Introduction Transgenic hGAS mice, which have high circulating progastrin concentrations show increased colonic epithelial proliferation and increased susceptibility to colorectal carcinogenesis. As signalling between colonic epithelial cells and their underlying myofibroblasts is known to regulate homeostasis and the colonic stem cell niche, we hypothesise that progastrin may regulate colonic carcinogenesis by exerting effects upon both myofibroblasts and epithelial cells. We have therefore assessed the numbers of pericryptal myofibroblasts in the colonic mucosa of hGAS mice and have investigated the effects of treating a human colonic myofibroblast cell line with exogenous recombinant progastrin.
Methods hGAS mice were backcrossed onto a C57BL/6 genetic background and epithelial mitotic index and crypt length were assessed in the distal colon on a cell positional basis using H&E sections. The numbers of total and proliferating myofibroblasts were assessed by dual immunohistochemistry with anti-α-smooth muscle actin or vimentin antibodies (to stain myofibroblasts); and anti-Ki67 antibody (to stain proliferating cells). CCD18co human colonic myofibroblasts were treated with 0-10 nM progastrin, gastrin-17 (G-17) or glycine-extended gastrin (G-Gly) for 18 h and proliferation was assessed using a luminescence-based assay. Signalling pathways were investigated by pre-treating cells with inhibitors for the CCK-2 receptor (YM022), Protein kinase C (RO-32-0432), MAP kinase (PD98059) or PI3 kinase (LY294002).
Results Consistent with previous observations in hGAS mice on the FVB/N genetic background the percentage of colonic epithelial mitotic cells was increased by 90% in hGAS mice (p<0.001), which also showed a 22% increase in colonic crypt length (p<0.01). The percentage of colonic mucosal cells which were myofibroblasts was increased in hGAS mice by α-smooth muscle actin and vimentin staining (26% and 25%, respectively, p<0.001); but no Ki67 staining was observed in myofibroblasts. 0.1 nM PG maximally increased proliferation of CCD18co cells by 86% (p<0.001) and this was inhibited by RO-032-0432 (p<0.001). G-17 and G-Gly did not affect CCD18co cell proliferation.
Conclusion Progastrin overexpressing hGAS mice show increased numbers of colonic pericryptal myofibroblasts as well as increased colonic epithelial proliferation. Progastrin also stimulates the proliferation of colonic myofibroblasts in vitro via Protein kinase C pathway signalling. Progastrin therefore appears to increase colonic susceptibility to carcinogenesis by affecting both stromal and epithelial compartments.