Abstract 4849: The Dual Rho-GEF Kalirin Promotes Vascular Smooth Muscle Cell Migration and Proliferation
Vascular smooth muscle cell (SMC) migration and proliferation are regulated by signaling through the Rho family GTPases, RhoA and Rac, which are activated by GTP exchange factors (GEFs). One such Rho-GEF was recently associated with human atherosclerosis by genetic epidemiology: kalirin, a 340 kDa protein containing both Rac- and RhoA-GEF domains. We tested the hypothesis that SMC kalirin promotes Rac or RhoA signaling, and thereby atherogenic SMC activity. By immunoblotting (IB) and quantitative immunofluorescence microscopy, we found that kalirin is expressed abundantly in SMCs, and is up-regulated 1.6±0.2-fold (p<0.03) in the media of atherosclerotic, as compared with normal arteries. To test kalirin’s role in regulating SMC signaling, we compared 3 independent lines of SMCs from congenic WT and kalirin−/+ mice (with 45±5% of WT kalirin levels), as well as SMCs transfected with non-targeting or kalirin-targeting siRNA (43±9% protein knockdown). We assessed Rac and RhoA activation by G-LISA (Cytoskeleton, Inc.), and by IB for (respectively) phospho-Thr423-p21-activated kinase (activated Rac effector), and phospho-Ser695-MYPT1 of myosin light chain phosphatase (targeted only by RhoA-activated kinase). In response to FBS or PDGF, SMCs with kalirin deficiency ((−/+) or RNAi-mediated) showed reduction in the activation of Rac (by 50±10%, p<0.05), but not RhoA. Moreover, SMC migration toward FBS or PDGF (8–15-fold/basal, Transwell chambers) was reduced by 30 –50% in kalirin-deficient SMCs (p<0.05). FBS-induced proliferation (assessed by SMC counting) was also reduced in kalirin−/+ SMCs, by 20±5% (p<0.05), even though ERK activation was not altered. To test kalirin action on SMCs in vivo, we denuded the common carotid artery endothelium with a 0.36-mm wire, a procedure we have shown (by bone marrow transplant) produces neointimal hyperplasia comprising SMCs derived only from the arterial media. Neointimal area 4 wk after wire injury was 60±10% less (p<0.05) in kalirin−/+ (0.012±0.003 mm2) than in WT mice (0.03±0.01 mm2) matched for gender, age, and weight, even though medial area was equivalent. We conclude that kalirin is an important Rac-GEF in SMCs, and promotes SMC migration and proliferation in a manner that may prove pro-atherogenic.