title: Extracellular Calcium-Sensing Receptor Inhibition of Intestinal EpithelialTNF Signaling Requires CaSR-Mediated Wnt5a/Ror2 Interaction.
abstract: Tumor necrosis factor alpha (TNFα) 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 TNFα secretion and then activated endogenous CaSR. CaSR activation inhibited TNFα secretion, which in RAW264.7 cells knockdown of CaSR by short-interfering RNA (siRNA) duplex reversed. LPS-stimulated NFκB promoter activity in RAW264.7 cells was inhibited by CaSR activation with Ca(2+) 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 Ca(2+) reduced TNFR1 protein expression ∼70%. TNFα/INFγ addition to high resistance T84 monolayers reduced transepithelial resistance 50% within 4 h. CaSR activation (3 mM Ca(2+)) together with rhWnt5a (200 ng/ml) prevented this reduction while Wnt3a addition had no effect. LPS-stimulated TNFα 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 NFκB activity and reduce TNFα secretion from macrophages and stroma while Wnt5a/Ror2 engagement on intestinal epithelia reduces TNFR1 expression, allowing TNFα signaling to be titrated. Our results also suggest that canonical Wnt signaling may enhance TLR4 stimulation of TNFα secretion from murine macrophages.
authors: Department of Physiology, Queen's University Kingston, ON, Canada.
authors: Jacqueline C
authors: JC
authors: Kelly
authors: P
authors: P
authors: Lungchukiet
authors: R John
authors: RJ
authors: Macleod
references: Dev Cell. 2008 Jan;14(1):132-9
references: 18160346
references: Am J Physiol. 1996 Feb;270(2 Pt 1):C418-28
references: 8779903
references: Biochem Biophys Res Commun. 2008 Aug 29;373(3):445-9
references: 18588857
references: Am J Physiol Gastrointest Liver Physiol. 2008 Oct;295(4):G748-59
references: 18703641
references: Am J Physiol Gastrointest Liver Physiol. 2006 Mar;290(3):G496-504
references: 16474009
references: Dev Dyn. 2010 Jan;239(1):1-15
references: 19530173
references: Am J Physiol Gastrointest Liver Physiol. 2007 Mar;292(3):G753-66
references: 17138967
references: Blood. 2006 Aug 1;108(3):965-73
references: 16601243
references: Am J Gastroenterol. 2000 Feb;95(2):359-67
references: 10685736
references: Gastroenterology. 2004 Feb;126(2):451-9
references: 14762782
references: Carcinogenesis. 2009 Jan;30(1):88-92
references: 19017685
references: J Immunol. 2003 Dec 1;171(11):6164-72
references: 14634132
references: Dev Cell. 2008 Jan;14(1):120-31
references: 18160348
references: Dig Dis Sci. 2003 May;48(5):870-6
references: 12772782
references: J Coll Physicians Surg Pak. 2005 Oct;15(10):597-600
references: 19810294
references: Cell Mol Immunol. 2004 Feb;1(1):57-62
references: 16212922
references: Am J Pathol. 2005 Feb;166(2):409-19
references: 15681825
references: Cell. 2006 May 5;125(3):523-33
references: 16678096
references: J Nutr. 2008 Sep;138(9):1658-63
references: 18716166
references: Am J Physiol Renal Physiol. 2008 Feb;294(2):F345-54
references: 18032544
references: Nat Rev Mol Cell Biol. 2004 Sep;5(9):739-51
references: 15340381
references: FASEB J. 2006 Dec;20(14):2562-4
references: 17077282
references: Bone. 2010 May;46(5):1416-23
references: 20149906
references: Nat Rev Mol Cell Biol. 2009 Jul;10(7):468-77
references: 19536106
references: Traffic. 2010 Oct;11(10):1265-71
references: 20477987
references: Arch Intern Med. 2009 Feb 23;169(4):391-401
references: 19237724
references: J Biol Chem. 2002 Aug 16;277(33):30337-50
references: 12036954
references: Trends Cell Biol. 2010 Jun;20(6):346-54
references: 20359892
references: Methods Mol Biol. 2010;626:117-30
references: 20099125
references: EMBO J. 2010 Jan 6;29(1):41-54
references: 19910923
references: J Clin Invest. 2007 Mar;117(3):703-11
references: 17318263
references: J Clin Invest. 2008 Feb;118(2):560-70
references: 18219394
references: Biochim Biophys Acta. 2000 Apr 12;1484(2-3):87-92
references: 10760459
references: Am J Physiol Gastrointest Liver Physiol. 2007 Jul;293(1):G403-11
references: 17463182
references: Am J Physiol Renal Physiol. 2002 Nov;283(5):F963-70
references: 12372772
references: BMC Neurosci. 2010 Mar 09;11:33
references: 20214800
references: N Engl J Med. 1997 Oct 9;337(15):1029-35
references: 9321530
references: Oncogene. 2004 Mar 15;23(11):1985-97
references: 15021886
references: Gastroenterology. 2006 Oct;131(4):1153-63
references: 17030185
references: J Clin Invest. 2000 May;105(9):1299-305
references: 10792005
references: Gastroenterology. 2010 Sep;139(3):869-81, 881.e1-9
references: 20580720
references: Am J Physiol Gastrointest Liver Physiol. 2010 Nov;299(5):G1187-97
references: 20829523
references: Mol Cell Biol. 2000 Apr;20(7):2326-33
references: 10713156
references: FASEB J. 2010 Nov;24(11):4599-612
references: 20667980
references: Physiol Rev. 2001 Jan;81(1):239-297
references: 11152759
references: Arterioscler Thromb Vasc Biol. 2008 Mar;28(3):504-10
references: 18174455