Abstract 871: Dual Regulation of Actin Dynamics by Cofilin and SSH1L in PDGF-stimulated Vascular Smooth Muscle Cells
Platelet-derived growth factor (PDGF) plays a central role in vascular healing, in large part by stimulating smooth muscle cell (SMC) migration by a reactive oxygen species (ROS)-dependent mechanism. In atherosclerosis or restenosis, vascular dysfunction also involves migration of VSMC, which in turn requires rapid turnover of actin filaments. In other cell types, the ADF/cofilin family of actin binding proteins stimulates disassembly and severance of actin and seems to play an essential role in maintaining and protruding lamellipodia at the leading edge of migrating cells. Cofilin activity is negatively regulated by LIM kinases (LIMK) through phosphorylation at Ser-3 and is reactivated by phosphatases. We hypothesized that PDGF regulates cofilin activation in VSMC. In human aortic smooth muscle cells (HASMC), PDGF stimulated incorporation of non-muscle G-actin into the actin cytoskeleton. With regard to the signaling pathways responsible, we found that while PDGF induced LIMK activation at 10 min, cofilin was dephosphorylated between 5 and 120 min, with maximum at 30 min (0.61 ± 0.01 vs. 0.23 ± 0.06 relative densitometric units, p<0.01), in a process partially reversed by the flavoenzyme/NADPH oxidase inhibitor diphenylene iodonium (47.3% ± 14% inhibition, vs. PDGF stimulated, p=0.05), suggesting that PDGF simultaneously activates a cofilin phosphatase. To identify the responsible phosphatase, we first showed that the recently described Slingshot-1L (SSH1L) is expressed in HASMC. SSH1L levels are regulated negatively by confluence and positively by serum. Of importance, PDGF incubation (12 h) upregulated SSH1L mRNA (0.70 ± 0.05 x 105 vs. 1.6 ± 0.02 x 105 copies/108 18S, p<0.01) and acutely stimulated SSH1L activity (282.2 ± 94.5 vs. 496.0 ± 45.9 pmoles PO4/protein unit) after 30 min. Finally, the inhibition of SSH1L expression by siRNA completely abolished PDGF-induced cofilin dephosphorylation (115% ± 10% p=0.003 vs. scrambled siRNA). Taken together, our results suggest that PDGF participates in actin dynamics by ROS-dependent dual regulation of cofilin activity via LIMK and SSH1L.