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27

COLONIC EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CaR) INHIBITS PROLIFERATION BY STIMULATING WNT5A AND SIAH2-MEDIATED BETA-CATENIN DEGRADATION

I Pacheco, D Pieris, C Spencer, RJ MacLeod
GIDRU, Dept of Physiology, Queen's University, Kingston, Ontario

To understand the basis of how Ca²⁺ supplementation reduces the onset of colon cancer we activated the extracellular calcium-sensing receptor (CaR) on subconfluent adenocarcinoma-derived Caco-2 and HT-29 cells. Proliferation was assessed by MTT assay and the Wnt target Cyclin D. High Ca²⁺ (5 mM) reduced MTT absorbance 80% (P<0.05) in comparison with growth promoting 0.05 mM Ca²⁺ and substantially reduced cyclin D1 transcripts (RT-PCR). High Ca²⁺ (3 mM to 5 mM, 18 h) stimulated upregulation (10´) of transcripts of the noncanonical ligand (tumour suppressor)Wnt5a. Western blot analysis revealed increased Wnt5a protein. Transient transfection of Caco-2 and HT-29 cells with Wnt5a plasmid reduced TCF/Topflash activity, a beta-catenin reporter, to amounts equivalent to addition of 3 mM to 5 mM Ca²⁺. Reduction in TCF/Topflash was CaR-mediated as it was prevented by transient transfection with a dominant negative CaR (R185Q). Total cellular beta-catenin (Western blots) was also reduced by CaR activation in these cells. Addition of high Ca²⁺ to subconfluent Caco-2 cells upregulated transcripts for Siah1 and Siah2, E3-ubiquitin ligases known to stimulate beta-catenin degradation. Transient transfection with dominant negative Siah2 reversed the CaR-mediated inhibition of TCF/Topflash reporter activity. CaR activation increased production of phosphorylated GS3K. Because both Caco-2 and HT-29 cells express truncated APC, we induced wild type APC in HT-29 cells to determine if CaR-mediated inhibition of Wnt signalling required APC. Zn²⁺-inducible wild type APC HT-29 cells robustly expressed the CaR (RT-PCR and Western blots), but CaR-activation did not inhibit the TCF/Topflash reporter. We conclude that CaR-mediated inhibition of beta-catenin signalling in colonic cell lines requires mutated APC and the induction of Wnt5a and Siah2. Our current results suggest that Ca²⁺ supplementation may be more efficacious following initial APC mutations.

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