TNFα stimulates mucin secretion, but not mucin synthesis, in human gallbladder epithelial cells

Abstract

Introduction: Gallbladder mucin hypersecretion occurs early in the pathogenesis of gallstone disease but the mediators which cause this response have not been identified. That one or more biliary constituents is responsible has been suggested by the finding that ligation of the cystic duct in cholesterol fed animals prevents mucin hypersecretion. In attempting to identify the mediator(s) involved, we considered the inflammatory cytokine TNFct since (a) inflammatory changes in the gallbladder mucosa have been shown to precede mucin hypersecretion, (b) TNFct is a constituent of bile and (c) TNFct has been shown to stimulate mucin secretion from epithelial cells in other tissues. The aim of this study was to investigate the effect of TNFtx on mucin secretion and gene expression in human gallbladder epithelial cells. Methods: Gallbladder epithelial cells derived from a well-differentiated gallbladder carcinoma were grown to confluence in DMEM containing 10% fetal bovine serum (FBS). Cells were incubated with TNFct (10-100 ng/ml) for 30 min, 2 h and 24 h. To study mucin secretion, cells were preincubated with 3H glucosamine and radioactive mucin was precipitated from the medium and quantified using standard protocols. To determine the effect of TNFct on mucin gene expression, RNA was isolated from treated cells and mRNA levels for the primary gallbladder mucin genes MUC5B and MUC3 were determined after hybridization of slotblots to fluorescein-labeled cDNA probes. Mucin mRNA levels were normalized to levels of glyceraldehyde 3-phosphate dehydrogenase mRNA. In some cases, experiments were repeated with primary cultures of gallbladder epithelial cells grown on collagen IV coated plates in MEM containing 10% FBS. Results: At 24 h, TNFcc (50 ng/ml) caused a nearly two-fold increase in mucin secretion from both the epithelial cell line cultures (1.7 +/0.14 fold) and the epithelial cell primary cultures (1.8 +/0.22 fold). The effects of other concentrations and incubation times examined were similar and were equivalent in both cell preparations. Pre-incubation of TNFoc with neutralizing anti-TNFct antibodies abolished the increase in mucin secretion, however, the increase was unchanged by translation and transcription inhibitors. By contrast, TNFct had no effect on transcript levels of either MUC5B or MUC3 at any concentration tested. Condusions: Treatment of gallbladder epithelial cells with the inflammatory mediator TNFcc causes the release of pre-synthesized mucin but does not stimulate transcription of the MUC5B and MUC3 genes or lead to increased mucin synthesis. Other biliary constituents are likely to be responsible for the sustained mucin hypersecretion which precedes stone formation.