AKAP9 regulation of microtubule dynamics promotes Epac1-induced endothelial barrier properties.
ABSTRACT: Adhesive forces at endothelial cell-cell borders maintain vascular integrity. cAMP enhances barrier properties and controls cellular processes through protein kinase A bound to A-kinase anchoring proteins (AKAPs). It also activates exchange protein directly activated by cAMP (Epac1), an exchange factor for Ras-related protein 1 (Rap1) GTPases that promotes cadherin- and integrin-mediated adhesion through effects on the actin cytoskeleton. We demonstrate that AKAP9 facilitates the microtubule polymerization rate in endothelial cells, interacts with Epac1, and is required for Epac1-stimulated microtubule growth. AKAP9 is not required for maintaining barrier properties under steady-state conditions. Rather, it is essential when the cell is challenged to make new adhesive contacts, as is the case when Epac activation enhances barrier function through a mechanism that, surprisingly, requires integrin adhesion at cell-cell contacts. In the present study, defects in Epac-induced responses in AKAP9-silenced cells were evident despite an intact Epac-induced increase in Rap activation, cortical actin, and vascular endothelial-cadherin adhesion. We describe a pathway that integrates Epac-mediated signals with AKAP9-dependent microtubule dynamics to coordinate integrins at lateral borders.
Project description:Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.
Project description:<h4>Purpose</h4>To investigate whether Epac1 is key to Compound 49b's regulation of zonula occluden 1 (ZO-1) and occludin levels in human retinal endothelial cells (REC) and in an Epac1 vascular-specific conditional knockout mouse retina.<h4>Methods</h4>Primary REC were grown in normal (5 mM) or high glucose (25 mM). Some cells were treated with a novel ?-adrenergic receptor agonist, Compound 49b. Additional dishes were treated with Epac1 siRNA or Compound 49b + Epac1 siRNA. Protein levels of ZO-1, occludin, VEGF, and protein kinase C zeta (PKCz) were measured by Western blotting. Cell permeability was measured in REC grown in normal or high glucose and treated with Compound 49b, a specific Epac 1 agonist (8-CPT-2'-O-Me-cAMP), or VEGF. Epac1 floxed and cdh5-Cre mice were bred to generate Epac1 knockout mice in vascular endothelial cells. Immunofluorescence was done on retinal flatmounts from the floxed and Cre-Lox mice for occludin and ZO-1. Western blotting was also done for both proteins in whole retinal lysates from the mice.<h4>Results</h4>High glucose significantly reduced ZO-1 and occludin protein levels, which was associated with reduced cell adhesion. Compound 49b increased endothelial cell barrier protein levels through active Epac1. Knockout of Epac1 in vascular endothelial cells substantially reduced ZO-1 and occludin staining in retinal flatmounts, as well as protein levels.<h4>Conclusions</h4>Compound 49b increased ZO-1 and occludin protein levels, likely leading to decreased permeability.
Project description:Vascular endothelial (VE)-cadherin is a cell-cell adhesion molecule involved in endothelial barrier functions. Previously, we reported that cAMP-Epac-Rap1 signal enhances VE-cadherin-dependent cell adhesion. Here, we further scrutinized how cAMP-Epac-Rap1 pathway promotes stabilization of VE-cadherin at the cell-cell contacts. Forskolin induced circumferential actin bundling and accumulation of VE-cadherin fused with green fluorescence protein (VEC-GFP) on the bundled actin filaments. Fluorescence recovery after photobleaching (FRAP) analyses using VEC-GFP revealed that forskolin stabilizes VE-cadherin at cell-cell contacts. These effects of forskolin were mimicked by an activator for Epac but not by that for protein kinase A. Forskolin-induced both accumulation and stabilization of junctional VEC-GFP was impeded by latrunculin A. VE-cadherin, alpha-catenin, and beta-catenin were dispensable for forskolin-induced circumferential actin bundling, indicating that homophilic VE-cadherin association is not the trigger of actin bundling. Requirement of alpha- and beta-catenins for forskolin-induced stabilization of VE-cadherin on the actin bundles was confirmed by FRAP analyses using VEC-GFP mutants, supporting the classical model that alpha-catenin could potentially link the bundled actin to cadherin. Collectively, circumferential actin bundle formation and subsequent linkage between actin bundles and VE-cadherin through alpha- and beta-catenins are important for the stabilization of VE-cadherin at the cell-cell contacts in cAMP-Epac-Rap1 signal-activated cells.
Project description:Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.
Project description:Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host-pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.
Project description:Background and Aims:Activation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed. Methods:Endothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2'-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling. Results:Epac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3K?, PI3K?, and PI3K? but not PI3K? isoforms. The western blot data demonstrate Epac agonist activates PI3K? and PI3K? isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner. Conclusion:Our data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation. Chemical Compounds Used In This Study:8-pCPT-2'-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).
Project description:Evidence exists that cAMP stabilizes the endothelial barrier, in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties, similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (EPac/Rap 1) signaling enhanced transendothelial electrical resistance and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin 5. Ultrastructural analysis of the intercellular contact zone architecture documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contact sites displaying complex interdigitations of cell borders, in which membranes of neighboring cells were closely apposed over comparatively long distances; in addition, they were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP, whereas F/R-mediated Rac1 activation and barrier enhancement were not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement, and contact zone reorganization. These results indicate that, in microvascular endothelium, Rac1-mediated alterations in contact zone architecture contribute to cAMP-induced barrier stabilization.
Project description:Vascular endothelial (VE)-cadherin homophilic adhesion controls endothelial barrier permeability through assembly of adherens junctions (AJs). We observed that loss of VE-cadherin-mediated adhesion induced the activation of Src and phospholipase C (PLC)?2, which mediated Ca(2+) release from endoplasmic reticulum (ER) stores, resulting in activation of calcineurin (CaN), a Ca(2+)-dependent phosphatase. Downregulation of CaN activity induced phosphorylation of serine 162 in end binding (EB) protein 3. This phospho-switch was required to destabilize the EB3 dimer, suppress microtubule (MT) growth, and assemble AJs. The phospho-defective S162A EB3 mutant, in contrast, induced MT growth in confluent endothelial monolayers and disassembled AJs. Thus, VE-cadherin outside-in signaling regulates cytosolic Ca(2+) homeostasis and EB3 phosphorylation, which are required for assembly of AJs. These results identify a pivotal function of VE-cadherin homophilic interaction in modulating endothelial barrier through the tuning of MT dynamics.
Project description:Tight regulation of cadherin-mediated intercellular adhesions is critical to both tissue morphogenesis during development and tissue homeostasis in adults. Cell surface expression of the cadherin-catenin complex is often directly correlated with the level of adhesion, however, examples exist where cadherin appears to be inactive and cells are completely non-adhesive. The state of p120-catenin phosphorylation has been implicated in regulating the adhesive activity of E-cadherin but the mechanism is currently unclear. We have found that destabilization of the microtubule cytoskeleton, independent of microtubule plus-end dynamics, dephosphorylates p120-catenin and activates E-cadherin adhesion in Colo 205 cells. Through chemical screening, we have also identified several kinases as potential regulators of E-cadherin adhesive activity. Analysis of several p120-catenin phosphomutants suggests that gross dephosphorylation of p120-catenin rather than that of specific amino acids may trigger E-cadherin adhesion. Uncoupling p120-catenin binding to E-cadherin at the membrane causes constitutive adhesion in Colo 205 cells, further supporting an inhibitory role of phosphorylated p120-catenin on E-cadherin activity.
Project description:Alcohol intoxication can increase inflammation and worsen injury, yet the mechanisms involved are not clear. We investigated whether acute alcohol intoxication increases microvascular permeability and investigated potential signaling mechanisms in endothelial cells that may be involved.Conscious rats received a 2.5 g/kg alcohol bolus via gastric catheters to produce acute intoxication. Microvascular leakage of intravenously administered fluorescein isothiocyanate (FITC)-conjugated albumin (FITC-albumin) from the mesenteric microcirculation was assessed by intravital microscopy. Endothelial-specific mechanisms were studied using cultured endothelial cell monolayers. Transendothelial electrical resistance (TER) served as an index of barrier function, before and after treatment with alcohol or its metabolite acetaldehyde. Pharmacologic agents were used to test the roles of alcohol metabolism, oxidative stress, p38 mitogen-activated protein kinase (MAPK), myosin light-chain kinase (MLCK), rho kinase (ROCK), and exchange protein activated by cAMP (Epac). VE-cadherin localization was investigated to assess junctional integrity. Rac1 and RhoA activation was assessed by ELISA assays.Alcohol significantly increased FITC-albumin extravasation from the mesenteric microcirculation. Alcohol also significantly decreased TER and disrupted VE-cadherin organization at junctions. Acetaldehyde significantly decreased TER, but inhibition of alcohol dehydrogenase or application of a superoxide dismutase mimetic failed to prevent alcohol-induced decreases in TER. Inhibition of p38 MAPK, but not MLCK or ROCK, significantly attenuated the alcohol-induced barrier dysfunction. Alcohol rapidly decreased GTP-bound Rac1 but not RhoA during the drop in TER. Activation of Epac increased TER, but did not prevent alcohol from decreasing TER. However, activation of Epac after initiation of alcohol-induced barrier dysfunction quickly resolved TER to baseline levels.Our results suggest that alcohol intoxication increases microvascular permeability to plasma proteins. The data also suggest the endothelial-specific mechanism involves the p38 MAPK, Rac1, and reorganization of VE-cadherin at junctions. Last, activation of Epac can quickly resolve alcohol-induced endothelial barrier dysfunction.