Negative Crosstalk Between the Extracellular Calcium Receptor and -Catenin in Colonic Epithelium

OSVALDO REY; WENHAN CHANG; DANIEL BIBLE; NORA ROZENGURT; ENRIQUE ROZENGURT

Congreso; Digestive Diseases Week; 2012

American Gastroenterological Association

Background: Signaling pathways activated by G protein-coupled receptors (GPCRs) expre- ssed in the gastrointestinal (GI) tract play a critical role in the regulation of multiple functions of the digestive system, including cell proliferation, differentiation, inflammation and promo- tion of colorectal cancer (CRC). The extracellular Ca2+-sensing receptor (CaSR), a member of the C family of GPCRs, is expressed in many tissues and organs not directly involved in the control of Ca2+ homeostasis, including both surface and crypt epithelial cells in rodent and human colons. Interestingly, the expression of the CaSR is greatly reduced or completely lost in CRC again suggesting that signaling pathways activated by this receptor negatively control cellular proliferation and CRC development. However, there is no definitive evidence for a physiological role of the CaSR in the regulation of normal and/or abnormal intestinal epithelial cell proliferation In Vivo. Results: In order to examine the role of the CaSR in colonic epithelial cell proliferation, we used a recently developed Casr intestinal-specific knock out mice model. Our results demonstrate that genetic ablation of this receptor led to hyperproliferation of colonic epithelial cells (Ki67 labeling), expansion of the proliferative zone, increase in the size of colonic crypts (length and number of cells/crypt) and enhanced -catenin nuclear localization, a regulator of genes that control intestinal cell proliferation and differentiation. To elucidate the mechanism(s) by which CaSR inhibits colonic cell proliferation, we examined multi-site phosphorylation of -catenin, a post-translational modification that plays a critical role in its degradation, nuclear translocation and transcrip- tional activity. We demonstrate that stimulation of the CaSR by either extracellular Ca2+ (5 mM) or the allosteric activator R-568 (100 nM) produced a striking and time-dependent (30 min to 4 h) decrease (≈ 60%) in the phosphorylation of -catenin at Ser552 and Ser675 in NCM-460 human-colon derived epithelial cells expressing the CaSR. Concomitantly, we detected by immunofluorescence a reduced nuclear reactivity of the phosphoSer552 antibody as well as a marked redistribution of -catenin to the plasma membrane. These changes in phosphorylation and intracellular distribution were associated with a 40% downregulation of -catenin mediated transcriptional activation (TCF/LEF-1 luciferase reporter TOP-Flash assay). Conclusion: These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cells proliferation In Vivo and suggest that the mechanism involves regulation of -catenin phosphorylation.