Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB106

Poster Communications

Guardian goblet cells protect the human large intestinal stem cell niche via muscarinic receptor-coupled, ROS/calcium-dependent mucus secretion.

C. Kam1, N. Pelaez-Llaneza1, A. parris1, A. lee1, V. jones1, M. R. williams1

1. School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom.

Mucus barrier formation is compromised in human intestinal disease. In Muc2 knock-out mice, direct contact of luminal bacteria with the gut epithelium causes inflammation and colon cancer. Recent studies of differentiated murine goblet cells (1,2) point to a pivotal role for ROS and calcium signalling in excitation-mucus secretion coupling. The aim of the current study was to investigate a potential role for ROS and calcium signalling in cholinergic-induced mucus secretion by goblet progenitor cells (GPCs) located in the human colonic stem cell niche. Methods: Human colonic crypt cultures were generated from colorectal tissue samples obtained at surgical resection (NRES approval). Intracellular calcium was monitored by Fura2 imaging. Transcript expression was assessed by RT-PCR. Protein localisation was visualised by fluorescence immunolabelling and changes in protein expression/localisation were quantified by image analysis of immunofluorescence intensity. Results: The muscarinic receptor agonist oxotremorine (1 μM) stimulated calcium signals throughout the intestinal stem cell niche of human colonic crypts. The human colonic epithelium (N>5 subjects) expressed transcripts for: MAChR subtypes 1, 3 & 5; InsP3R subtypes 1-3; RYR subtypes 1-3; TPC1 and TPC2. Immunolabelling confirmed the expression of their protein counterparts in Muc2+ GPCs located adjacent to LGR5+ stem cells. The peak amplitude of the muscarinergic calcium signal was inhibited by more than 80% (P<0.01, n>30 crypts, N>20 subjects) by TPC1/2 antagonists NED19 (100 μM) and diltiazem (100 μM). Expression of the ROS generator, NADPH oxidase 1 (NOX1) by GPCs was demonstrated by immunofluorescence labelling. A range of NOX1 inhibitors (DPI, 5 μM; apocynin, 100 μM; VAS2870, 56 μM), or the antioxidant n-acetylcysteine (5 mM), suppressed the peak amplitude of the muscarinergic calcium signal generation by at least 50% (P<0.01, n>10 crypts, N>3 subjects for all treatments). The Muc2-content (i.e. immunofluorescence intensity) of GPCs was reduced by approximately 50% following stimulation with muscarinic receptor agonists (N=9 subjects, n≥40 crypts and n≥500 GPCs); this was accompanied by the appearance of Muc-2 protein in the crypt lumen. Inhibition of muscarinergic calcium signals by pre-incubation with BAPTA-AM (66 mM), NED-19, or DPI not only inhibited Muc-2 secretion, but promoted an accumulation of intracellular Muc2 content. Finally, cholinergic input promoted an increase in Muc2+ progenitor cell nuclei exhibiting mitotic figures. Conclusions: Cholinergic input to the human large intestinal stem cell niche maintains the population of goblet progenitor cells and promotes NOX/ROS-dependent calcium mobilisation via intracellular TPC channels. Increased cytosolic calcium stimulates Muc2 secretion into the crypt lumen which guards against invading luminal bacteria.

Where applicable, experiments conform with Society ethical requirements