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EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CASR)-MEDIATED WNT5A SECRETION INHIBITED BETA-CATENIN SIGNALING OF EPITHELIAL CELLS INDEPENDENT OF TYROSINE KINASE RECEPTOR ROR2

I Pacheco, M Hayes, R John MacLeod
GIDRU, Department of Physiology, Queen's University, Kingston, Ontario

BACKGROUND: Molecular determinants of chemoprotection by calcium supplementation against colon cancer are currently unknown. Wnt5a, a Wnt family member, will signal through Frizzleds 2,4,7 or Ror2. It is not known in the gut which receptors Wnt5a uses. Wnt5a protein expression in primary Dukes B colon cancer identified a subgroup of patients with good prognosis (Cancer Res. 2005; 65(20): 9142-6).
METHODS: Western blots of Wnt5a protein from HT-29 or sub-confluent Caco-2 lysates and conditioned medium after CaSR activation was performed. Interfering RNA used to determine involvement of CaSR. Tcf signaling was measured by TOPFLASH luciferase in cells transiently transfected with wild type or dominant negative Ror2. Role of APC integrity on Wnt5a induction of Siah2 assessed by RT-PCR and Western blot of inducible full length compared with truncated APC cell lines.
RESULTS: Extracellular Ca²⁺ activation of CaSR stimulated Wnt5a secretion. CaSR activation or Wnt5a transfection of these cells inhibited Tcf/TOPFLASH reporter equivalently and reduced cellular beta-catenin. Cells transfected with siRNA against CaSR reduced CaSR transcripts and protein, blocked Ca²⁺-stimulated Wnt5a secretion, and reversed Ca²⁺-mediated inhibition of Tcf/TOPFLASH reporter. Wnt5a inhibition of Tcf/TOPFLASH reporter was not increased by overexpression of Ror2 or reversed by dominant negative Ror2-GPI constructs. CaSR and Wnt5a inhibition of Tcf/TOPFLASH reporter was reversed by dominant negative E-ubiquitin ligase Siah2. CaSR activation or Wnt5a transient transfection of truncated APC cells increased Siah2 transcripts and protein.
CONCLUSION: We conclude that CaSR-mediated secretion of Wnt5a in an autocrine manner inhibits beta-catenin signaling in intestinal cells. The Wnt5a inhibition of beta-catenin signaling is independent of endogenous Ror2 by requires substantial increases of the E-ubiquitin ligase, Siah2.

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