The Effect Of Proton-Sensing Gpcrs On Cell Migration Of MEF

In this study, we focused on the effects of extracellular acification on intracellular signaling pathways and its underlying molecular mechanisms in mouse embryonic fibroblasts (MEFs). To our best knowledge, it is the first time to study the effects of extracellular acidic pH in MEFs. It was reported that extracellular acidification can stimulate a variety of intracellular signaling pathways and regulate cellular function through proton-sensing G protein-coupled receptors (GPCRs) or proten-sensing ion channels. By this regulatory mechanism, extracellular acidic pH plays an importment role in the development of some disease, such as cancer and asthma. The elucidation of mechanisms of extracellular acidification stimulating intracellular signaling pathways in a variety of cell types is of great importance for developing new targets of treatment and will provide valuable scientific evidence for the treatment and the medicinal development of related disease.This research used MEFs derived from OGR1-KO, GPR4-KO, TDAG8-KO and wild-type mice as subjects. The experiments of cell migration, cell viability assay (MTT), BrdU and [Ca2+]i measurement were used to detect the effects of extracellular acidification on intracellular signaling pathways in MEFs. Using real-time Taqman PCR, we examined the mRNA expression of OGR1, GPR4, TDAG8 and G2A in WT MEFs. Western blotting assay was performed to detect the proteins expression of OGR1, GPR4, TDAG8 and G2A in WT MEFs and the phosphorylation level of Aktl and p38MAPK protein after the stimulation of extracellular acidic pH in MEFs for researching intracellular signaling pathways stimulated by extracellular acidification. ANOVA (using SPSS 13.0 software) was performed to compare the differences between test groups and controls. Paired t-test was used to analyze the differences of theoretical values and actual values of pH 6.8-induced WT MEFs and OGR1-KO, GPR4-KO and TDAG8-KO MEFs, respectively. Values were considered significant at p< 0.05 (*).In the present study, we first examined the effects of extracellular acidic pH on MEFs migration and IGF-1, EGF and PDGF-induced MEFs migration. Our results showed that extracellular acidification induced the migration of MEFs, and specifically enhanced PDGF-induced migration of MEFs. Shortly after, we further examined whether extracellular acidification imparts its effect on the viability and proliferation of MEFs and research if the above results are correlated to the viability and proliferation of MEFs. The results of MTT and BrdU assays showed that extracellular acidification significantly inhibited the viability and proliferation of MEFs in the presence or absence of PDGF. By using specific inhibitors in cell migration assay, we found that extracellular acidification-induced MEFs migration was completely suppressed when cells were pre-incubated with PTX, a specific inhibitor for Gi protein with or without PDGF, however, YM254890, a specific inhibitor for Gq protein, has no effect on pH 6.8-induced cell migration with or without PDGF, suggesting that Gi protein, but not Gq protein, is involved in the course of extracellular acidification-induced cell migration of MEFs in the presence or absence of PDGF. Western-blotting experiments showed that the protein expression of p-p38MAPK at pH 6.8 was greater than at pH 7.6 in the presence or absence of PDGF.To certificate whether the increasing effects induced by pH 6.8 and PDGF on MEFs migration are additive or not, we analyzed all the results of 34-time independent migration assays in our study using the paired t-test. Statistical analysis certified that pH 6.8-and PDGF-induced increasing effects on MEFs migration were not additive (t=7.72, d.f.= 33, P=0.000), and when pH 6.8 and PDGF were mixed together, the number of migrating cells was approximately 50% greater than the sum of migrating cell numbers induced by pH 6.8 and PDGF alone, averagely. These findings verified that signaling pathways initiated by extracellular acidification interact with signaling pathways initiated by PDGF to facilitate directed cell migration. The results of [Ca2+]i measurement showed that extracellular acidification induced transient [Ca2+]i elevation, which was mediated by Gq/11 protein in MEFs.Experiments using qPCR, western blotting and OGR1-, GPR4-, and TDAG8-KO MEFs showed no known proton-sensing GPCRs or ion channels involved in the migration of MEFs and transient [Ca2+]i elevation induced by extracellular acidic pH.The present study concluded that extracellular acidification increased MEF migration either by itself, or synergistically with PDGF through activation of proton-sensing GPCRs/Gi/p38MAPK signal pathway, but not through proton-sensing GPCRs/Gq/Ca2+ signal pathway. Known proton-sensing GPCRs (OGR1, GPR4, TDAG8 and G2A) and ion channel TRPV1 didn’t regulate acidification-induced cell migration and [Ca2+]i elevation. Our results verified that others novel proton-sensing GPCRs exist in MEFs besides four known proton-sensing GPCRs.