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

Abstract

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of alpha(2C)-ARs from the transGolgi to the cell surface. The expression of alpha(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 muM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 microM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH(2)-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 microM). Rap1CA increased the activity of an alpha(2C)-AR promoter-reporter construct, which was inhibited by SP600125 (3 microM) or by the mutation of an AP-1 binding site in the alpha(2C)-AR promoter. Furthermore, forskolin (10 microM) or CMC (100 microM) increased the expression of the alpha(2C)-AR protein, and these effects were inhibited by SP600125 (3 microM). Therefore, cyclic AMP increases the expression of alpha(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 alpha(2C)-AR gene. By increasing the expression of cold-responsive alpha(2C)-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

“…Amaxa nucleofections (Amaxa, Gaithersburg, MD) were performed for delivery of plasmid DNA to human microVSM as previously described (12,19). Transfected cells were subsequently washed once with serum-depleted medium (0.5% Hams), quiesced in 0.5% Ham's for 48 -72 h, followed by solvent (DMSO, H 2O), forskolin (10 M), or 8-pCPT-2=-O-Me-cAMP (100 M) treatments for indicated times.…”

“…Visualization of proteins of interest was performed as previously described (12,19). Samples were separated on 10% SDS-PAGE for ␣ 2C-AR or 8% SDS-PAGE for filamin.…”

Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice

“…Endothelin stimulates ERK activation and subsequent AP-1 transcriptional activity (41). Cold-induced vasoconstriction is initiated by increased expression of cutaneous smooth muscle α 2C -adrenoceptors (α 2C -Ars), which is mediated through a cyclic AMP/Rap1/JNK/AP-1 signaling pathway (42). For the future, it remains to be demonstrated whether induction of vasoconstriction for both events also involves transcriptional induction of Myl9 via Junb.…”

The Epac1 Signaling Pathway Regulates Cl− Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

“…In most cell types, RhoA acts as an upstream signaling molecule to activate ROCK, which in turn is involved in the translocation of protein to the cell surface (47,48). A similar signaling module has been described in microvascular smooth muscle cells (49). In line with previously described ROCK functional data, we observed a significant reduction in colocalization of KCNN4c with WGA in T84WT cells pretreated with ROCK inhibitor, whereas 8-pCPT-2Ј-OMe-cAMP induced this colocalization to the apical surface in the absence of this inhibitor.…”