Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells

• Published in: American journal of physiology: Gastrointestinal and liver physiology 2007-07, Vol.293 (1), p.G403-G411
• Main Authors: MacLeod, R John, Hayes, Madeline, Pacheco, Ivan
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Adenomatous Polyposis Coli Protein - biosynthesis; beta Catenin - antagonists & inhibitors; Calcium; Calcium - pharmacology; Calcium Signaling - drug effects; Cell Line, Tumor; Cells; Cells, Cultured; Colonic Neoplasms - physiopathology; Colorectal cancer; Disease prevention; Humans; Nuclear Proteins - biosynthesis; Proteins; Proto-Oncogene Proteins - secretion; Receptor Tyrosine Kinase-like Orphan Receptors; Receptors, Calcium-Sensing - physiology; Receptors, Cell Surface - biosynthesis; Ubiquitin-Protein Ligases - biosynthesis; Wnt Proteins - secretion; Wnt-5a Protein
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.00119.2007

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.