CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucrase-isomaltase using Ror2 on intestinal epithelia

Abstract

Pacheco II, MacLeod RJ. CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucraseisomaltase using Ror2 on intestinal epithelia. Am J Physiol Gastrointest Liver Physiol 295: G748 -G759, 2008. First published August 14, 2008 doi:10.1152/ajpgi.00560.2007.-To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca 2ϩ , screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca 2ϩ (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSRactivated myofibroblasts to intestinal stem cell niche microenvironments.

Expression profiling of Wnt family of genes in normal and inflammatory bowel disease primary human intestinal myofibroblasts and normal human colonic crypt epithelial cells

“…26 The biological effects of Wnt5A in epithelial cells may depend on the availability of key receptors. 27,28 Thus, Wnt5A binding to Fz/LRP receptors may lead to canonical signaling.…”

“…Wnt5A can also signal through the ROR2 tyrosine kinase receptor, with activation of the JNK signaling pathway and polarized cell migration, as occurs in wound healing. 29 Restriction of ROR2 expression on the surface of colonic epithelial cells 26 would be expected to enable noncanonical Wnt5A-mediated signaling in this site in vivo, but lack of these receptors in stem cells would be expected to allow the canonical pathway to be activated. Recently, Wnt5A has also been reported to induce increased calcium-dependent cellcell adhesion, by promoting the association of b-catenin and E-cadherin complexes.…”

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts

“…These cells were originally cultured from a mucosal biopsy of human neonatal colon and share many of the structural and functional characteristics of in situ colonic subepithelial myofibroblasts, including a reversible stellate morphology, ␣-smooth actin expression, and the presence of multiple cell surface receptors (41). 18Co cells provide a model to elucidate physiological and pathophysiological functions of intestinal subepithelial myofibroblasts and, accordingly, have been used extensively to study colonic myofibroblast function in a variety of settings (22,31,38). 18Co cells were maintained at 37°C in DMEM supplemented with 10% FBS in a humidified atmosphere containing 10% CO 2-90% air.…”

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts

“…These cells share many of the structural and functional characteristics of in situ colonic subepithelial myofibroblasts, including a reversible stellate morphology, ␣-smooth actin expression, and the presence of multiple cell surface receptors (51). 18Co cells provide a model to elucidate physiological and pathophysiological functions of intestinal subepithelial myofibroblasts and, accordingly, have been used extensively to study colonic myofibroblast function in a variety of settings (22,33,42). 18Co cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO 2 and 90% air.…”