Copper transporter ATP7A interacts with IQGAP1, a Rac1 binding scaffolding protein: role in PDGF-induced VSMC migration and vascular remodeling

Abstract

Vascular smooth muscle cell (VSMC) migration contributes to neointimal formation after vascular injury. We previously demonstrated that copper (Cu) transporter ATP7A is involved in platelet-derived growth factor (PDGF)-induced VSMC migration in a Cu- and Rac1-dependent manner. The underlying mechanism is still unknown. Here we show that ATP7A interacts with IQGAP1, a Rac1 and receptor tyrosine kinase binding scaffolding proteins, which mediates PDGF-induced VSMC migration and vascular remodeling. In cultured rat aortic SMCs, PDGF stimulation rapidly promoted ATP7A association with IQGAP1 and Rac1 and their translocation to the lipid rafts and leading edge. Cotransfection assay revealed that ATP7A directly bound to NH₂-terminal domain of IQGAP1. Functionally, either ATP7A or IQGAP1 depletion using siRNA significantly inhibited PDGF-induced VSMC migration without additive effects, suggesting that IQGAP1 and ATP7A are in the same axis to promote migration. Furthermore, IQGAP1 siRNA blocked PDGF-induced ATP7A association with Rac1 as well as its translocation to leading edge, while PDGF-induced IQGAP1 translocation was not affected by ATP7A siRNA or Cu chelator. Overexpression of mutant IQGAP1 lacking a Rac1 binding site prevented PDGF-induced translocation of Rac1, but not ATP7A, to the leading edge, thereby inhibiting lamellipodia formation and VSMC migration. In vivo, ATP7A colocalized with IQGAP1 at neointimal VSMCs in a mice wire injury model, while neointimal formation and extracellular matrix deposition induced by vascular injury were inhibited in ATP7A mutant mice with reduced Cu transporter function. In summary, IQGAP1 functions as ATP7A and Rac1 binding scaffolding protein to organize PDGF-dependent ATP7A translocation to the lamellipodial leading edge, thereby promoting VSMC migration and vascular remodeling.

Keywords: copper transporter; migration; platelet-derived growth factor; vascular remodeling; vascular smooth muscle.

Fig. 1.

Platelet-derived growth factor (PDGF) stimulation promotes ATP7A binding to IQGAP1. A: PDGF stimulation promoted IQGAP1 association with ATP7A in a time dependent manner. Growth-arrested rat aortic smooth muscle cells (RASMCs) were stimulated with or without 50 ng/ml PDGF for indicated times (in minutes). Lysates were immunoprecipitated (IP) with anti-IQGAP1 antibody, followed by immunoblotting (IB) with anti-ATP7A and anti-IQGAP1 antibodies. Values represent means ± SD (n = 3) of 3 independent experiments. *P < 0.05 vs control cells. B: identification of domains of IQGAP1 necessary for binding to ATP7A. Schematic representation of the IQGAP1 constructs cloned into the pcDNA5/FRT expression vector and used for stable expression in this study. IQGAP1-C, IQGAP1-N, IQGAP1ΔCHD (ΔCHD), IQGAP1ΔWW (ΔWW), or IQGAP1ΔIQ (ΔIQ). Cell lysates from stably transfected CHO cells were IP with anti-ATP7A antibody, followed by IB with either anti-NH₂-terminal or COOH-terminal IQGAP1 antibodies (IQGAP1-N, IQGAP1-C). C, N, and NC indicate IQGAP1 COOH-terminus, NH₂ terminus, and negative control, respectively.

Fig. 2.

ATP7A and IQGAP1 are involved in vascular smooth muscle cell (VSMC) migration stimulated by platelet-derived growth factor (PDGF) or wound scratch. A: rat aortic smooth muscle cells (RASMCs) were transfected with ATP7A and/or IQGAP1 or control siRNA (30 nmol/l) for 48 h. Cell migration was assessed by the modified Boyden chamber assay after stimulation with or without 50 ng/ml PDGF for 8 h. B: wound-scratch assay was performed in confluent monolayers of RASMCs transfected with siRNA in the presence of PDGF (50 ng/ml). Images were captured immediately after rinsing at 0 h and at 8 h after the wounding in the cells. Bar graph represents averaged data, expressed as cell number per field. Values represent means ± SD (n = 3) for 3 independent experiments. *P < 0.05 vs control siRNA-treated cells stimulated with PDGF.

Fig. 3.

IQGAP1 is required for platelet-derived growth factor (PDGF)-induced recruitment of ATP7A to the leading edge in vascular smooth muscle cells (VSMCs). A–D: rat aortic smooth muscle cells (RASMCs) were transfected with ATP7A, IQGAP1, CTR1, or control siRNA for 48 h. For chelation of copper, RASMCs were treated with 200 µmol/l bathocuproine disulfonate (BCS) 48 h before treatment of PDGF. Growth-arrested RASMCs were stimulated with 50 ng/ml PDGF for 5 min (A and B). Confluent monolayer of RASMCs were scratched in the presence of 50 ng/ml PDGF for 18 h (C and D). Cells were costained with anti-ATP7A (green) and IQGAP1 (red) antibodies. Arrowheads point to the staining of leading edge. White small arrows indicate IQGAP1-expressed cells, while pink small arrows indicate to the IQGAP1 knockdown cells. Large arrows indicate to direction of migration. All fluorescence images were taken at 5 different fields/well and are representative of 3 different experiments.

Fig. 4.

Platelet-derived growth factor (PDGF) promotes Rac1 association with ATP7A in an IQGAP1-dependent manner. A: PDGF stimulation promotes IQGAP1 association with Rac1 in a time dependent manner. Growth-arrested rat aortic smooth muscle cells (RASMCs) were stimulated with or without 50 ng/ml PDGF for indicated times (minutes). B: RASMCs transfected with IQGAP1 siRNA or control siRNA for 48 h were stimulated with or without 50 ng/ml PDGF for 5 min. A and B: cell lysates were immunoprecipitated (IP) with anti-Rac1 antibody, followed by immunoblotting (IB) with anti-ATP7A, Rac1, or IQGAP1 antibodies. Values represent means ± SD (n = 3) of 3 independent experiments. *P < 0.05 vs control cells. C: RASMCs were transfected with IQGAP1 siRNA or control siRNA for 48 h. Growth-arrested RASMCs were stimulated with or without 50 ng/ml PDGF for 5 min. RASMCs were costained with anti-Rac1 (green) and IQGAP1 (red). Arrowheads point to the staining of leading edge.

Fig. 5.

IQGAP1 is required for platelet-derived growth factor (PDGF)-induced recruitment of ATP7A to the caveolae/lipid rafts in vascular smooth muscle cells (VSMCs). A: ATP7A and IQGAP1 are localized at caveolae/lipid rafts. Rat aortic smooth muscle cells (RASMCs) were fractionated by sucrose gradient centrifugation. Fractions from the top (fraction 1) to the bottom (fraction 13) were immunoblotted with antibodies as indicated. B: effect of IQGAP1 siRNA on PDGF-induced recruitment of ATP7A and Rac1 to caveolae/lipid rafts. RASMCs were transiently transfected with IQGAP1 siRNA or control siRNA for 48 h. Growth-arrested RASMCs were stimulated with 50 ng/ml PDGF for 5 min. Equal amounts of caveolae/lipid rafts fraction 4 and 5 were immunoblotted with antibodies as indicated. ATP7A, Rac1, IQGAP1, and PDGFR-β proteins semiquantified by normalizing to Cav1 protein. Values represent means ± SD (n = 3) of 3 independent experiments. *P < 0.05 vs control cells. C: for disruption of lipid rafts, RASMCs were treated with 10 mmol/l methyl-β-cyclodextrin 2 h before treatment of PDGF. Growth-arrested RASMCs were stimulated with 50 ng/ml PDGF for 5 min. Cells were costained with anti-ATP7A (green) and IQGAP1 (red) antibodies. Fluorescence images were taken at 5 different fields/well and are representative of 3 different experiments.

Fig. 6.

Rac1 binding site in IQGAP1 are required for platelet-derived growth factor (PDGF)-induced translocation of Rac1, but not ATP7A, to the leading edge, lamellipodia formation, and VSMC migration. A–D: rat aortic smooth muscle cells (RASMCs) were infected with Ad. IQGAP1 lacking binding site with Rac1 (Ad.IQGAP1ΔGRD) or Ad.null for 48 h. A and B: growth-arrested RASMCs were stimulated with 50 ng/ml PDGF for 5 min and were stained with either anti-ATP7A or Rac1 antibodies (green). Arrowheads point to the staining of leading edge. C: growth-arrested RASMCs were stimulated with 50 ng/ml PDGF for 5 min and stained with phalloidin to visualize lamellipodia. Cells with lamellipodia were expressed as percentage of total cell number. D: cell migration was assessed by the modified Boyden chamber assay after stimulation with or without 50 ng/ml PDGF for 8 h. Migrated cells were expressed as cell number per field. *P < 0.05 vs Ad.null-treated cells. Values represent means ± SD (n = 3) for 3 independent experiments.

Fig. 7.

ATP7A are involved in neointimal formation in response to vascular injury and colocalized with IQGAP1 in vivo. A: hematoxylin and eosin (H&E), Elastica van Gieson (EVG; delineates elastic laminae), or Masson trichrome staining of femoral arteries obtained from WT and ATP7A mutant mice at 3 wk after injury. B: quantitative morphometric analysis of vessel remodeling in WT and ATP7A mutant mice (means ± SE of three sections from each of six vessels). *P < 0.05 vs WT mice. C: ATP7A and IQGAP1 are highly expressed in neointimal VSMCs of wire-injured mouse femoral arteries. Immunofluorescence analysis for injured (3 wk after) femoral artery costained with anti-ATP7A (green) and IQGAP1 (red) antibodies.

Fig. 8.

Proposed model for role of IQGAP1 and ATP7A in platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) migration. PDGF promotes IQGAP1 translocation from the cytoplasm to the leading edge, thereby stimulating lamellipodia formation via recruiting ATP7A and Rac1, which in turn promotes directional VSMC migration involved in neointimal formation. ATP7A may transports copper to the secretory copper enzymes at the leading edge, which may promote extracellular matrix (ECM) remodeling and VSMC migration.