cAMP is a well-known intracellular signaling molecule that is capable of restoring diminished endothelial barrier function. Previous reports suggested that cAMP-induced barrier enhancement occurs through PKA (27
). In this study, however, we demonstrated a novel PKA-independent signaling pathway, the cAMP-Epac-Rap1 signaling pathway, involved in cAMP-induced barrier function based on the following observations. PGI2- and FSK-reduced endothelial permeability was insensitive to H89. A specific activator for Epac, 8-CPT-2′-O-Me-cAMP, reduced both basal and thrombin-increased permeability. Plasma leakage in response to VEGF was also inhibited by 8-CPT-2′-O-Me-cAMP in vivo. We found that the activation of Rap1 leads to decreased permeability. Not only all cAMP-elevating bio-ligands we tested but also FSK, dbcAMP, and IBMX activated Rap1. Consistently, cAMP-dependent Rap1 activation upon stimulation by these ligands involved Epac in the regulation of barrier function. A previous report showed that Rap1 is phosphorylated by PKA in neutrophils and platelets, although the function of phosphorylated Rap1 has not been elucidated (37
). So far, Epac is known to regulate several biological functions including integrin-dependent cell adhesion, insulin secretion, and calcium release through ryanodine-sensitive Ca2+
channels (reviewed in reference 5
). In addition to these Epac-mediated functions, we show, for the first time, that Epac-Rap1 signaling is important for regulation of endothelial barrier function.
AJ assembly contributes to the regulation of barrier function. Rap1 is involved in the formation and maintenance of AJ constituted by cadherin (23
). Recently, it has been reported that homophilic ligation of E-cadherin induced Rap1 activation, which may be responsible for maturation of AJ (20
). Consistently, suppression of endogenous Rap1 inhibits formation of E-cadherin-dependent cell adhesion (36
), suggesting the critical role of Rap1 in the establishment of cadherin-based cell-cell contacts. Here, we demonstrate that Rap1 also acts downstream of cAMP-Epac to potentiate VE-cadherin-dependent cell adhesion, thereby improving barrier function. In addition to cAMP-elevating ligands, S1P, which enhances AJ formation and barrier function (18
), also activated Rap1 (our unpublished data). Thus, Rap1 may play a crucial role in barrier function induced by various types of barrier-improving factors.
Our data and previous studies show that cAMP protects thrombin-induced endothelial barrier dysfunction. cAMP does not limit the effect of thrombin on the initial loss of endothelial barrier (32
). Instead, cAMP enhances the restoration of barrier function disrupted by thrombin. Recently, it was also reported that Cdc42 regulates the restoration of endothelial barrier function disrupted by thrombin (24
). Thus, cAMP-Epac-Rap1 signaling may facilitate the formation of VE-cadherin-based cell-cell contacts, cooperatively or in parallel with Cdc42.
Rap1 enhances integrin-dependent cell adhesion in a variety of hematopoietic cells by modulating the affinity and avidity of integrin (6
). Cell adhesion to VEC-Fc-coated dishes was augmented by Rap1 activation, suggesting that the homophilic binding of VE-cadherin is also likely ascribed to the affinity and avidity of VE-cadherin modulated by Rap1-triggered inside out signaling. Hogan et al. reported that Rap1 activity is required for the targeting of E-cadherin molecules into nascent cell-cell contact sites, which in turn leads to the maturation of E-cadherin-based cell-cell contacts (20
). Thus, cAMP-Epac-Rap1 signaling may also regulate the recruitment of VE-cadherin into maturing cell-cell contacts. Since downstream signaling of Rap1 that increases homophilic binding of VE-cadherin has not yet been characterized, the effector of cAMP-Epac-Rap1 signaling will need to be identified.
The actin cytoskeleton is a critical determinant of vascular integrity (10
). PGI2, FSK, and 8-CPT-2′-O-Me-cAMP induced cortical actin rearrangement in a Rap1-dependent manner. FSK-induced VE-cadherin-dependent cell adhesion was inhibited by cytochalasin D. Thus, Rap1 may promote VE-cadherin-dependent cell adhesion by inducing cortical actin rearrangement. AF-6 may act downstream of Rap1 to regulate the actin cytoskeleton, since it binds to GTP-bound Rap1 and the actin cytoskeleton regulator, profilin, and is localized at AJ (2
). Consistently, Canoe, the drosophila homolog of AF-6, and Rap1 function in the same molecular pathway during embryonic dorsal closure, which requires cell-cell contacts (3
). S1P promotes endothelial barrier function by inducing Rac-dependent cortical actin rearrangement. S1P also induces Rap1 activation (our unpublished data). A previous report indicates that Rac can function downstream of Rap1 in the processing of the amyloid precursor protein (28
). Taken together, Rac may act downstream of Rap1 to induce cortical actin rearrangement.
In conclusion, we have demonstrated that the cAMP-Epac-Rap1 signaling pathway promotes VE-cadherin-mediated cell adhesion and consequently improves endothelial barrier function.