Return to Table of Contents
CYCLIN D1 AND C-MYC MEDIATE PROTEINASE-ACTIVATED RECEPTOR 2 (PAR2)-INDUCED DNA SYNTHESIS
S Wen, H Wang, J Bjazevic, MK Jung, WK MacNaughton
Inflammation Research Network, University of Calgary, Calgary, Alberta
Background: Proteinase-activated receptor (PAR)-2 is a G-protein coupled receptor activated by the proteolytic activity of serine proteinases such as trypsin, tryptase and matriptase. PAR2 and its endogenous activators are highly expressed in a variety of tumours. The activation of PAR2 promotes proliferation in several colon cancer cell lines. However, the mechanisms whereby PAR2 induces cell proliferation are still unknown. Our preliminary findings showed that PAR2 increased cell proliferation and also DNA synthesis in the human epithelial cell line, A549. C-myc and cyclin D1 are important regulators of G1/S phase transition in the cell cycle. In the present study, we aimed to test whether c-myc and cyclin D1 are involved in PAR2-induced DNA synthesis.
Methods and Results: A549 epithelial cells were treated with the selective PAR2 activating peptide (PAR2-AP), SLIGRL-NH2 (50 ÁM). RT-PCR showed that the PAR2-AP significantly increased c-myc and cyclin D1 mRNA expression starting from 1 and 3 hr, respectively, after treatment. The protein levels of c-myc and cyclin D1 were also elevated as measured by densitometry of Western blots. To test whether beta-catenin, a known regulator of c-myc and cyclin D1, mediates PAR2-induced upregulation of c-myc and cyclin D1, the TOP-FLASH luciferase reporter assay was performed. We showed that b-catenin transcriptional activity was elevated 4-fold (p<0.01) with PAR2-AP treatment. Using another luciferase reporter construct, we showed that transcriptional activity of E2F was also elevated more than 2-fold with PAR2-AP treatment. Moreover, the downstream target gene of E2F, cyclin E, was also upregulated at both mRNA and protein level. Taken together, we have shown that activation of PAR2 in epithelial cells increased the expression of c-myc and cyclin D1, the transcriptional activation of E2F and DNA synthesis.
Conclusions: Thus, our study provides evidence of a new role for PAR2 and PAR2-activating proteinases in cell proliferation and tumorigenesis.