Effects of Endotoxin on Type 3 Inositol 1,4,5‐Trisphosphate Receptor in Human Cholangiocytes

Abstract

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.

Type 3 Inositol 1,4,5‐Trisphosphate Receptor Is Increased and Enhances Malignant Properties in Cholangiocarcinoma

“…A). In liver biopsies obtained from controls, quantitative confocal immunofluorescence showed that ITPR3 expression in cholangiocytes was low and was concentrated in the apical region, as has been described, whereas labeling of ITPR3 in cholangiocytes was significantly more intense in patients with hilar CCA or iCCA (Fig. A‐C).…”

“…Both cell proliferation and progression through the cell cycle depend on Ca 2+ signals in the nucleus in particular, so the nuclear‐targeted fluorescent calcium sensor GCaMP3 was used to specifically determine the role of ITPR3 in Ca 2+ signaling in the nucleus. MzCha1 cells were stimulated with extracellular adenosine triphosphate (ATP; 20 μM) because cholangiocytes express P2Y receptors that link to inositol trisphosphate (IP 3 )‐mediated Ca 2+ signaling . ATP consistently increased Ca 2+ in the nucleoplasm of control cells, but nuclear Ca 2+ signals were minimal to absent in ITPR3‐KO cells (Fig.…”

“…Several mechanisms have been reported for down‐regulation of ITPR3 in cholangiocytes, but factors that could increase ITPR3 are less clear. Increasing cholangiocyte proliferation has been attributed in part to activation of the sphingosine‐1‐phosphate receptor 2 (S1PR2), so we investigated whether S1PR2 affects ITPR3 expression.…”

“…MzCha1 cells were stimulated with extracellular adenosine triphosphate (ATP; 20 μM) because cholangiocytes express P2Y receptors that link to inositol trisphosphate (IP 3 )-mediated Ca 2+ signaling. (12,19) ATP consistently increased Ca 2+ in the nucleoplasm of control cells, but nuclear Ca 2+ signals were minimal to absent in ITPR3-KO cells (Fig. 2D,E).…”

“…Two well‐established normal human cholangiocyte (NHC) cell lines were used: i ) the H69 cells, which were kindly provided by Dr. Douglas M. Jefferson (Tufts University), and ii ) the NHC cell line . Four CCA cell lines were used: i ) the MzCha1 cell line, which was kindly provided by Dr. Greg Fitz (University of Texas Southwestern), ii ) the HuCCA1 cell line, which was kindly provided by Dr. Stitaya Sirisinha (Mahidol University), iii) the TFK‐1 cell line, which was kindly provided by Dr. Mario Strazzabosco (Yale University), and iv ) the HuCCT1 cell line, which was purchased from Japanese Collection of Research Bioresources Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan).…”