Encephalitogenic Myelin Proteolipid Fragment

(E) ECs plated on collagen (top panels) or fibronectin (bottom panels) were stimulated with vehicle control (Control) or O-Me-cAMP (O-Me) and stained with antibody to 5 or v

(E) ECs plated on collagen (top panels) or fibronectin (bottom panels) were stimulated with vehicle control (Control) or O-Me-cAMP (O-Me) and stained with antibody to 5 or v. 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. Introduction Adherens junctions (AJs) at endothelial cell-cell contacts regulate the barrier properties of the endothelium by controlling the infiltration of plasma components and cells into the tissue. They undergo continuous remodeling in resting monolayers and in response to agents that alter permeability. These events are primarily coordinated by vascular endothelial (VE) cadherin and its associated cytoplasmic proteins, cytoskeletal-based contractile forces, and small GTPases.1 Endothelial integrins promote cell adhesion, spreading, migration, and survival, and, in concert with AJs, also contribute to barrier integrity.2,3 Although well known to bind at the cell-matrix RIP2 kinase inhibitor 2 interface, integrins also localize to endothelial junctions, where they may regulate barrier properties.4 cAMP is a well-known secondary messenger that enhances barrier properties, and its principal target is protein kinase A (PKA), which increases barrier function by reducing actomyosin contractility.2 PKA interacts with A-kinase anchoring proteins (AKAPs), a family of scaffolding proteins that reside in certain subcellular sites to spatially and temporally compartmentalize cAMP signaling.5 In addition, cAMP activates exchange protein directly activated by cAMP (Epac) proteins, which are guanine exchange factors for Ras-related protein 1 (Rap) GTPases that, in limited cases, transduce their signals by interacting with AKAP complexes.6,7 Epacs regulate several cellular functions, ranging from cell-cell and cell-matrix interactions, exocytosis, and cellular Ca2+ handling to gene expression.8 In endothelial cells (ECs), Epac1 activation enhances barrier function by increasing RIP2 kinase inhibitor 2 VE-cadherin adhesion and cortical actin, and opposes the effects of edemagenic agents and Rho GTPase activation.8 Recent work suggests that Epac interacts with microtubules (MTs) and the microtubule binding protein MAP1A,9,10 and enhances microtubule growth in ECs.11 Many aspects of cell-cell and cell-matrix adhesion require reorganization of actin and MTs at cortical sites. In contrast to the well-described relationship between cadherins and integrins with the actin cytoskeleton, the role of MTs in regulating these complexes is only beginning to be elucidated.12 MTs are highly dynamic structures. Commonly, the minus ends of MTs anchor at RIP2 kinase inhibitor 2 the centrosome and Golgi, while the plus ends establish transient interactions with sites of cell-to-cell and focal adhesions. This facilitates the delivery of cargo to maintain a gradient of AJ components and induces the turnover RIP2 kinase inhibitor 2 of focal adhesions. Microtubule dynamics, microtubule linkage to actin, and their capture at cortical sites are regulated by plus-end-binding proteins (+TIPs) such as EB1, CLIP-170, and CLASPs, which transiently bind to the plus ends of growing MTs.13 There is evidence that AKAP9 participates in microtubule remodeling. AKAP9 exists as both long isoforms and a short isoform called Yotiao.14 The long isoforms (350-450 kDa) localize to the centrosome and Golgi in interphase cells and promote microtubule regrowth,15 and recent studies have shown that they confer microtubule nucleating activity at the Golgi.16 However, the contribution of AKAP9 to the regulation of microtubule dynamics is not well understood, and the biological role of these large isoforms in cellular responses remains largely unexplored. We tested the hypothesis that AKAP9 and Epac1 interact functionally to enhance the barrier properties of the endothelium through effects on microtubule dynamics. Methods Antibodies and reagents Rabbit anti-AKAP917 was a gift from Drs Lei Chen and Robert Kass (Columbia University, New York, NY); anti-dynein light chain18 was a gift from Kerry S. Campbell (Fox Chase Cancer Center, Philadelphia, PA); and anti-Glu tubulin19 Rabbit Polyclonal to EPHA3 was a gift from Dr G. G. Gunderson (Columbia University). Antibodies obtained from commercial sources were: VE-cadherin (Beckman Coulter); EB1 and GM130 (BD Biosciences); -tubulin (Abcam); integrin V3 (CD51/61) and 51 (CD94e; Chemicon); Yotiao (Invitrogen); platelet-endothelial cell adhesion molecule-1 (PECAM-1), -catenin, and p-120 (BD Biosciences); Rap1 (Santa Cruz Biotechnology); flag (mouse monoclonal, clone M2), -tubulin, V5, and -actin (Sigma-Aldrich); pericentrin (Covance); and Epac1 (Cell Signaling Technology). The reagents fibronectin, collagen type IV, phalloidin, nocodazole, RGD (Arg-Gly-Asp), RGE (Arg-Gly-Glu) peptide, and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich; hVE-Cadherin-Fc was from R&D Systems; sphingosine-1-phosphate (S1P) was from Calbiochem; and 8-pCPT-2for 30 minutes at 4C. Cleared lysates were incubated for 20 minutes.