Submitted on May 19, 2006
Accepted on November 26, 2006
The Extracellular Calcium-Sensing Receptor (CaSR) reciprocally regulates secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts
Dinithi Peiris1, Ivan Pacheco2, Craig Spencer2, and R John MacLeod3*
1 Gastrointestinal Diseases Research Unit, Kingston General Hospital, Dept of Physiology, Queen's University, Kingston, Canada
2 Dept of Physiology, Queen's University, Kingston, Canada
3 GIDRU/ Dept of Physiology, Queen's University, Kingston, Canada
* To whom correspondence should be addressed. E-mail: rjm5{at}post.queensu.ca.
To understand whether post-prandial extracellular Ca2+ (Ca2+o) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co) and observed increases in Bone Morphogenetic Protein 2 (BMP-2) transcripts. The current experiments demonstrate regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and reveal a new property of BMP-2 on intestinal barrier. Activation by Ca2+o or spermine, GdCl3, neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from normal human colon stimulated both synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with siRNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC50 of Ca2+o (5.7 mM vs 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required PI-3 kinase activation (assessed by P-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded post-confluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased transepithelial resistance (TER) of low and high resistance T84 epithelial monolayers. CaSR stimulation of the T-84 epithelia and the colonic myofibroblasts downregulated the BMP family antagonist, Noggin, as assessed by RT-PCR and Western blots. Together our data suggest that the CaSR mediates the effective concentration of BMP-2 in the intestine which leads to enhanced repair and barrier development.
