The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Bartosz J Zieba,Mykhaylo V Artamonov,Li Jin,Ko Momotani,Ruoya Ho,Aaron S Franke,Ronald L Neppl,Andra S Stevenson,Alexander S Khromov,Magdalena Chrzanowska-Wodnicka,Avril V Somlyo

doi:10.1074/jbc.m110.205062

Abstract

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Highlights

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC20) phosphorylation and force
We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity
Our findings demonstrate the presence of a ␤-adrenergicand PGI2-activated cAMP-mediated activation of Epac

Summary

EXPERIMENTAL PROCEDURES

Tissue Screen and Western Blots—Buffers and Western blot analysis are described in the supplemental Experimental Procedures. Cells Culture and Cell Transfection—Rat aortic (R518), human bronchi, and mouse fundus SM cells were isolated and maintained as described in the supplemental Experimental Procedures. Mouse embryonic fibroblast cells were isolated from Rap1B knock-out mice [34] as described previously [35]. Rap and RhoA Activation Assays—The GTP loading status of RhoA and Rap in SM cells and tissues were assessed using Rhotekin and RalGDS assays, respectively [36, 37] as detailed in the supplemental Experimental Procedures. Expression of Smooth Muscle Marker Genes—Subconfluent R518 aortic cells were serum-starved for 24 h, treated with 50 ␮M 007 for 13 h, and lysed in TRIzol reagent (Invitrogen), and RNA was isolated following the manufacturer’s instructions. If the data proved highly non-Gaussian, a log transform (log(1 ϩ value)) was used to normalize the data before statistical analysis

RESULTS

Findings

DISCUSSION