Cholangiocarcinoma (CCA) is the second most common malignancy arising in the liver. It carries a poor prognosis, in part because its pathogenesis is not well understood. The type 3 inositol 1,4,5‐trisphosphate receptor (ITPR3) is the principal intracellular calcium ion (Ca2+) release channel in cholangiocytes, and its increased expression has been related to the pathogenesis of malignancies in other types of tissues, so we investigated its role in CCA. ITPR3 expression was increased in both hilar and intrahepatic CCA samples as well as in CCA cell lines. Deletion of ITPR3 from CCA cells impaired proliferation and cell migration. A bioinformatic analysis suggested that overexpression of ITPR3 in CCA would have a mitochondrial phenotype, so this was also examined. ITPR3 normally is concentrated in a subapical region of endoplasmic reticulum (ER) in cholangiocytes, but both immunogold electron microscopy and super‐resolution microscopy showed that ITPR3 in CCA cells was also in regions of ER in close association with mitochondria. Deletion of ITPR3 from these cells impaired mitochondrial Ca2+ signaling and led to cell death. Conclusion: ITPR3 expression in cholangiocytes becomes enhanced in CCA. This contributes to malignant features, including cell proliferation and migration and enhanced mitochondrial Ca2+ signaling.

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.

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. T he bile salt export pump (BSEP) is a member of the adenosine triphosphate-binding cassette superfamily of transporters. 1 It is primarily expressed in the liver, where it localizes to the canalicular membrane of hepatocytes. BSEP/Bsep is the major determinant of bile saltdependent bile secretion, and secretes monovalent conjugated bile acids from hepatocytes. 2 Genetic deficiencies of BSEP lead to progressive cholestatic liver injury 3,4 and are risk factors for hepatocellular carcinoma. 5 BSEP/Bsep expression is highly regulated by the nuclear farnesoid X receptor (FXR, NR1H4) which heterodimerizes with the retinoid X receptor (RXR, NR2B), and binds to an inverted repeat 1 element in the BSEP promoter. 6 BSEP/ Bsep is also regulated by the liver receptor homolog 1(Lrh-1), and its expression is decreased in hepatocyte-specific Lrh Ϫ/Ϫ mice. 7 However, Bsep expression is still preserved in both Fxr Ϫ/Ϫ and Lrh Ϫ/Ϫ mice, 7-9 suggesting that additional transcriptional factors regulate its expression.Nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in maintaining redox homeostasis by regulating the expression of many phase I and phase II

Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis, 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 Ca release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor TLR4, which links to activation of 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 kb deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. 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 patients with severe alcoholic hepatitis CONCLUSION: Stimulation of TLR4 via LPS activates NF-κB to downregulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or alcoholic hepatitis. This article is protected by copyright. All rights reserved.