Jittima Weerachayaphorn

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.

Nuclear factor erythroid 2–related factor 2 is a positive regulator of human bile salt export pump expression†‡

The bile salt export pump (BSEP) is the major determinant of bile salt–dependent bile secretion, and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor. However, BSEP expression, though reduced, is retained in the livers of Fxr−/− mice, indicating that additional transcriptional factors may regulate its expression. Nuclear factor erythroid 2–related factor 2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant‐responsive elements that regulate many hepatic phase I and II enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two musculo‐aponeurotic fibrosacroma (Maf) recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study, we assessed whether Nrf2 plays a role in human BSEP expression and if this might be mediated by MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP messenger RNA expression by approximately seven‐fold in HepG2 cells and protein by approximately 70% in human hepatocytes. Small interfering RNAs lowered NRF2 expression in HepG2 cells and prevented the up‐regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose‐dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. Chromatin immunoprecipitation assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter. Conclusion: Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury. (HEPATOLOGY 2009.)

Nuclear factor-E2-related factor 2 is a major determinant of bile acid homeostasis in the liver and intestine

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.

Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice

BACKGROUND & AIMS Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown. METHODS We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated. RESULTS Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced α-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix. CONCLUSIONS Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction.

Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes

BACKGROUND & AIMS Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3. METHODS The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases. RESULTS We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders. CONCLUSIONS The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

Type 2 inositol trisphosphate receptor gene expression in hepatocytes is regulated by cyclic AMP

The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.

Dysregulated microRNA expression profiles in cholangiocarcinoma cell-derived exosomes

Aim: Cholangiocarcinoma (CCA) is a malignant tumor of bile duct epithelial cells. The prognosis of CCA is poor due to lack of effective therapeutic targets and detection at an advanced stage. Exosomes are secreted nano‐sized vesicles and contribute to the malignancy of several cancers via transferring their miRNAs between cells. Thus, exosomal miRNAs may serve as new therapeutic targets and potential biomarkers for CCA. Main methods: Exosomes were isolated from three different CCA cell lines and normal human cholangiocyte cells, followed by miRNA profiling analysis. Potential role of dysregulated miRNA was investigated by knockdown experiment. Key findings: We found that 38 and 460 miRNAs in CCA exosomes were significantly up‐ and down‐regulated, respectively. Of these differentially expressed miRNAs, the hsa‐miR‐205‐5p and miR‐200 family members were markedly up‐regulated for 600–1500 folds, whereas the miR‐199 family members and their clustered miRNA, hsa‐miR‐214‐3p, were down‐regulated for 1000–2000 folds. The expression patterns of these representative exosomal miRNAs were similar to those observed in all types of CCA cells. The target genes of the top ten most up‐ and down‐regulated miRNAs are significantly associated with well‐characterized cancer‐related pathways. Consistently, knockdown of the most up‐regulated miRNA, miR‐205‐5p, reduced KKU‐M213 cell invasion and migration. Significance: We have demonstrated the distinct miRNA signatures in exosomes released from CCA cells, compared to normal human cholangiocyte cells. These exosomal miRNAs may have the potential to be novel therapeutic targets and biomarkers for CCA.

Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5‐trisphosphate receptor

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It may result in several types of liver problems, including impaired liver regeneration (LR), but the mechanism for this is unknown. Because LR depends on calcium signaling, we examined the effects of NAFLD on expression of the type II inositol 1,4,5‐trisphosphate receptor (ITPR2), the principle calcium release channel in hepatocytes. ITPR2 promoter activity was measured in Huh7 and HepG2 cells. ITPR2 and c‐Jun protein levels were evaluated in Huh7 cells, in liver tissue from a rat model of NAFLD, and in liver biopsy specimens of patients with simple steatosis and nonalcoholic steatohepatitis (NASH). LR was assessed in wild‐type and Itpr2 knockout (Itpr2–/–) mice following 67% hepatectomy. Cell proliferation was examined in ITPR2‐knockout HepG2 cells generated by the CRISPR/Cas9 system. c‐Jun dose dependently decreased activity of the human ITPR2 promoter. c‐Jun expression was increased and ITPR2 was decreased in fat‐loaded Huh7 cells and in livers of rats fed a high‐fat, high‐fructose diet. Overexpression of c‐Jun reduced protein and mRNA expression of ITPR2 in Huh7 cells, whereas knockdown of c‐Jun prevented the decrease of ITPR2 in fat‐loaded Huh7 cells. ITPR2 expression was decreased and c‐Jun was increased in liver biopsies of patients with steatosis and NASH compared to controls. ITPR2‐knockout cells exhibited less nuclear calcium signaling and cell proliferation than control cells. LR assessed by Ki‐67 and proliferating cell nuclear antigen was markedly decreased in Itpr2–/– mice. Conclusion: Fatty liver induces a c‐Jun‐mediated decrease in ITPR2 in hepatocytes. This may account for the impaired LR that occurs in NAFLD. (Hepatology 2018;67:560‐574).

Effects of endotoxin on type 3 inositol 1,4,5‐trisphosphate receptor in human cholangiocytes

Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll‐like receptor 4 (TLR4), which links to activation of nuclear factor‐κB (NF‐κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF‐κB, and promoter activity was inhibited by p65/p50. Nested 0.5‐ and 1.0‐kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF‐κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF‐κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF‐κB to down‐regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.