INTERACTION BETWEEN TRPM7 AND EPIDERMAL GROWTH FACTOR RECEPTOR MEDIATES VASCULAR SMOOTH MUSCLE CELL ACTIVATION AND PROLIFERATION

Abstract

Objective: Epidermal growth factor (EGF) binds to EGF receptor (EGFR) and stimulates multiple vascular signaling pathways. Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein (channel+enzyme, termed chanzyme) playing an important role in vascular homeostasis. Previous studies in renal cells demonstrated interactions between EGFR and TRPM6, a homologue of TRPM7. Here we questioned the importance of TRPM7 in EGF-mediated effects on vascular smooth muscle cell (VSMC) function. Design and method: Primary cultured VSMCs were isolated from humans and rodent models. Rat VSMCs (rVSMCs) and human VSMCs (hVSMCs) were stimulated with EGF (50ng/ml) in the absence/presence of gefitinib (1 μM), PP2 (10 μM), 2APB (30 μM) and NS8593 (40 μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aorta were isolated from wild type (WT), TRPM7 kinase-deficient (TRPM7+/-kinase), and TRPM7 kinase-dead (TRPM7R/R) mice. Protein expression was investigated by immunoblotting. Intracellular Ca2+ and Mg2+ were assessed using fluorescence microscopy and flow cytometry respectively. EGFR-TRPM7 interaction was explored by proximity ligation assay (PLA) and immunoprecipitation. VSMC migration and proliferation were assessed by wound-healing assay and CFSE proliferation studies. Results: In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), p < 0.05 vs control; effects abolished by gefitinib and PP2. EGF-induced Mg2+ and Ca2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg2+) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) in rVSMCs 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, primarily in the cell membrane. VSMCs from TRPM7+/-kinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7+/-kinase showed reduced wall thickness. Conclusions: Our findings demonstrate important cross talk between EGFR and TRPM7. EGF/EGFR is both upstream and downstream of TRPM7. This interaction, which involves c-Src, regulates VSMC migration and proliferation. We identify a novel EGFR:TRPM7 pathway that may play an important role in vascular function and remodeling.