The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts.
To understand whether postprandial extracellular Ca(2+) (Ca(o)(2+)) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co cells) and observed increases in bone morphogenetic protein (BMP)-2 transcripts. The present experiments demonstrated that regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and revealed a new property of BMP-2 on the intestinal barrier. Activation by Ca(o)(2+), spermine, GdCl(3), or neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from the normal human colon stimulated both the synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with short interfering RNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC(50) of Ca(o)(2+) (5.7 vs. 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3 h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required phosphatidylinositol 3-kinase activation (as assessed by phospho-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded postconfluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased the transepithelial resistance of low- and high-resistance T-84 epithelial monolayers. CaSR stimulation of T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist Noggin, as assessed by RT-PCR and Western blot analysis. 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.
Peiris
Dinithi
D
Gastrointestinal Diseases Research Unit, Kingston General Hospital, and Department of Physiology, Queen's University, Kingston, Ontario, Canada.
Pacheco
Ivan
I
Spencer
Craig
C
MacLeod
R John
RJ
D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D018938:Caco-2 Cells; D002118:Calcium / Q000819:agonists / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D009355:Neomycin / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D044169:Receptors, Calcium-Sensing / Q000819:agonists / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D000099176:Noggin Protein
D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D002118:Calcium; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D009355:Neomycin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases; D000099176:Noggin Protein; C525718:BMP2 protein, human; C525809:BMP4 protein, human