PKA and Epac synergistically inhibit smooth muscle cell proliferation.
Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2'-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2'-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.
Hewer
Richard C
RC
Bristol Heart Institute, University of Bristol, Bristol, UK.
Sala-Newby
Graciela B
GB
Wu
Yih-Jer
YJ
Newby
Andrew C
AC
Bond
Mark
M
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects*; D051419:Paxillin / Q000378:metabolism; D010590:Phalloidine / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D051419:Paxillin; D034741:RNA, Small Interfering; D010590:Phalloidine; D004917:Erythromycin; C057513:erythromycin propionate-N-acetylcysteinate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins; D000111:Acetylcysteine