Abstract 5682: Novel Role of Copper Transporters CTR1 and ATP7A in PDGF-induced Vascular Smooth Muscle Cell Migration and Vascular Remodeling

Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration primary through the PDGF receptor-beta (PDGFR), which contributes to vascular remodeling in vivo. Previous study shows that copper chelation inhibits neointimal formation in response to injury. However, underlying mechanisms remain unknown. Intracellular copper levels are tightly regulated by the copper importer CTR1 which localizes at plasma membrane (PM) and the copper exporter ATP7A which traffics between trans-Golgi network (TGN) to PM. Here we show that both CTR1 and ATP7A are expressed in cultured VSMC. Co-immunoprecipitation assay shows that ATP7A binds to PDGFR basally and PDGF stimulation rapidly promotes their association within 5 min, which is prevented by knockdown of CTR1 with siRNA (85%). Proteomic analysis in caveolae/lipid raft (C/LR) fraction reveals that ATP7A is localized in C/LR where PDGFR is found. PDGF stimulation promotes recruitment of ATP7A to C/LR fraction, which is inhibited by CTR1 siRNA (95%). Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) shows that C/LR contains high amount of copper (139.7 (C/LR) vs. 17.9 (non-C/LR) ppb/mg proteins). In response to wound injury or PDGF stimulation, ATP7A translocates from TGN to PM at the leading edge where it colocalizes with PDGFR and the LR marker cholera toxin subunit B in actively migrating VSMCs, which is prevented by CTR1 siRNA or C/LR disruptor methyl-beta cyclodextrin. Note that this ATP7A translocation is associated with decrease in copper levels in C/LR, suggesting increase in copper efflux in C/LR during active VSMC migration. Functionally, PDGF-induced VSMC migration is significantly inhibited by knockdown of CTR1 or ATP7A with siRNAs (84% and 68%, respectively) as well as treatment with cell permeable copper chelator TTM (50.2%) and cell impermeable copper chelator BCS (39.1%). Moreover, ATP7A expression is dramatically increased in the neointima in a mouse wire injury model. In summary, CTR1-dependent translocation of the copper exporter ATP7A to C/LR and its binding to PDGFR plays an important role in PDGF-induced VSMC migration, which may contributes to vascular remodeling in response to injury and development of atherosclerosis. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).