Abstract

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. We recently demonstrated that cAMP-sensitive PKA and Epac signalling pathways synergise to inhibit VSMC proliferation but the underlying mechanisms are not understood.

Using PKA and Epac-selective agonists and microarray analysis we tested the hypothesis that PKA and Epac block VSMC proliferation via coordinate regulation of immediate early genes required for cell-cycle progression. Epac agonist significantly enhanced PKA-dependent inhibition of EGR1 mRNA (4.6±0.38 fold for PKA vs 13.8±1.09 fold for PKA+EPAC, p<0.05) and protein expression (1.9±0.18 fold for PKA vs 7.1±0.8 fold for PKA+EPAC, p<0.05). This inhibition was replicated by elevating endogenous cAMP with forskolin (4.8±1.9 fold, p<0.05), adenosine (3.4±0.7, p<0.05), A2B receptor agonist BAY60-6583 (4±1.2 fold, p<0.05) or Cicaprost (3.7±0.5 fold, p<0.05). Importantly, dominant-negative EGR1 inhibited VSMC proliferation.

cAMP-dependent inhibition (3.9±0.8 fold, p<0.05) of EGR1 transcription (reporter gene activity) was associated with a potent stimulation of CREB activity (225±72 fold, p<0.05). However, expression of dominant-negative CREB did not prevent cAMP-dependent inhibition of EGR1 expression. cAMP-mediated EGR1 repression was also associated with MAPK inhibition. However, inhibition of EGR1 transcription preceded MAPK inhibition. cAMP retains the ability to inhibit EGR1 expression in MAPK-blocked cells, demonstrating MAPK and CREB independent inhibition of Egr1 expression by cAMP. Inhibition of EGR1 transcription was associated with a significant (to 21.4±6.4%, p<0.05) inhibition of SRF activity, suggesting a role for SRF inhibition in cAMP-mediated EGR1 repression and inhibition of proliferation.

In summary, our results demonstrate that PKA and Epac synergise to inhibit VSMC proliferation by rapidly inhibiting EGR1 expression via a mechanism that probably involves inhibition of SRF activity. This also provides a mechanistic explanation for the CREB and MAPK-independent anti-mitogenic effects of cAMP in VSMC.