G protein coupled receptors are coupled to extracellular signal regulated kinase (ERK) activation via stimulation of the epidermal growth factor receptor (EGFR) in a number of tissues. Regulation of ERK1/2 activity is thought to play a central role in the control of intestinal cell proliferation and differentiation. The aim of the present study was to investigate the cell signaling pathways that link muscarinic acetylcholine receptor stimulation to ERK1/2 activation and proliferation in human colonic epithelial cells.
METHODS: HT29 cells were cultured in DMEM supplemented with 5 % FCS (5 % CO2/37 °C). Cell preparations were stimulated by carbachol (100 µM) in the presence or absence of cell signaling inhibitors. Epidermal growth factor receptor and (EGFR) and ERK1/2 phosphorylation was assessed by immunoblotting and/or immunocytochemical techniques based on using phospho-specific antibodies. Shedding of membrane associated TGFα was assessed by immunocytochemistry. Cell proliferation was assessed by 3H thymidine incorporation (during 24 hours).
RESULTS: Carbachol (100 µM, 10 mins) stimulated cytoplasmic ERK1/2 and translocation into the nucleus; maximal cytoplasmic stimulation was noted at 1 hour. These effects were mimicked by calcium ionophores (e.g. ionomycin 1 µM) and abolished by inhibitors of calcium signal generation (e.g. BAPTA-am, 25 µM; caffeine, 5 mM). Carbachol-induced ERK activation was reduced by 50 % in the presence of BB94 (5 µM) (metalloprotease inhibitor), TIMP 3 (0.5 µM) (Tissue Inhibitor of metalloproteases), anti-TGFα (10 ng/ml), anti-EGFR (10 ng/ml; to prevent EGF ligand binding to the receptor) and tyrophostin (1 µM) (EGFR kinase inhibitor). No inhibition was seen in the presence of TIMP 1 and 2 (0.5 µM). Similar observations were made regarding carbachol induced-TGFα shedding, and -EGFR phosphorylation. Carbachol induced a 40 % increase in HT29 cell 3H thymidine incorporation. This effect was abolished in the presence of BB94 (5 µM), anti-TGFa (10 ng/ml) and anti-EGFR (10 ng/ml).
Carbachol induced calcium signals mediate metalloproteinase dependent shedding of TGFα and downstream activation of mitogenic epidermal growth factor receptor signals in HT29 colonic epithelial cells. These observations suggest that neuronal-epithelial cell interactions regulate the proliferative status of the colonic epithelium.
This work was supported by BBSRC, Humane Research Trust, Mason Medical Research Trust and Nuffield Foundation