Abstract 14512: MARCKS Regulates Small GTPase Rac1 and Cdc42 Activity and is Indispensable for PDGF Induced Vascular Smooth Muscle Cell Migration
Background: Vascular smooth muscle cell (VSMC) migration is a key event in the development of intimal hyperplasia, which limits the durability of all revascularization procedures. The myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin associated protein and major substrate of protein kinase C. MARCKS mRNA expression is increased in animal models of intimal hyperplasia. We hypothesize that MARCKS potentiates VSMC migration through regulation of the actin cytoskeleton.
Methods and Results: Human coronary artery smooth muscle cells (CASMC) were transfected with siRNA targeting MARCKS. VSMC migration as determined by the wound healing assay was significantly attenuated (75% reduction, p<0.05) after MARCKS knockdown. Cell morphology was examined with immunofluorescent staining of cortactin followed by laser confocal imaging. Active actin polymerization along the dorsal ruffles and lamellipodia was found much less prominent in the MARCKS knockdown cells, and the majority of cortactin failed to be recruited to the cell peripheral areas. The small GTPase family members Rac1 and Cdc42 are known to be the key regulators of actin dynamics controlling the dorsal ruffle and lamellipodia formation. GTPase pull-down assay demonstrated that Rac1 and Cdc42 failed to be activated after MARCKS knockdown. Additionally, the substrates of Rac1 and Cdc42, PAK1 and ERK1/2, were not phosphorylated in response to PDGF stimulation in MARCKS knockdown cells. The actin-binding domain of MARCKS is also known to bind the phospholipid PIP2; PIP2 is a key upstream factor implicated in Rac1 and Cdc42 activation. The proportion of membrane associated PIP2 was determined using live cell imaging (Zeiss 510 LCMS). Membrane bound PIP2 was significantly decreased (50% reduction, p<0.001) after MARCKS knockdown.
Conclusions: MARCKS knockdown attenuates VSMC migration. MARCKS knockdown prevents the activation of the small GTPases Rac1 and Cdc42 through decreasing the membrane associated PIP2, in turn preventing the phosphorylation of their substrates PAK1 and ERK 1/2. MARCKS is a potential target for therapies to prevent VSMC migration and the formation of intimal hyperplasia.
- © 2012 by American Heart Association, Inc.