Bristol Heart Institute, University of Bristol, Bristol, UK.
J Mol Cell Cardiol. 2011 Jan;50(1):87-98. doi: 10.1016/j.yjmcc.2010.10.010. Epub 2010 Oct 30.
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.
环磷酸腺苷信号促进健康血管中的血管平滑肌细胞静息和血管损伤后的血管修复。环磷酸腺苷通过尚未完全了解的机制抑制血管平滑肌细胞增殖。我们研究了蛋白激酶 A (PKA) 和 Epac 信号在环磷酸腺苷诱导的血管平滑肌细胞增殖抑制中的作用。环磷酸腺苷介导的生长停滞依赖于 PKA。然而，用 6-苯甲酰-cAMP 选择性激活 PKA 并没有抑制血管平滑肌细胞的增殖，这表明需要其他途径。使用选择性环磷酸腺苷类似物 8-CPT-2'-O-Me-cAMP 激活 Epac 不会影响高度磷酸化的视网膜母细胞瘤 (Rb) 蛋白水平，Rb 蛋白是 G1-S 期转变的标志物，也不会影响 BrdU 掺入，尽管 Epac 效应物 Rap1 被激活。然而，6-苯甲酰-cAMP 和 8-CPT-2'-O-Me-cAMP 协同抑制 Rb 高度磷酸化和 BrdU 掺入，表明这两种途径都需要抑制生长。与此一致的是，组成型激活的 Epac 增加 Rap1 活性，并与 6-苯甲酰-cAMP 协同抑制血管平滑肌细胞增殖。PKA 和 Epac 协同抑制 ERK 和 JNK 的磷酸化。先前与环磷酸腺苷介导的生长停滞相关的星状形态的诱导也依赖于 PKA 和 Epac 的激活。用 Rap1GAP 或 siRNA 沉默抑制 Rap1 并不能否定 forskolin 诱导的 Rb 高度磷酸化、BrdU 掺入或星状形态的抑制。这一数据首次表明，Epac 通过一种独立于 Rap1 的机制与 PKA 协同作用，介导环磷酸腺苷诱导的血管平滑肌细胞生长停滞。这项工作突出了 Epac 在血管平滑肌细胞中环磷酸腺苷依赖性生长停滞中的主要作用。
