Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle α_2C-adrenoceptors

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of α_2C-adrenoceptors (α_2C-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of α_2C-ARs from the trans Golgi to the cell surface. The expression of α_2C-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 μM), the selective EPAC activator, 8-pCPT-2′-O-Me-cyclic AMP (CMC; 100 μM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH₂-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 μM). Rap1CA increased the activity of an α_2C-AR promoter-reporter construct, which was inhibited by SP600125 (3 μM) or by the mutation of an AP-1 binding site in the α_2C-AR promoter. Furthermore, forskolin (10 μM) or CMC (100 μM) increased the expression of the α_2C-AR protein, and these effects were inhibited by SP600125 (3 μM). Therefore, cyclic AMP increases the expression of α_2C- ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the α_2C-AR gene. By increasing the expression of cold-responsive α_2C-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.