Actin Polymerization In Differentiated Vascular Smooth Muscle Cells Requires Vasodilator-Stimulated Phosphoprotein (VASP)

Abstract

Our group has shown that alpha agonists and phorbol esters increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that actin polymerization is linked to contractility. However, the underlying mechanisms are still largely unknown. Inhibition of actin filament elongation by cytochalasin-D treatment decreases contractility without changing the level of myosin light chain phosphorylation in this tissue, suggesting that actin filament elongation processes are necessary for smooth muscle contraction. The enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is associated with actin filament elongation in non-muscle systems. In this study, we evaluated the possible functions of Ena/VASP in dVSMC. Among Ena/VASP proteins, only VASP is highly expressed in ferret aorta. High resolution 3-D deconvolved fluorescent images of immunostained freshly dissociated aorta cells show that VASP partially colocalizes with both alpha-actinin and vinculin, markers of dense bodies and dense plaques in dVSMC. Profilin, which is known to associate with monomeric G-actin and VASP to facilitate actin filament elongation also colocalizes with both alpha-actinin and vinculin, potentially identifying both the dense bodies and the dense plaques as hot spots of actin polymerization. Differential centrifugation and imaging data indicate that VASP may undergo subtle conformational or/and positional changes in response to stimuli. The EVH1 domain of VASP is known to be responsible for targeting VASP to its sites of action. Introduction of an expressed EVH1 domain of Ena/VASP, made as a chimeric protein with the TAT transduction tag, acted as a decoy to inhibit stimulus-induced increases in actin polymerization. In contrast, introduction of the EVH1 mutant F78S, which does not bind target poly-Pro sequences, had no effect. Thus, VASP may be involved in actin filament assembly at dense bodies and dense plaques in dVSMC. Support: NIH P01 HL86655.