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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 (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-?B (NF-?B). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-?B, 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-?B subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-?B 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-?B 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.

SUBMITTER: Franca A

PROVIDER: S-EPMC6351171 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Franca Andressa A Carlos Melo Lima Filho Antonio A Guerra Mateus T MT Weerachayaphorn Jittima J Loiola Dos Santos Marcone M Njei Basile B Robert Marie M Xavier Lima Cristiano C Vieira Teixeira Vidigal Paula P Banales Jesus M JM Ananthanarayanam Meenakshisundaram M Leite M Fatima MF Nathanson Michael H MH

Hepatology (Baltimore, Md.) 20181231 2

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 Ca<sup>2+</sup> releas ...[more]

PMID: 30141207

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Project description:Background & aimsMost 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.MethodsThe 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.ResultsWe 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.ConclusionsThe 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.

Dataset Information

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

Publications

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

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