Forkhead Transcription Factors Inhibit Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

Md Ruhul Abid,Kiichiro Yano,Shaodong Guo,Virendra I Patel,Gautam Shrikhande,Katherine C Spokes,Christiane Ferran,William C Aird

doi:10.1074/jbc.m502149200

Abstract

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy. Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead proteins may play a role in VSMC biology. Under in vitro conditions, platelet-derived growth factor (PDGF)-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 stimulated phosphorylation of FoxO in human coronary artery smooth muscle cells via MEK1/2 and/or phosphatidylinositol 3-kinase-dependent signaling pathways. PDGF-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 treatment resulted in the nuclear exclusion of FoxO, whereas PDGF-BB alone down-regulated the FoxO target gene, p27(kip1), and enhanced cell survival and progression through the cell cycle. These effects were abrogated by overexpression of a constitutively active, phosphorylation-resistant mutant of the FoxO family member, TM-FKHRL1. The anti-proliferative effect of TM-FKHRL1 was partially reversed by small interfering RNA against p27(kip1). In a rat balloon carotid arterial injury model, adenovirus-mediated gene transfer of FKHRL1 caused an increase in the expression of p27(kip1) in the VSMC and inhibition of neointimal hyperplasia. These data suggest that FoxO activity inhibits VSMC proliferation and activation and that this signaling axis may represent a therapeutic target in vasculopathic disease states.

Highlights

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy
platelet-derived growth factor (PDGF)-BB, tumor necrosis factor (TNF)-␣, and insulin-like growth factor (IGF)-1 Induce Phosphorylation of FoxO Proteins in coronary artery smooth muscle cells (CASMC)—To determine whether PDGF-BB, TNF-␣, and IGF-1 modulate the phosphorylation of forkhead proteins in VSMC, we carried out Western blot analyses of CASMC treated in the absence or presence of the cytokine/ growth factor
CASMC transduced with adenoviruses expressing either ␤-galactosidase (Adv), TM-FKHRL1, or WT-FKHRL1 were analyzed for percentile distribution of G0/G1 (B), S (C), and G2/M (D) cell cycle phases

Summary

EXPERIMENTAL PROCEDURES

Materials—PD98059, LY294002, and wortmannin were purchased from Calbiochem. Recombinant human PDGF-BB, TNF-␣, and IGF-1 were purchased from R&D Systems (Minneapolis, MN). After three washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature. The cells were washed once with and incubated in Opti-MEM I (without phenol red) for 30 min and transduced at a multiplicity of infection of 20 in SmGM medium for 12 h, grown in fresh medium for another 10 h, and serum-starved in SmBM for 16 –72 h as indicated. The cells were incubated with anti-FKHR, anti-FKHRL1, or anti-HA antibody (1:100) in 200 ␮l of blocking buffer for 1 h at room temperature, followed by fluorescein isothiocyanate-conjugated secondary anti-rabbit IgG (1:100) in 200 ␮l of blocking buffer for 1 h. The cells were incubated with DAPI-containing mounting medium Vectashield (Vector Laboratories, Burlingame, CA) for 10 min at room temperature, and representative images were captured with a Nikon Eclipse E800 microscope and a Spot digital camera. Trypan blue exclusion method was used to count the number of viable cells where indicated (55)

Forkhead Signaling in Vascular Smooth Muscle Cells

RESULTS

DISCUSSION