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  • Co operation or synergy between

    2019-07-11

    Co-operation or synergy between PKA and Epac has been recently reported in PCCL3 thyroid cell line in which cAMP is pro-mitogenic, in Kifunensine to VSMC [19]. Our study demonstrates for the first time that PKA and Epac also synergise to inhibit cell proliferation in a cell type where cAMP in anti-mitogenic. However, other studies have indicated both pro- [36] and anti-proliferative [20] properties of Epac signalling independent of PKA, highlighting the complex and highly cell-type specific nature of this signalling network. There is evidence that the growth-inhibitory effect of cAMP in VSMC is dependent on the prolonged rather than transient PKA activation [28]. However, our data demonstrates that stimulation with a PKA-selective cAMP analogue fails to induce growth arrest, despite inducing robust PKA activation that persists for at least 16h and is actually greater than that induced by forskolin at later time points. Furthermore, we show that Epac activation did not further increase PKA activity. This data indicates that synergy between PKA and Epac signalling does not occur at the level of PKA activation. Several studies have demonstrated a role for Epac signalling in the activation of Rap1, with an additional role for PKA signalling [12], [37], [38]. This suggests that Rap1 may act as a common target for PKA and Epac in some cell types, raising the possibility of synergy between PKA and Epac acting at the level of Rap1 activation. However, our data demonstrates that PKA signalling did not enhance Epac-dependent Rap1 activation in VSMC but instead decreased it, indicating that PKA actually antagonised Epac-dependent Rap1 activation. Rap1 activation is therefore an unlikely target for the synergy between these two pathways. In accordance with this, specific and complete inhibition of Rap1 activity with Rap1GAP did not reverse the effects of cAMP-elevation on cell proliferation, demonstrating that cAMP-dependent growth inhibition is Rap1-independent in VSMC. Rap1-independence was further confirmed using siRNA-mediated silencing of Rap1 A/B. This clearly implicates a role for other currently unidentified Epac-effector proteins in cAMP-dependent growth arrest. Although many studies demonstrate a role for Rap1 signalling in mediating the effects of Epac activation in other cell types [18], several studies have also described Rap1-independent effects of Epac, similar to those described in this study [33], [39]. Future research, beyond the scope of the current study, should focus on identifying and characterising other Epac-effector proteins to advance our understanding of the mechanisms underlying cAMP-dependent signalling. Taken together, this data indicates that the synergy between PKA and Epac signalling must occur at a level further downstream and not at the level of PKA or Epac activity itself. Our data also clearly demonstrates that cAMP-induced stellate morphology in VSMC, with the associated loss of actin stress fibres and focal adhesions, is dependent upon activation of both PKA and Epac signalling suggesting that synergy may occur at the level of cytoskeleton organisation. Both PKA and Epac have been previously implicated in cytoskeleton regulation in other cell types by targeting numerous downstream proteins involved in the regulation of actin-polymerisation, including VASP, RhoA, Rac1 and Merlin [18], [40], [41], [42]. cAMP-dependent growth inhibition in VSMC is associated with this induction of stellate morphology and loss of cytoskeleton integrity suggesting a possible causative role. Numerous studies have demonstrated a critical requirement forcytoskeleton integrity and organisation for G1-S phase progression [34], [43], suggesting that the cytoskeleton may act as a nexus for PKA and Epac-dependent signals, ultimately controlling VSMC proliferation. It remains to be determined whether cytoskeleton remodelling represents the primary mechanism for cAMP-dependent growth arrest in VSMC. However, several studies have demonstrated that mitogenic signalling through the ERK1/2 pathway is dependent on cytoskeleton integrity. We investigated whether inhibition of members of the MAPK pathway could be a target for synergy between PKA and Epac signalling since they have been implicated in cAMP-mediated growth arrest in some cell types [24], [44], but not others [45], [46]. For example, Epac signalling has been reported to regulate JNK signalling in HEK-293T cells while elevated cAMP has also been shown to inhibit ERK activation [24], [33]. Whether cAMP inhibits VSMC proliferation via ERK1/2 in VSMC remains controversial [44], [45]. However, elevation of endogenous cAMP with forskolin in our experiments resulted in inhibition of both basal and serum stimulated ERK and JNK phosphorylation. CPT and BNZ stimulation synergised to inhibit ERK and JNK phosphorylation, indicating that coordinated signalling of PKA and Epac mediates inhibition of these MAPKs and may contribute to the anti-proliferative effects of cAMP in VSMC.