1. University of South Florida, Tampa FL, USA.
Background: Cyclic AMP influences many facets of signaling in vascular smooth muscle cells (VSMCs) including those mediated by protein kinase A (PKA) and those operating through exchange proteins activated by cAMP (EPACs). Activation of EPACs has been suggested to promote VSMC migration and to increase arterial neointima development; however, the molecular mechanisms of EPACs in VSMCs still remain unclear. The goal of this study was to identify mechanisms of action of EPACs related to vascular growth, and the hypothesis was that EPACs operate via distinct, kinase-mediated mechanisms to control VSMC migration and proliferation.
Methods and results: Using a wounding scrape injury on confluent rat primary VSMCs, the direct EPAC activator CPT transiently stimulated migration after 6 hours followed by inhibition of migration after 16 hours which persisted through 22 hours. This prolonged and stable inhibition of cell migration by EPAC was potentiated by pharmacologic blockade of MAPK, PI3K, Rac, PKC or PKG. In-Cell Western analysis on intact adherent cells was then used to identify differential phosphorylation of the actinregulatory protein vasodilator-activated serum phosphoprotein (VASP), as VASPSer157 and VASPser239 have been suggested as respective readouts of PKA and PKG and to have capacities to regulate cell growth. CPT (10μM) alone had no effect on either phosphoVASP species, but in the presence of a PKA inhibitor VASPser239 phosphorylation was significantly elevated. Involvement of focal adhesion proteins implicated in cell motility (focal adhesion kinase (FAK), paxillin (PAX)), were then assessed with/without EPAC stimulation. CPT increased PAX expression but failed to markedly alter FAKTyr397 phosophorylation. Finally, CPT alone had minimal effects on VSMC proliferation yet along with blockade of Rac, PI3K or MAPK or following direct activation of PKA proliferation was significantly decreased.
Conclusions: These data confirm that PKA and EPAC work cooperatively to primarily inhibit VSMC migration likely through mechanisms largely independent of VASP. Multiple kinases appear to be involved in this EPAC regulation of VSMC migration. Collectively these data suggest that EPAC regulates migration of VSMCs in time- and concentration-dependent manner, yet its precise mechanisms of action and its full influence on cellular proliferation remain to be fully characterized.
Keywords: CPT, cyclic AMP, EPAC, migration, MAPK, PI3K, PKA, PKC, PKG, proliferation, Rac, VASP