Extracellular calcium-sensing receptor inhibition of intestinal epithelial TNF signaling requires CaSR-mediated Wnt5a/Ror2 interaction

Tumor necrosis factor alpha (TNF alpha) and its receptor TNFR1 play a central role in the development of colitis-associated colon cancer. To understand a role for the extracellular calcium-sensing receptor (CaSR) and its non-canonical Wnt mediators, Wnt5a/Ror2, we used reductionistic systems. We added lipopolysaccharide (LPS) to mouse peritoneal macrophages, RAW264.7 cells, a murine macrophage cell line, and 18Co colonic myofibroblasts, to stimulate TNF alpha secretion and then activated endogenous CaSR. CaSR activation inhibited TNF alpha secretion, which in RAW264.7 cells knockdown of CaSR by short-interfering RNA (siRNA) duplex reversed. LPS-stimulated NF kappa B promoter activity in RAW264.7 cells was inhibited by CaSR activation with Ca2+ or other polyvalent CaSR agonists. Reducing CaSR expression with siRNA duplex prevented this inhibition. Following LPS addition to CaSR-HEK cells or RAW264.7 macrophages, CaSR stimulation deneddylated Cullin1. Wnt5a added to HT-29 cells which overexpressed Ror2 or T84 monolayers treated with 3 mM Ca2+ reduced TNFR1 protein expression similar to 70%. TNF alpha/INF gamma addition to high resistance T84 monolayers reduced transepithelial resistance 50% within 4 h. CaSR activation (3 mM Ca2+) together with rhWnt5a (200 ng/ml) prevented this reduction while Wnt3a addition had no effect. LPS-stimulated TNFa secretion from RAW264.7 cells was not effected by rhWnt5a but increased 10-fold by Wnt3a. Together our results suggest that following LPS challenge, CaSR activation will inhibit NF kappa B activity and reduce TNF alpha secretion from macrophages and stroma while Wnt5a/Ror2 engagement on intestinal epithelia reduces TNFR1 expression, allowing TNF alpha signaling to be titrated. Our results also suggest that canonical Wnt signaling may enhance TLR4 stimulation of TNF alpha secretion from murine macrophages.