Slingshot phosphatase regulation of cofilin activation during vascular smooth muscle cell migration and neointima formation following vascular injury.

摘要

Vascular smooth muscle cell (VSMC) migration is a vital component of neointima formation following vascular injury. Rearrangement of the actin cytoskeleton is required for cell migration. Cofilin, an actin binding protein, mediates actin filament disassembly, replenishing actin monomers for polymerization at the leading edge of migrating cells. Cofilin is activated upon dephosphorylation of a conserved N-terminal serine by the slingshot (SSH) family of phosphatases (SSH1, SSH2 and SSH3), but the importance of the differential roles of each specific SSH isoform in cofilin activation and VSMC migration is unknown. Our preliminary findings demonstrated that cofilin is critical for VSMC migration and that the three SSH isoforms are expressed in both cultured VSMCs and carotid artery tissue. Thus, we hypothesized that the SSH isoforms may differentially control cofilin activation during VSMC migration and that this regulation is important during neointimal formation. The first aim defined the role of each of the SSH isoforms in their regulation of cofilin dephosphorylation and PDGF-induced migration in cultured VSMCs using small interference RNA (siRNA) to specifically target each of the SSH isoforms. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, while SSH3 siRNA had no effect. Only SSH1 siRNA significantly attenuated VSMC migration in response to wound healing and PDGF-induced migration, establishing a crucial role for SSH1-mediated cofilin activation in VSMC migration. The second aim evaluated the expression of isoform specific SSH-mediated regulation of cofilin during neointima formation. To test this, rat carotid arteries were injured with a Fogarty 2F catheter and assessed at 4, 7 and 14 days post-injury. Immunoblot analysis revealed an increase in cofilin and SSH1 expression at 14 days post-injury (vs. non-injured), and immunohistochemistry (IHC) staining of injured arteries localized cofilin and SSH1 expression in the neointima. Taken together, these studies demonstrate that SSH1-dependent regulation of cofilin is necessary for VSMC migration and may potentially contribute to neointima formation following vascular injury in vivo.