Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells.
To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.
MacLeod
R John
RJ
Department of Physiology, Queen's University, 76 Stuart Street, Kingston, Ontario, Canada. rjm5@post.queensu.ca
Hayes
Madeline
M
Pacheco
Ivan
I
D000230:Adenocarcinoma; D025601:Adenomatous Polyposis Coli Protein / Q000096:biosynthesis; D002118:Calcium / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000503:physiopathology*; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis; D051153:Wnt Proteins / Q000378:metabolism*; D000071818:Wnt-5a Protein; D051176:beta Catenin / Q000037:antagonists & inhibitors; D000098563:Seven in Absentia Proteins
D025601:Adenomatous Polyposis Coli Protein; C095550:CASR protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D051176:beta Catenin; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D002118:Calcium