose-dependently protected DNA in oxidative damage HUVECs model. These results suggested that FPYLRH could significantly attenuate the H2O2-induced stress injury in HUVECs and might be used as a potential natural antioxidant in the functional food industries.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

Solute Carrier Organic Anion Transporter Family Member 3A1 Is a Bile Acid Efflux Transporter in Cholestasis.

BACKGROUND & AIMS: Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. METHODS: We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. RESULTS: Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. CONCLUSIONS: We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.

Mechanisms of bile acid mediated inflammation in the liver.

Bile acids are synthesized in the liver and are the major component in bile. Impaired bile flow leads to cholestasis that is characterized by elevated levels of bile acid in the liver and serum, followed by hepatocyte and biliary injury. Although the causes of cholestasis have been extensively studied, the molecular mechanisms as to how bile acids initiate liver injury remain controversial. In this chapter, we summarize recent advances in the pathogenesis of bile acid induced liver injury. These include bile acid signaling pathways in hepatocytes as well as the response of cholangiocytes and innate immune cells in the liver in both patients with cholestasis and cholestatic animal models. We focus on how bile acids trigger the production of molecular mediators of neutrophil recruitment and the role of the inflammatory response in this pathological process. These advances point to a number of novel targets where drugs might be judged to be effective therapies for cholestatic liver injury.

Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.

Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis.

BACKGROUND & AIMS: Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. METHODS: We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. RESULTS: Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. CONCLUSIONS: Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

All-trans-retinoic acid improves cholestasis in α-naphthylisothiocyanate-treated rats and Mdr2-/- mice.

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-ß and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.

Adult sea lamprey tolerates biliary atresia by altering bile salt composition and renal excretion.

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 µM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72-hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418-2426).

Elevated hepatic multidrug resistance-associated protein 3/ATP-binding cassette subfamily C 3 expression in human obstructive cholestasis is mediated through tumor necrosis factor alpha and c-Jun NH2-terminal kinase/stress-activated protein kinase-signaling pathway.

UNLABELLED: Multidrug resistance-associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4- and 4.6-fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7-fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract-binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein-binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. CONCLUSIONS: Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1.

Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.

UNLABELLED: Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor ß1 (Tgf-ß1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α-smooth muscle actin (α-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin-ß1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.

Aryl hydrocarbon receptor and NF-E2-related factor 2 are key regulators of human MRP4 expression.

Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.

Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.

Cholesterol 7alpha-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRalpha by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1alpha plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.