Involvement Of Lysophosphatidic Acid Receptors In LPA-Induced BGC-803Cell Migration

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator, which elicits a variety of biological functions mainly through G-protein coupled receptors. Although LPA is shown to stimulate proliferation and motility via LPA receptors, LPAR1and LPAR3in several cancer cell lines, but the role of LPA receptors in gastric cancer cells is still being unknown. However, several researches reported that LPAR2play an important role in the carcinogenesis of gastric cancer, but there is no report to show the LPAR3involvement in the carcinogenesis. For this reason, we examined LPA receptors (LPAR1, LPAR2and LPAR3) in BGC-803cells along with real time PCR method.Among these receptors, LPAR2and LPAR3were shown to be highly expressed in BGC-803cells, a human gastric cancer cell line. Transient transfection with LPAR2and LPAR3siRNAs were observed to reduce LPAR3mRNA in BGC-803cells and eliminate the LPA-induced cell migration. The results suggest that the LPAR3involves LPA-induced BGC-803cell migration. Our main findings were as follows:1. LPA significantly increased cellular migration.2. Ki-16425, antagonist of LPA1and LPA3receptors suppressed the LPA-induced migration of BGC-803cells. LPA-induced migration of BGC-803cell depends on LPA1and LPA3receptors.3. We discovered that LPA-induced cell migration is reduced by adding pertussis toxin (PTX). This result shows that Gi protein more involved in LPA-induced cell migration than Gq protein (Fig.3.4). 4. Results of real time-PCR method shown that LPAR3receptor predominantly expressed in human gastric cell line BGC-803cells among other receptors.5. LPAR2and LPAR3play an important roles in the LPA-induced cell migration, their expression was silenced by LPAR2and LPAR3-specific shRNAs in the BGC-803cells (Figures4.6,4.7and4.8). RT-PCR analysis showed that shLPAR3expression was decreased by53%compared with the control (Fig.4.4). However shLPAR2expression was decreased less than shLPAR3(Fig.4.5). As mentioned, shLPAR3expression was more decreased in the BGC-803cells, suggesting that LPAR3may be important in LPA-induced cell migration.6. Silencing LPAR2-3expression by shLPAR2and shLPAR3the LPA-induced cell migration of BGC-803were completely inhibited. Silencing LPAR3expression significantly decreased the LPA-induced migration of BGC-803cells compared with silencing LPAR2in BGC-803cells (Fig.4.9). The results demonstrated that LPA-induced migration of BGC-803gastric cancer cells was mainly mediated by LPAR3.7. The cell migration induced by LPA in shLPAR2-transfected BGC-803cells was significantly inhibited by PTX. However, shLPAR3-transfected cell migrating activity in the presence of PTX is not significant (Fig.4.10). These results suggest that PTX-sensitive G proteins involved in BGC-803cell migration.