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5 REGULATED BMP-2 SECRETION FROM COLONIC MYOFIBROBLASTS MEDIATED BY THE EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CAR) PROMOTES INTESTINAL EPITHELIAL BARRIER DEVELOPMENT D Pieris, I Pacheco, C Spencer, RJ MacLeod To understand whether postprandial extracellular Ca2+ (Ca2+o) changes were related to intestinal epithelial homeostasis, we performed array analysis on CaR-expressing colonic myofibroblasts and observed increases in BMP-2 transcripts. The current experiments reveal that regulated secretion of BMP-2 occurs in response to CaR activation of these cells and reveal a new property of BMP-2 on intestinal barrier. CaR activation of 18Co cells or primary isolates of myofibroblasts from normal human colon by Ca2+o, or other agonists spermine, GdCl3 or neomycin sulfate stimulated both synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with dominant negative CaR (R185Q) increased the EC50 of Ca2+o (5.7 mM vs 2.3 mM). Upregulation of BMP-2 transcipts and secretion occurred within 3 h of CaR activation and was prevented by actinomycin D. Other Gq/11 agonists ADP or endothelin did not stimulate BMP-2 synthesis. CaR-mediated BMP-2 synthesis and secretion required PI-3 kinase activation (assessed by P-Akt generation) but was not affected by PKC, JNK, p38 MAPK, Erk1&2 or EGF receptor kinase inhibitors. New biological roles for this secreted BMP-2 were suggested by our findings that exogenous BMP-2 accelerated restitution compared with CaR activation alone in wounded postconfluent Caco-2 cells (um/h: 105±11 vs 67±18, P<0.05). Exogenous BMP-2 increased transepithelial resistance (TER) of T84 epithelial cells grown on Transwells. The dose-responsive increase was rapid. Within 24 h of application the TER was larger compared with nonstimulated cells (ohms/cm2: 718±19 vs 337±22, P<0.05, n=12). CaR stimulation of both T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist, noggin, as assessed by RT-PCR and Western blots. Together our data suggest that the CaR mediates the effective concentration of BMP-2 in the intestine, which leads to enhanced repair and barrier development.
GIDRU, Dept of Physiology, Queen’s University, Kingston, Ontario