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050 PROTEINASE-ACTIVATED RECEPTOR 2 (PAR2) INCREASES CELL PROLIFERATION THROUGH CYCLIN D1 H Wang, T Harrison, WK MacNaughton Proteinase-activated receptor (PAR)-2 is a G-protein coupled receptor that is activated by the proteolytic activity of serine proteinases such as trypsin and tryptase. PAR2 plays important roles in physiological and pathophysiological processes, and has recently been shown to promote colon cancer cell proliferation. However, the mechanisms whereby this occurs are still unknown. Cyclin D1 is an important regulator of DNA synthesis in the cell cycle. We aimed to test whether cyclin D1 is involved in PAR2-induced cell proliferation. We used the human epithelial cell line, A549, which was treated with the selective PAR2 activating peptide (PAR2-AP), SLIGRL-NH2 (50 µM). RT-PCR showed that exposure of cells to the PAR2-AP significantly increased cyclin D1 mRNA expression by 100% by 3 hours after treatment (p<0.05). Using the TOP-FLASH luciferase reporter assay for the transcriptional activity of beta-catenin, which is elevated in many forms of colon cancer and is a known regulator of cyclin D1, we also showed that beta-catenin activity was elevated 4-fold (p<0.01) with PAR2-AP treatment. Cell cycle analysis by flow cytometry showed a slight increase of the G2/M phase by 12 hours after the stimulation with PAR2-AP. Moreover, using the BrdU-labeling method, we found that exposure of cells to the PAR2-AP significantly increased DNA synthesis 12 hours after treatment.
Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta
In conclusion, we have shown that activation of PAR2 in epithelial cells increased cyclin D1 and DNA synthesis by stimulating the transcriptional activity of beta-catenin. Thus, our study provides evidence of a new role for PAR2 and PAR2-activating proteinases in cell proliferation and tumorigenesis.
H Wang is supported by a CAG-CCFC Fellowship