Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein

Lihong Wang,Hailong Cao,Liping Liu,Bangmao Wang,W.Allan Walker,Sari A Acra,Fang Yan

doi:10.1074/jbc.m114.553800

Abstract

The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

Highlights

These results suggest that p40-stimulated activation of EGF receptor (EGFR) mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury
It has been demonstrated that LGG and Lactobacillus planetarium inhibit the adherence of enteropathogenic Escherichia coli to the intestinal epithelial cells through induction of MUC2 and MUC3 mucin production [13]
One of the mechanisms by which probiotics protect the host from pathogenic bacterial invasion is through stimulating mucin gene expression, synthesis, and secretion [34]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatment—The LS174T cell line (ATCC CL-188TM) is a human colon cancer cell line that exhibits characteristics of normal colonic mucosal cells, including microvilli prominent in secretory cells and the presence of intracytoplasmic mucin vacuoles. Preparation of Cellular Lysates for Western Blot Analysis— LS174T cells were solubilized in cell lysis buffer containing 1% Triton X-100, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and a proteinase inhibitor mixture (Sigma-Aldrich) and incubated for 1 h on ice. The scraped suspensions were centrifuged at 14,000 rpm for 15 min at 4 °C, and the protein concentration was determined using a BCA protein assay kit (Pierce Thermo Scientific). PAS Assay—Colon epithelial cells isolated from mice, mucosal lysates from colon organ culture, and LS174T cells were disrupted in PBS using Tissuelyser to obtain soluble proteins. Tissue sections and cultured cell slides were incubated with an rabbit anti-MUC2 antibody (Santa Cruz Biotechnology, Inc.) for 48 h at 4 °C, followed by a FITC-conjugated (Jackson ImmunoResearch) or HRP-labeled goat anti-rabbit IgG antibodies at room temperature for 1 h.

RESULTS

Findings

DISCUSSION