At one time the biological effects of cAMP were thought
At one time, the biological effects of cAMP were thought to be mediated exclusively by Protein Kinase A (PKA). Consistent with this, PKA inhibitors have been shown to reverse the effects of cAMP-elevating agents on VSMC proliferation . However, we recently reported that cAMP-mediated inhibition of Skp2 expression, a key regulator of G1-S phase progression, could only be partially reversed by a PKA inhibitor . Other studies demonstrated PKA-independent effects of cAMP analogues on cell-cycle regulatory proteins in other cell types , . These observations indicate the involvement of additional PKA-independent cAMP-sensitive pathways involved in cell-cycle regulation. The recent identification of a new family of cAMP-sensitive proteins, the Epac family (Epac 1 and 2) established the existence of a distinct PKA-independent signalling pathway , , . Epac 1 and 2 both contain N-terminal cAMP-binding domains and C-terminal guanine-nucleotide exchange factor (GEF) domains allowing them to function as cAMP-binding proteins with intrinsic GEF activity that can couple cAMP levels to the activation of members of the Ras-like family of GTPases such as Rap1 , . Numerous highly cell-type specific functions for Epac proteins have been proposed, including integrin-mediated cell adhesion, cell–cell junctions, migration and apoptosis , ,  as well as having both pro- and anti-proliferative properties , , depending on the cell type under investigation. However, the role of the Epac signalling pathway in cAMP-mediated inhibition of VSMC proliferation is not known.
Discussion The growth-inhibitory properties of cAMP in VSMC have been recognised for many years . Elevated cAMP levels arrest aa-dutp in G1 phase of the cell cycle, at least in part through inhibition of G1-regulatory proteins Cyclin D1 and Skp2 , . However, upstream signalling mechanisms underlying these anti-proliferative effects have remained elusive. Several studies demonstrated a clear role for PKA, at a time when this was the only known cAMP-sensitive protein. For example, PKA inhibition completely reversed cAMP inhibition of VSMC proliferation and injury-induced neointima formation in vivo and our results are consistent with this essential role of PKA in cAMP-mediated growth arrest. Our data now demonstrates that PKA activation is essential for cAMP-mediated growth arrest but alone is unable to induce these effects, indicating a requirement for an additional cAMP-sensitive pathway. The recent identification of the Epac family of cAMP-sensitive GEFs demonstrated the existence of a distinct signalling pathway directly activated by cAMP . Characterisation of Epac proteins in different cell types shows diverse and highly cell-type specific functions, reflecting the specific effects of cAMP in those cells. These include integrin-dependent adhesion, cell–cell junction formation, apoptosis, cardiac hypertrophy, cell differentiation, cytoskeleton rearrangements, gene expression and cell proliferation . However, the function of Epac signalling in cAMP-mediated growth arrest of VSMC remained unknown. We show that Epac1 but not Epac2 is expressed in VSMC together with the Epac-effector proteins Rap1A and Rap1B. Furthermore, we show that Rap1 is activated in response to forskolin or by an Epac-selective cAMP analogue, demonstrating that this pathway is present and functional in VSMC. The development of cAMP analogues, selective for PKA or Epac , has provided invaluable tools for the analysis of PKA and Epac signalling and we confirm their specificity in VSMC. Using these analogues, we show for the first time that activation of either pathway alone had little effect on proliferation, while dual activation potently suppressed it. This indicates that Epac plays a major role, in synergy with PKA, in mediating cAMP-dependent growth inhibition of VSMC. This novel function for Epac in VSMC was confirmed using adenovirus-mediated expression of a constitutively activated truncation of Epac. Activation of Epac in this way, independent of PKA stimulation, had no effect of cell proliferation. However, co-stimulation of PKA potently suppressed proliferation and Rb-hyperphosphorylation, again demonstrating a synergistic interaction between PKA and Epac signalling in mediating VSMC growth arrest. In order to further confirm the role of Epac in cAMP-mediated growth inhibition in VSMC, we employed siRNA-mediated gene silencing (data not shown). Interestingly, Epac siRNA failed to reverse forskolin-induced growth arrest despite efficient silencing of Epac protein expression. However, great care must be taken when interpreting data from Epac silencing experiments since Epac is known to control both cAMP-dependent and cAMP-independent signalling pathways . This considered, Epac silencing does not equate to inhibition of Epac activity. For example, Epac has recently been shown to control JNK signalling in a cAMP-independent manner  and consistent with this, we find that Epac silencing also inhibits JNK activity in VSMC (data not shown). These cAMP-independent functions of Epac complicate analysis of Epac silencing data and probably explain the inability of Epac silencing to reverse cAMP-mediated growth arrest. Future development of specific pharmacological Epac antagonists should facilitate this type of functional analysis of Epac and allow both in vitro and in vivo confirmation of the role of Epac in VSMC growth regulation.