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OVEREXPRESSION OF b-CATENIN ENHANCES PROTEINASE-ACTIVATED RECEPTOR-1-INDUCED CELL PROLIFERATION IN COLON CELLS

H Wang, MD Hollenberg, WK MacNaughton
Mucosal Inflammation Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta

BACKGROUND: Proteinase-activated receptors (PARs) are G protein-coupled receptors that are activated by serine proteinases that cleave the extracellular N-terminus to reveal a new N-terminus, which then acts as a "tethered ligand" to activate the receptor. PAR-1, a recognized receptor for thrombin, plays an important role in wound healing and inflammation, and is also involved in tumour progression, as is b-catenin, a crucial tumourogenic determinant in many colon cancers. To date, however, an interaction between PAR-1 and b-catenin has yet to be reported. We hypothesized that overexpression of b-catenin would affect the response of colonic cells to the activation by PAR-1.
METHODS AND RESULTS: RKO, a PAR-1-expressing human colonic cancer cell line possessing a low level of b-catenin, was stably transfected with a constitutively activated b-catenin construct to yield the RKO-b-catenin cell line (RKO_b-cat). When stimulated with the PAR-1-activating peptide, TFLLR-NH₂ (PAR-1-AP), RKO_b-cat cells showed a 2-fold increase (P<0.5) in sensitivity with respect to the characteristic PAR-1-induced calcium influx signal, compared with the RKO parental cell line. Furthermore, the PAR-1-AP concentration-response curve for RKO_b-cat cells was shifted to the left compared with the wild-type RKO cells. RT-PCR showed that overexpression of b-catenin in RKO_b-cat cells significantly increased PAR-1 mRNA expression. Moreover, the PAR-1-AP at a concentration of 100 µM, also stimulated RKO_b-cat, but not RKO, cell proliferation, as measured by both cell counting and WST-1 reagent. However, PAR-1-AP treatment did not affect the activity of a
b-catenin reporter construct (TOP) in a transient transfection assay, implying that PAR-1 activation had no effect on the transcriptional activity of
b-catenin.
CONCLUSIONS: Our data suggest that expression of an activated b-catenin mutant at an early stage of colon cancer may sensitize cells to a PAR-1-mediated increase in invasiveness and tumourigenesis, now recognized to be driven by tumour-derived proteinase like matrix metalloproteinase-1 (Cell 2005;120:303).

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