Abstract MP48: EGF Regulates VSMC Migration And Proliferation Through Crosstalk Between TRPM7 And EGFR

Abstract

Epidermal growth factor (EGF), signals throught the EGF receptor (EGFR) and plays an important role in the pathogenesis of vascular remodeling. Transient receptor potential melastatin 7 (TRPM7) is a channel bound to a kinase domain important for Mg 2+ , Zn 2+ and Ca 2+ homeostasis. Cancer patients treated with EGFR inhibitors develop hypomagnesemia, suggesting a relationship between EGFR and TRPM7. Here we investigated the role of TRPM7 in EGF signaling in vascular smooth muscle cell (VSMC) from humans (hVSMC) and rats (rVSMC). VSMCs were stimulated with EGF (50ng/ml) for 5min and 24h with/without pretreatment of gefitinib (1μM), PP2 (10μM), 2APB (30μM) and NS8593 (40μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aortas were isolated from wild type (WT), TRPM7-deficient (TRPM7 +/Δkinase ) and kinase-dead (TRPM7 R/R ) mice. Protein expression was assessed by immunoblotting. Ca 2+ and Mg 2+ were assessed using Cal-520 and Mg-green probes respectively. EGFR/TRPM7 interaction was investigated by proximity ligation assay (PLA), immunoprecipitation and confocal microscopy. VSMC migration and proliferation were examined by wound healing and CFSE proliferation assays. In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), (p<0.05); effects abolished by gefitinib and PP2. EGF-induced Mg 2+ and Ca 2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg 2+ ) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) through c-Src, EGFR and TRPM7, p<0.05. Cell migration (26%) and proliferation (17%) were enhanced by EGF, and reduced by inhibitors of EGFR, TRPM7 and ERK1/2, p<0.05. EGF induced TRPM7-EGFR interaction (51%), which was reduced by gefitinib (34%) and PP2 (25%). VSMC from TRPM7 +/Δkinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7 R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7 +/Δkinase showed reduced wall thickness (35%). Our findings demonstrate that interaction between EGFR/TRPM7 is a key process underlying EGF-induced VSMC migration and growth. This novel EGF-c-Src-EGFR-TRPM7 pathway may play an important role in vascular remodeling.