Abstract
Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC. Furthermore, elevated cAMP inhibited mitogen-induced nuclear-translocation of MKL1 and MKL2 in VSMCs but not ECs. Forskolin also significantly inhibited serum response factor (SRF)-dependent reporter gene (SRE-LUC) activity and mRNA expression of pro-proliferative and pro-migratory MKL1/2 target genes in VSMCs but not in ECs. In ECs, MKL1 was constitutively nuclear and MKL2 cytoplasmic, irrespective of mitogens or cAMP. Pharmacological or siRNA inhibition of MKL1 significantly inhibited the proliferation and migration of VSMC and EC. Our new data identifies and important contribution of MKL1/2 to explaining the strikingly different response of VSMCs and ECs to cAMP elevation. Elucidation of these pathways promises to identify targets for specific inhibition of VSMC migration and proliferation.
Highlights
Elevation of cAMP inhibits vascular smooth muscle cells (VSMCs) proliferation by downregulating multiple key cell-cycle intermediates[4, 14,15,16,17] but the upstream signalling mechanisms are incompletely characterised
In this study we investigated the regulation of the serum response factor (SRF) co-factors, Megakaryoblastic leukemia 1 (MKL1) and Megakaryoblastic leukaemia 2 (MKL2), by cAMP signalling in VSMCs and ECs and the role of this mechanism in regulation of cell proliferation and migration
We demonstrate that cAMP-induced actin-depolymerisation prevents mitogen-induced nuclear localisation of MKL1 and 2 in VSMCs and that this mechanism underlies, at least in part, the anti-mitogenic and anti-migratory effects of cAMP in these cells
Summary
Elevation of cAMP inhibits VSMC proliferation by downregulating multiple key cell-cycle intermediates[4, 14,15,16,17] but the upstream signalling mechanisms are incompletely characterised. Significant change in F- or G-actin (Fig. 2f) Taken together, these data demonstrate that elevated cAMP induces rapid loss of F-actin stress fibres, increased actin monomer levels and a condensed stellate morphology in VSMCs but not in ECs. Divergent effects of elevated cAMP on SRF-dependent gene expression in VSMCs and ECs.
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