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EXTRACELLULAR CALCIUM-SENSING RECEPTOR STIMULATES SECRETION OF THE WNT ANTAGONIST, DKK-1, FROM COLONIC MYOFIBROBLASTS TO INCREASE TRANSEPITHELIAL RESISTANCE
I Pacheco, RJ MacLeod
GIDRU/Dept of Physiology, Queen’s University, Kingston, Ontario
AIM: To determine if CaSR activation of colonic myofibroblasts contributed to intestinal epithelial homeostasis, we stimulated the CaSR on a human colonic myofibroblast cell line (18Co) and primary culture colonic myofibroblasts (hCMF) with high Ca2+ and screened for Wnt antagonists and Dkk family members by RT-PCR. Furthermore, the effect of the Wnt antagonist, Dkk-1, on epithelial barrier formation and recovery from acute damage was elucidated.
Materials and METHODS: Cells were transiently transfected with siRNA against CaSR and 48h later treated with low Ca2+ (0.5 mM), or high Ca2+ (3 or 5 mM) and Dkk-1 levels were measured by ELISA. The effect of CaSR activation in colonic epithelial cells on the expression of LRP6/Kremmen1 was assessed by rtPCR. The effect of rhDkk1 on barrier recovery from “Ca2+-switch” using 2 mM EDTA (5min) was assessed by measuring changes in transepithelial resistance (TER) of T84 monolayers seeded on Transwells.
RESULTS: High Ca2+ treatment time-dependently stimulated the transcript and secretion of Dkk1 from 18Co and hCMF. Transient transfection with siRNA duplexes against the CaSR reduced the secreted Dkk-1 to constitutive levels. Addition of the soluble Wnt receptor rmFzd8/Fc chimera to scavenge canonical Wnt3a that could stimulate Dkk1 secretion did not alter the kinetics of Dkk-1 secretion. Other CaSR agonists (poly-L-Arginine, Spermine and neomycin) increased Dkk1 secretion from 18Cos to levels similar to high calcium. Other GPCR agonists ADP, angiotensin and thrombin did not affect Dkk-1 transcript and secretion. The CaSR-mediated secretion of Dkk-1 was reduced to basal levels by EGFR kinase and Src inhibitors. High calcium challenge had no effect on Dkk-1 secretion from colonic epithelial cell lines. However, CaSR activation of HT-29, HT-29-APC, DLD-1, and T-84 cell lines increased LRP6 expression. CaSR activation in HT-29 and T84 cell lines up-regulated Kremen-1 transcript. Ca2+-switch experiments showed that T-84 cells treated with high Ca2++Dkk-1 (25ng/mL) recovered with greater TER within 24 h than cells treated with high Ca2+ alone (1900±10 vs 2440±25 OMEGA/cm2; p<0.05).
CONCLUSIONS: We conclude that CaSR activation of 18Co myofibroblasts regulates Dkk1 production by stimulating EGFR, and Src pathways. CaSR mediates increases of LRP6/Kremen1 on epithelia and the ligand, Dkk1 from the subepithelia. These findings support a new paracrine model mediated by the CaSR that promotes intestinal barrier recovery.