cAMP Signaling in Leukocyte Transendothelial Migration

【摘要】  The migration of leukocytes across the vascular endothelium is crucial for immunosurveillance as well as for inflammatory responses. Uncontrolled leukocyte transendothelial migration results in pathologies such as asthma, rheumatoid arthritis, and atherosclerosis. The molecular mechanisms that regulate leukocyte transendothelial migration involve signaling downstream of intracellular messengers such as cAMP, calcium, phosphoinositol lipids, or reactive oxygen species. Among these, cAMP is particularly intriguing because it is generated in both leukocytes and endothelial cells and regulates leukocyte chemotaxis as well as endothelial barrier function. In addition, physiological stimuli that induce cAMP production generate both pro- and antiinflammatory signals, underscoring the complexity of cAMP-driven signaling. This review discusses our current knowledge of the control of leukocyte transendothelial migration by two main cAMP effectors: protein kinase A and the Rap exchange factor Epac ( E xchange p rotein directly a ctivated by c AMP).

In this review we discuss the role of cAMP in the control of leukocyte transendothelial migration. Focusing on two major targets of cAMP, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), we provide an overview of the current knowledge on the regulation of leukocyte transendothelial migration by cAMP-driven signaling in both leukocytes and endothelium.

【关键词】  Epac PKA migration leukocytes endothelial cells

Introduction

A hallmark of chronic inflammation is the uncontrolled and excessive migration of leukocytes from the peripheral blood into the tissues. This leukocyte extravasation is tightly regulated by bidirectional signaling in both leukocytes and vascular endothelium. In the initial phase of the inflammatory response, locally produced proinflammatory cytokines trigger the activation of circulating white blood cells, followed by their reversible tethering and rolling over activated endothelium, a process controlled by selectins. Leukocytes subsequently firmly adhere to the endothelium through surface integrins and acquire a polarized shape. In the final phase of this process, leukocytes migrate across the vessel wall into the underlying tissues. This involves the regulated disassembly of endothelial junctional complexes to create GAPs to allow leukocyte passage.

The molecular mechanisms that control leukocyte transendothelial migration (TEM) involve extensive signaling mediated by intracellular messengers such as cAMP and calcium, as well as by phosphoinositol lipids, small GTPases, reactive oxygen species, and protein tyrosine kinases. All of this results in coordinated remodeling of the actin cytoskeleton, activation of integrins, and phosphorylation and transient inactivation of endothelial junctional proteins, allowing efficient TEM. 1

cAMP regulates a wide range of cellular processes, including differentiation, secretion, gene transcription, regulation of cell shape, cytoskeletal remodeling, proliferation, apoptosis, adhesion, and migration. 2 A large number of extracellular stimuli, including hormones, neurotransmitters, and growth factors, induce intracellular cAMP production upon binding to their cognate G protein-coupled receptors that trigger the activation of one of the several isoforms of adenylate cyclase. 3 In turn, phosphodiesterases (PDEs) degrade cAMP, preventing its diffusion in the cell, to ensure specific activation of nearby signaling complexes. 4 The different cellular responses to cAMP may be explained by (1) localized production and degradation of cAMP, regulated by adenylate cyclases and PDEs 3,4; (2) the interaction of the cAMP effector Protein Kinase A (PKA) with specific A kinase anchoring proteins (AKAPs) that control the subcellular localization of PKA signaling; 5 and (3) the existence of a variety of additional cAMP effectors such as Epac (Exchange protein directly activated by cAMP), PDZ-GEFs, and cyclic nucleotide-gated channels ( Figure 1 ). 6-8 Of the latter two, information on their cAMP selectivity, mode of activation, and role in cAMP signaling is currently limited.

Figure 1. cAMP effector proteins. Schematic representation of the domain structure of currently known cAMP effectors. The indicated domains are: cAMP, cAMP-binding domain; cNMP, cyclic nucleotide-binding domain; DEP, Dishevelled, Egl-10, Plekstrin domain; REM, Ras-exchanger motif; RA, Ras-association domain; AKAP, A-Kinase anchoring protein-binding domain; C, region of interaction with catalytic subunit; GEF, guanine nucleotide exchange factor; PDZ, PDZ domain; CaM, CaM-binding domain.

Since their discovery in the 1950s it has become clear that cAMP and the agonists that trigger its production are important immunomodulators. cAMP-generating stimuli, such as prostaglandins (PGEs), serotonin (5-HT), ß 2 -adrenergic agonists, and adenosine, are found at sites of inflammation, eg, in atherosclerotic lesions. 9,10 This has led to the identification of some cAMP-inducing stimuli and phosphodiesterases as successful therapeutic targets for the treatment of asthma, chronic obstructive pulmonary disorder (COPD), rheumatoid arthritis, atherosclerosis, and cancer. 11,12 However, recent clinical data have shown that currently available therapies are often not efficient enough or may even induce undesired effects. For instance, nonsteroidal antiinflammatory drugs (NSAIDs) that block production of prostaglandins and are commonly used in the treatment of inflammatory conditions were recently found to increase coronary events in certain circumstances. Similarly, therapeutic use of ß 2 -adrenergic agonists to reduce plasma leakage in chronic airway diseases is limited, because vascular endothelium becomes desensitized by these agents. 13

The limited effectiveness of therapies directed at components of cAMP signaling pathways might be attributable to the fact that physiological stimuli that induce cAMP production can exert both pro- and antiinflammatory effects. Most of them modulate common denominators of inflammatory disorders, such as vascular leakage and influx of inflammatory cells into injured tissues. One such agonist is 5-HT, which prevents vascular leakage and stimulates leukocyte chemotaxis. 14,15 Similarly, PGE2 reduces activation of macrophages by suppressing the expression of cytokines, including tumor necrosis factor (TNF)-, interleukin (IL)-12, and interferon (INF)-, and chemokines such as monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1ß, and IL-8. 16,17 Conversely, PGE2 increases the levels of circulating IL-6, an inflammatory cytokine. 18 It is unclear which factors control the pro- or antiinflammatory effects of cAMP, but these may include the duration and strength of the stimulus as well as the cell type involved, expressing a specific repertoire of cAMP-responsive effectors.

In this review we discuss the role of cAMP in the control of leukocyte transendothelial migration. Focusing on two major targets of cAMP, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), we provide an overview of the current knowledge on the regulation of leukocyte transendothelial migration by cAMP-driven signaling in both leukocytes and endothelium.

cAMP Effectors

The ubiquitously expressed and extensively studied serine/threonine kinase PKA is a heterotetramer, which combines two catalytic (C) with two regulatory (R) subunits. 19 Two PKA isozymes have been originally identified, PKA type I and type II, which differ in their regulatory subunits, RI and RII, respectively. Different isoforms of the regulatory subunit (RI, RIß, RII, RIIß) and of the catalytic subunit (C, Cß, C, PrKX) have been identified by molecular cloning. The regulatory subunits are differentially expressed and can form homo- and heterodimers, further increasing complexity but likely also specificity of cAMP signaling. 20 On binding of two cAMP molecules by each regulatory subunit, the inactive tetramer dissociates into one dimer of R subunits and two active catalytic subunits that phosphorylate various target proteins ( Figure 1 ).

Subcellular localization and compartmentalization of PKA is mainly determined by members of the functionally related, but structurally diverse, family of AKAPs. 5 These target PKA to specific substrates and various subcellular regions. In addition, AKAPs serve to colocalize PKA with phosphatases and PDEs. 5 Such a spatial regulation of PKA is essential to generate specific cellular responses to cAMP, including cell adhesion and migration.

Epac

The small GTPase Rap1 is activated by growth factors, adhesion molecules, and cytokines and has been implicated in the regulation of growth, secretion, integrin-mediated adhesion, neuronal differentiation, and morphogenesis. 21 Like most other small GTPases, Rap1 acts as a molecular switch, cycling between a GDP-bound inactive and a GTP-bound active state. This cycle is regulated by GTPase-activating proteins (GAPs), which enhance the hydrolysis of the bound GTP, and by guanine nucleotide-exchange factors (GEFs), which facilitate the release of the bound GDP and promote the binding of GTP. Epac is an exchange factor for Rap that is activated by cAMP. 6,7 By means of the Epac-selective cAMP analog 8-pCTP-2-O-Me-cAMP, 22 Epac was shown to regulate a variety of cellular processes previously attributed to PKA. These include E- and VE-cadherin-mediated cell-cell adhesion, integrin-mediated adhesion, monocyte chemotaxis, Ca ++ -induced exocytosis, and Fc -receptor-mediated phagocytosis. 1,14,21,23-26

The two Epac variants, Epac1 and Epac2, have a different domain structure ( Figure 1 ) and show tissue-specific expression. Epac1 is expressed in kidney, ovary, thyroid, and, at relatively low levels, in leukocytes. 6,14 Epac2 is predominantly expressed in the brain and in the adrenal gland. Both Epac1 and Epac2 contain a C-terminal catalytic region, which comprises a CDC25 homology domain responsible for the nucleotide exchange of Rap, a Ras-association domain (RA), and a Ras exchange motif (REM) necessary for the stability of the GEF domain. The N-terminal part of Epac is the inhibitory regulatory region (RR) and contains a DEP (Dishevelled, Egl, Plekstrin) domain, responsible for membrane localization, and a cAMP-binding domain that shares homology with the cAMP-binding domains of the PKA regulatory subunit. cAMP induces a conformational change of Epac, disrupting the intramolecular interaction between the N-terminal and the catalytic domain. 21,22 As a consequence, Epac becomes active and stimulates Rap activation. Epac2 contains a second, low affinity cAMP-binding domain which is not required for cAMP-induced activation of Epac2. Its function is, so far, elusive. 27

cAMP-Mediated Signaling in Leukocyte Adhesion

The first steps in leukocyte TEM, selectin-dependent rolling over the vascular wall and subsequent firm adhesion to the endothelial surface, are blocked by cAMP-elevating agents through inhibition of stimulus-induced L-selectin shedding and upregulation of the Mß2 integrin. 28,29 Likewise, elevation of cAMP levels also reduces fMLP-induced neutrophil adhesion to vascular endothelium by blocking the surface expression of Mß2 integrin. 30 Pharmacological inhibition of PKA reverses the cAMP-induced inhibition of 4ß1 and Mß2 integrin surface expression, indicating that PKA regulates adhesion through the modulation of integrin surface expression. 31,32 Interestingly, intracellular levels of cAMP decrease on neutrophil adhesion. This could be reversed by blocking antibody to the ß2-integrin. Thus, a feedback loop may exist in which a reduction of cAMP activates integrins, while activation of integrins leads to a further decrease of intracellular cAMP levels.

PKA may also downregulate integrin activation through modulation of the actin cytoskeleton. Rovere et al demonstrated that activation of PKA promotes T cell deadhesion by disassembly of the actin cytoskeleton, dissociating integrins ( Mß2) from cytoskeletal anchoring proteins. 33 In line with this, PKA was also found to inhibit the small GTPase RhoA, a critical regulator of actin-based contractility. 34 Stimulation with a membrane-permeable cAMP analogue resulted in reduced RhoA activation and inhibition of 4ß1-dependent adhesion of lymphocytes to vascular cell adhesion molecule (VCAM)-1 and of Mß2-dependent adhesion of neutrophils to fibrinogen. These effects were attenuated by selective PKA inhibitors. In conclusion, cAMP activation of PKA appears to negatively regulate leukocyte adhesion by regulating the shedding of L-selectin and the stimulus-induced upregulation and activation of integrins ( Figure 2 ).

Figure 2. Schematic overview of cAMP signaling in polarized leukocytes. cAMP-elevating agonists such as serotonin (5-HT), adenosine, or prostaglandins (PGE) bind to their cognate Gs-coupled receptors (GPCR), activating adenylate cyclase (AC) and the production of cAMP. cAMP activates PKA and Epac. A, Activation of PKA results in inhibition of cell surface expression of integrins, by blocking the mobilization of intracellular pools, resulting in reduced adhesion (1). Similarly, PKA regulates expression of selectins inhibiting rolling of leukocytes over the endothelium (2). PKA also modulates leukocyte adhesion through the regulation of integrin anchorage to the actin cytoskeleton, by inhibiting RhoA (3). Finally, PKA signaling inhibits integrin activation during initiation of adhesion; however, PKA is necessary for stable, sustained adhesion mediated by clustered integrins (4, 5). B, Epac1 is activated in the uropod and activates Rap1, which, together with its effector RAPL, translocates to the leading edge, locally activating integrins that mediate leukocyte adhesion and motility.

Epac1-Rap1

The situation appears quite different for the Epac1-Rap1 pathway. There is a large body of evidence corroborating the role of the small GTPase Rap1 in the stimulation of integrin-mediated adhesion. In lymphocytes, Rap1 stimulates Lß2-dependent adhesion. 35 Moreover, Rap1 increases the affinity of the IIbß3 integrin for fibrinogen in megakaryocytes, 36 and Rap1 is required for cell adhesion through Lß2 and 4ß1 integrins induced by integrin-activating antibodies and manganese ions in Jurkat cells. 37 Conversely, inhibition of Rap1 though expression of a RapGAP or of the dominant negative Rap1N17 inhibits the ability of Lß2 and 4ß1 integrins to bind their ligands. 38,39

A role for Epac1 in integrin-mediated adhesion was first reported in ovarian carcinoma cells, in which the Epac1-Rap1 pathway mediates 5ß1- and vß3-dependent adhesion to fibronectin. 23 Similarly, Epac1-Rap1 signaling mediates 3ß1-dependent adhesion of different types of adherent cells (eg, keratinocytes) to laminin. 40 Interestingly, recent studies suggest that Epac1 stimulates integrin-mediated adhesion of leukocytes. Basoni et al reported that TGF-ß induced a loss of the Epac1 transcript in U937 monocytic cells, which was paralleled by reduced activation of Rap1 and of Mß2. 41 We found that Epac1 activates ß1 integrins and promotes 4ß1 and 5ß1 (but not Mß2) integrin-mediated adhesion of U937 cells to fibronectin and promotes adhesion of primary monocytes to vascular endothelium under flow. 14 Finally, also in CD34 + hematopoietic progenitor cells, cAMP-mediated Rap1 activation results in increased 4ß1-mediated adhesion. 42 Together, these data indicate that Epac1-Rap1 signaling stimulates leukocyte adhesion, primarily through the activation of ß1 integrins ( Figure 2 ).

cAMP-Mediated Signaling in Leukocyte Directional Migration

cAMP or cAMP-generating agonists can exert opposite effects on leukocyte transendothelial migration. For example, PGE2 suppresses transendothelial migration of T lymphocytes and monocytes. 43,44 Similarly, stimulation of adenylate cyclase or inhibition of PDE attenuates chemotaxis of neutrophils and eosinophils. 45,46 These inhibitory effects of cAMP on leukocyte migration have been attributed to PKA, because competitive inhibitors of this enzyme reverse cAMP-induced inhibition of lymphocyte migration. 47,48

However, PKA was also shown to promote migration of leukocytes, which correlated with increased Mß2 cell-surface expression and adhesion, and with decreased fMLP-induced actin polymerization in neutrophils. 49,50 Stimulation of motility may occur through enhanced cell polarity as PKA inhibition was found to abrogate chemokine-induced lymphocyte polarization. 51 The observation that a gradient of PKA inhibitor stimulates neutrophil migration suggests that cAMP-induced leukocyte motility may require a polarized distribution of PKA activity within the cell. Several studies have shown that stimulation of neutrophils with chemotactic agents causes a small but consistent rise in cAMP, whereas the concentration of cAMP that inhibits chemotaxis is much higher. 52,53 Thus, PKA may exert inhibitory and stimulatory effects on cell migration, depending on the type of cAMP-inducing stimulus and the level and/or intracellular distribution of cAMP.

Epac1-Rap1

Constitutively active Rap1 induces lymphocyte polarization through its effector RAPL, independent of spatial cues such as adhesion or chemokine gradients. 54 GTP-bound active Rap1 localizes to the leading edge, whereas wild-type, GTP, and GDP-bound Rap1 is also present at a perinuclear area. 55,56 Similarly, chemokine stimulation of Rap1 is able to induce the translocation of RAPL from the perinuclear region to the leading edge, where it colocalizes with Lß2 integrin. In turn, targeting of Lß2 to the leading edge of the cell requires activation of Rap1 and RAPL. 57,58 This finding suggests that on activation, the Rap1-RAPL complex moves to the cells? leading edge and locally activates integrins, which initiates further cell polarization and migration.

In line with this, activation of Epac1 also induces monocyte polarization. Epac1 localizes to the perinuclear region of polarized migrating cells, which suggests that Epac1 activates Rap1 in a perinuclear area where activated Rap1 may associate with RAPL. 14,58 Expression of constitutively active Rap1 in T lymphocytes stimulates cell migration on immobilized intercellular adhesion molecule-1 (ICAM-1) and VCAM-1, even in the absence of chemokines. 54 Conversely, inhibition of Rap1 by RapGAP significantly blocked the ability of B lymphocytes to migrate toward SDF-1. 59 The role of the Rap1-RAPL complex in leukocyte migration was recently underscored in an in vivo study demonstrating that lymphocytes from RAPL-deficient mice showed impaired chemokine-stimulated transendothelial migration under flow and a lack of proper homing to lymphoid tissues. 57

In line with these findings, we showed that Rap1 activation by Epac1, downstream of the serotonin receptor, promotes SDF-1-induced migration of monocytic U937 cells. 14 Interestingly, Goichberg et al reported that also in CD34 + hematopoietic progenitor cells, cAMP-induced activation of Rap1 downstream of the PGE2 receptor results in increased transendothelial migration. Moreover, cAMP-induced Rap1 signaling promotes homing of CD34 + cells to the bone marrow. 42 Together, these data underscore the important stimulatory role of (localized) cAMP signaling in leukocyte adhesion and directional migration.

cAMP Signaling in Vascular Endothelium

The vascular endothelium forms a continuous monolayer and is a physical barrier to solutes, macromolecules, and leukocytes from blood to tissue. Inflammation is characterized by upregulation of adhesion molecules on the endothelium and by increased vascular permeability, induced by cytokines such as TNF-. 60 However, although increased permeability may facilitate leukocyte TEM, it is important to underscore the key role of leukocyte integrins and their ligands on the endothelium mediating strong adhesion and subsequent transmigration. 61,62

There is evidence for three different pathways by which leukocytes can cross the endothelium: transcytosis, which involves caveolin-mediated migration of leukocytes through the endothelial cell body, 63 transmigration at tricellular corners, 64 and paracellular transmigration through tight- and adherens junctions. 62,65 It is generally believed that the paracellular pathway is most commonly used, although cell-type specificity may play a role in choice of the mode of transmigration. 65,66 Particularly relevant in the context of paracellular leukocyte extravasation are the VE-cadherin-based adherens junctions ( Figure 3 ). VE-cadherin is a homophilic transmembrane adhesion molecule that associates to ß-or -catenin, which are dynamically connected to the actin cytoskeleton through -catenin. 67 Paracellular transmigration occurs through intercellular gaps between endothelial cells, which form upon the regulated and focal disassembly of VE-cadherin-mediated adherens junctions, induced by leukocyte adhesion-induced signaling. 1,65 Yet, leukocyte TEM is generally accepted not to compromise the endothelial barrier leading to increased permeability. 68 This is most likely because of tight contact between transmigrating cells and the endothelium, for instance through homotypic adhesion molecules such as CD31/PECAM1 or CD99, which are expressed on both cell types. 69,70

Figure 3. Control of endothelial cell-cell contact by PKA and Epac. cAMP-elevating agonists bind to Gs-coupled receptors, triggering the activation of adenylate cyclase (AC), production of cAMP, and the activation of PKA and Epac. A, PKA inhibits myosin-based contractility through (1) phosphorylation of myosin light chain kinase (MLCK), decreasing its activity; (2) inhibition of RhoA activity; and (3) through stabilization of microtubules (MT), thus reducing the activity of MT-associated RhoA-specific GEFs. In addition, PKA regulates Rac1 signaling (4) and phosphorylates filamin, affecting cortical actin organization (5). Finally, PKA stabilizes endothelial tight junctions (TJ) through phosphorylation of vasodilatator-stimulated phosphoprotein (VASP) (6). B, Potential effectors of Epac1 regulating endothelial integrity are: RIAM which binds to VASP (1); ARAP3, a GEF for RhoA (2); vinculin, supporting endothelial adherens junctions through association with -catenin (; 3); TIAM-1 and Vav2, GEFs for Rac1 (4) and AF-6 which associates with nectin and p120 catenin and reduce VE-cadherin (VE-cad) endocytosis, thus promoting cell-cell adhesion (5). ß indicates ß-catenin.

It is well established that increases in intracellular cAMP promote endothelial barrier integrity. Observations that cAMP-raising agents decrease endothelial permeability date back to the 1980s, when Stelzner et al showed that forskolin and cholera toxin, both being strong activators of adenylate cyclase, reduced the transfer of macromolecules, such as albumin, across endothelial monolayers. In addition, these investigators demonstrated, using the PKA antagonist Rp-cAMPS, that PKA mediated the increase in endothelial barrier function induced by cAMP. 71 The work of Stelzner et al initiated a series of studies in clustered cells, isolated microvessels, intact tissues, and organs on the protective effect of ß-adrenergic agonists and PGEs. These agents were found to decrease endothelial permeability induced by stimuli such as thrombin, neutrophil-derived hydrogen peroxide, and the inflammatory mediator bradykinin. 72,73

Subsequent studies implicated PKA in the protective effect of cAMP on endothelial barrier function, mainly based on the use of selective PKA inhibitors such as H89 and on expression of the PKA inhibitor (PKI) gene. 74,75 The mechanism by which PKA controls endothelial barrier function is not entirely clear, although a number of effectors have been identified that are all involved in the direct or indirect regulation of the actin cytoskeleton ( Figure 3 ). These will be briefly discussed below.

MLCK

Myosin-based contractility, critical for the maintenance of endothelial integrity, is positively regulated by myosin phosphorylation. Myosin light chain (MLC) phosphorylation is controlled by MLC kinase (MLCK) and MLC phosphatase (MLCP). 76 PKA phosphorylates MLCK, decreasing its affinity for calmodulin, which is essential for MLCK activation. 77 Moreover, PKA is also able to indirectly regulate MLCK through the phosphorylation and inhibition of phospholipase C. Consequently, Ca 2+ release is reduced and the formation of calmodulin complexes is inhibited. 78 Accordingly, forskolin as well as cAMP analogues partially inhibit the basal and thrombin-induced phosphorylation of MLC. 79 In addition, inhibition of PKA results in increased colocalization of MLCK with actin and enhanced thrombin-induced endothelial permeability. 80 Thus, PKA activation may exert its protective effect on the endothelial barrier through the inhibition of actomyosin-based contractility.

RhoGTPases

Activation of the small GTPase RhoA induces endothelial permeability through the stimulation of actin polymerization and actomyosin-driven contraction. PKA is able to phosphorylate RhoA and to inhibit RhoA activation in endothelial cells, thus counteracting the permeability-inducing contractile force. 1,76,81 Similar to RhoA, Rac1 is another GTPase which is important in the control of the actin cytoskeleton and endothelial barrier function. PKA has inhibitory and stimulatory effects on Rac1 activation in endothelial cells. 82 More specifically, PKA activation was reported to counteract the Clostridium sordelli lethal toxin (LT)-mediated Rac1 inhibition and to attenuate LT-induced endothelial permeability. 83 These findings implicate Rac1, as well as RhoA, in the PKA-mediated control of endothelial permeability.

Extracellular Signal Regulated Kinase

Liu et al showed that inhibition of PKA transiently enhances the activities of both ERK1/2 and of its upstream activator Raf-1 in pulmonary vascular endothelial cells. 84 This activation of the Raf-1-ERK1/2 pathway results in the phosphorylation of the cytoskeletal protein caldesmon, stress fiber formation, and in increased endothelial permeability. The phosphorylation of caldesmon plays an important role in the regulation of smooth muscle contraction by modulating the dynamics of actin filament organization. 85 Thus, caldesmon phosphorylation downstream of Raf-1/ERK1/2 signaling may contribute to the cytoskeletal reorganization that is induced by inhibition of PKA activity. Data from Liu et al also suggest that basal PKA activity is sufficient to suppress the ERK signaling pathway and inhibit Raf-1-Erk1/2-mediated endothelial gap formation. This provides additional evidence for the role of PKA in promoting endothelial barrier function through cytoskeletal reorganization.

Actin-Binding Proteins

PKA directly phosphorylates actin-binding proteins such as dematin, adducin, filamin, and vasodilator-stimulated phosphoprotein (VASP). 86,87 Filamin is particularly interesting in the control of endothelial permeability, because it regulates the distribution of F-actin between cortical actin and actin stress fibers. 88 PKA constitutively phosphorylates filamin in unstimulated endothelial cells, which increases the capacity of filamin to crosslink actin filaments. 87 Another actin-binding protein phosphorylated by PKA, VASP, stabilizes newly formed actin filaments. 89 VASP phosphorylation induces its localization to tight junctions in endothelial and epithelial cells, where it associates with the tight junctional protein ZO-1, and colocalizes with JAM-A and occludin. 90,91 VASP phoshorylation by PKA is required for restoration of proper barrier function in epithelial cells, as shown by Ca 2+ -switch experiments. 90 Moreover, expression of VASP deletion mutants decreases endothelial permeability. 91 Thus, PKA may promote endothelial integrity through the phosphorylation of VASP, which stimulates its interactions with tight junction proteins and mediates subsequent stabilization of barrier function.

Microtubules

The crosstalk between microtubules and the actin cytoskeleton is critical for the control of endothelial permeability. Depolymerization of the microtubule network with nocodazole results in the disruption of cortical actin, increased MLC phosphorylation, induction of RhoA activation, formation of stress fibers, and dissociation of endothelial cell-cell junctions. 92 Elevation of cAMP attenuates the increase in permeability of human pulmonary endothelium induced by microtubule disassembly. In addition, PKA blocks nocodazole-induced stress fiber formation, RhoA activation, and decreased MLC phosphorylation. Moreover, pretreatment of endothelial cells with forskolin attenuated nocodazole-induced MT depolymerization. 93 There is also evidence that PKA phosphorylates stathmin, reducing its MT-destabilizing activity. 94 These data indicate another mechanism for PKA-mediated endothelial barrier protection that involves stabilization of the microtubule cytoskeleton, resulting in inhibition of RhoA activity, MLC phosphorylation and actomyosin contractility ( Figure 3 ).

Epac1-Rap1

Recently, it was shown that Rap1, independently of its effect on integrin-mediated adhesion, promotes cadherin-mediated adhesion and antagonizes hepatocyte growth factor-induced disruption of adherens junctions in MDCK cells. Conversely, inhibition of Rap1 activity resulted in loss of epithelial cell-cell contact. 95 Two other groups corroborated these findings by showing that E-cadherin interacts with the RapGEF C3G and that Rap1 mediates proper targeting of E-cadherin and stabilization of E-cadherin complexes at the plasma membrane. 96,97

Rap1 plays a similar role in the stabilization of endothelial junctions ( Figure 3 ). Cullere et al found that cAMP-activated Epac1 markedly enhances endothelial barrier function, which is paralleled by an increase in cortical actin and a redistribution of adherens and tight junction proteins to cell-cell contacts. 24 In addition, activation of Epac1-Rap1 signaling blocked thrombin-induced endothelial permeability through the inhibition of RhoA. 24,25 Fukuhara et al found that activation of Epac1-Rap1 signaling by PGI2 results in rearranged cortical actin, accumulation of VE-cadherin at adherens junctions, and increased endothelial barrier function. 25 These effects were blocked by overexpression of Rap1GAP.

These data were further supported by Kooistra et al who, using Epac1-specific siRNA, showed that Rap1 activation by Epac1, but not by Epac2, was responsible for the increase in endothelial adherens junctions. 98 They also demonstrated that cytoskeletal rearrangements induced by the Epac1-Rap1 pathway were independent from the formation of VE-cadherin-mediated adherens junctions, because actin remodelling was still present in sparse cultures. Finally, Wittchen at al demonstrated that activation of the Epac1-Rap1 pathway not only promotes endothelial barrier function, but also inhibits transendothelial migration of differentiated HL60 cells. 99 In contrast, Cullere et al did not observe any blocking effect on neutrophil migration on endothelial Epac1 stimulation. This discrepancy is currently unexplained.

The downstream effectors of Rap1 that regulate its effects on cadherin-mediated cell-cell contact remain to be identified. Potential candidates include the Rac1 GEFs Vav2 and Tiam1, which localize to sites of cell-matrix contact after Rap1 activation in fibroblasts. 100 Similarly, the CDC42 GEF FRG acts downstream of Rap1 in the control of E-cadherin-mediated cell-cell adhesion, 97,99 and the RhoA GEF ARAP3 is also regulated by Rap1. Other potentially relevant effectors are cytoskeleton-associated regulatory proteins such as RIAM, which binds profilin as well as Ena/VASP, 101 and vinculin, which relocalizes to endothelial adherens junction after Rap1 activation and subsequently supports endothelial cell-cell contact by linking cadherins with actin through -catenin. 102 Finally, Cullere et al suggested that Epac1-Rap1 regulates endothelial integrity through the actin-binding protein AF-6/afadin. Rap1 is known to associate with AF-6, and the complex interacts with p120 catenin. This promotes the binding of p120 catenin to E-cadherin, reduces E-cadherin endocytosis, and induces formation of adherens junctions 103 ( Figure 3 ).

Concluding Remarks

Leukocyte transendothelial migration involves a large number of molecules and signaling events in both the migrating leukocyte and in the endothelium. cAMP is an important and potent regulator of both leukocyte chemotaxis and endothelial barrier function, and the discovery of the Epacs has significantly complicated existing models on the role of cAMP in leukocyte transendothelial migration. Earlier findings based on the use of cell-permeable nondiscriminating cAMP analogues need to be reevaluated, because these compounds will activate both Epac and PKA. Current available data clearly show that PKA and Epac both are relevant in leukocytes as well as in endothelial cells and act, most likely, through parallel pathways. Important determinants for the outcome of cAMP signaling appear to be the subcellular localization of cAMP production, the local concentration of cAMP, and the relative expression level of different cAMP effectors such as PKA or Epac. These findings lead to new questions on the balance between Epac- and PKA-mediated events in both leukocytes and endothelial cells, on potential redundancy between signaling through distinct cAMP targets, and on crosstalk with other signaling pathways. Finally, we cannot exclude that, as with Epac, additional cAMP targets will be identified that are relevant for leukocyte chemotaxis.

Given the protective role of cAMP-mediated signaling in inflammation, it is perhaps not surprising that inhibition of cAMP-breakdown by PDEs is an important means to counter inflammatory responses, with relevance for asthma and COPD as well as for atherosclerosis. 12 The promising results obtained with second generation phosphodiestarase inhibitors 12 further underscore the importance of detailed molecular and mechanistic insight in cAMP targets and cAMP-driven signaling.

In conclusion, despite more than 50 years of research on the "classical second messenger" cAMP, delineating its effects in various aspects of cellular biology remains a challenging area. The role of cAMP in leukocyte adhesion and motility and its pronounced barrier promoting effects in endothelial and epithelial cells make cAMP-driven signaling pathways key targets for more specific and successful therapies aimed at the control of inflammatory responses.

Acknowledgments

The authors thank D. Roos for critical reading of the manuscript.

Sources of Funding

M.L. was funded by project no. 3.2.00.58 from the Netherlands Asthma Foundation. M.F.-B. was funded by project 112 from the Landsteiner Foundation for Blood Transfusion. P.L.H. is a fellow of the Landsteiner Foundation for Blood Transfusion.

Disclosures

None.

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作者单位：Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands.