Intercellular Adhesion Molecule (ICAM)-1, But Not ICAM-2, Activates RhoA and Stimulates c-fos and rhoA Transcription in Endothelial Cells This information is current as of September 25, 2021. Paul W. Thompson, Anna M. Randi and Anne J. Ridley J Immunol 2002; 169:1007-1013; ; doi: 10.4049/jimmunol.169.2.1007 http://www.jimmunol.org/content/169/2/1007 Downloaded from References This article cites 38 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/169/2/1007.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Intercellular Adhesion Molecule (ICAM)-1, But Not ICAM-2, Activates RhoA and Stimulates c-fos and rhoA Transcription in Endothelial Cells1 Paul W. Thompson,*† Anna M. Randi,‡ and Anne J. Ridley2*† ICAM-1 and -2 are integrin-binding Ig superfamily adhesion molecules that are important for leukocyte transmigration across endothelial monolayers. ICAM-1 cross-linking is known to activate the small GTPase RhoA and induce stress fiber formation in endothelial cells, but ICAM-2 signaling has not been investigated. In this study, we compare ICAM-1 and ICAM-2 signaling and localization in HUVECs. Although ICAM-1 and ICAM-2 both localize with the actin-binding protein moesin in apical microvilli, only ICAM-1 colocalizes with moesin after cross-linking. Unlike ICAM-1, ICAM-2 does not activate RhoA or alter actin cytoskel- etal organization. Interestingly, ICAM-1 stimulates transcription of c-fos, a known early response gene. In addition, it up-regulates Downloaded from rhoA expression, suggesting that it activates a positive feedback pathway after RhoA activation. These results indicate that in endothelial cells, ICAM-1, but not ICAM-2, rapidly stimulates signaling responses involving RhoA. The Journal of Immunology, 2002, 169: 1007–1013. he vascular endothelium serves as a selective blood/tissue sponses (7). In contrast, in vitro studies indicate that ICAM-2 barrier, controlling the extravasation of solutes and mac- contributes to leukocyte transendothelial migration under non- http://www.jimmunol.org/ T romolecules and the passage of leukocytes into surround- inflammatory conditions and therefore could play a role in ing tissues. Leukocyte transmigration is dependent upon the ex- leukocyte recirculation (8, 9). Although lymphocyte homing to pression of adhesion receptors on the apical surface of the lymph nodes was apparently unimpaired in ICAM-2-deficient endothelium, including Ig superfamily members such as VCAM-1 mice (10), it has recently been reported that ICAM-2 plays an and ICAM-1 (1, 2). ICAM-2 is closely related to ICAM-1, but only important role in mediating DC transmigration across resting has two Ig-like extracellular domains, compared with the five Ig endothelium (5), and thus it may contribute to the tissue domains of ICAM-1 (3). Both were identified through their ability recruitment of a subset of hemopoietic cell types. ␤ to bind the same ligand, the 2 integrin LFA-1 (CD11a/CD18), As well as being critical for leukocyte adhesion, ICAM-1 can which is expressed on all leukocytes (3, 4). Recently, ICAM-2 has activate signaling pathways. Ab-induced cross-linking of ICAM-1 by guest on September 25, 2021 also been reported to bind to another ligand, dendritic cell (DC)3- on rat brain-derived endothelial cell lines has been reported to specific ICAM-grabbing nonintegrin, a C-type lectin expressed induce the activation of the Src tyrosine kinase and phosphoryla- only on DCs (5). tion of the cytoskeletally associated proteins focal adhesion kinase, In resting endothelial cells, ICAM-1 is expressed at very low paxillin, p130Cas and cortactin, as well as the activation of the levels whereas ICAM-2 is expressed abundantly. Proinflamma- small GTPase Rho (11–13). In the same cell lines, ICAM-1 cross- tory cytokines such as TNF-␣ induce a large increase in ICAM-1 levels (1) and a concomitant decrease in ICAM-2 linking induced actin cytoskeletal reorganization, which was levels on the surface of endothelial cells (6). ICAM-1 is shown to involve Rho, intracellular calcium signaling, and p38 essential for stable adhesion and transmigration of leukocytes in mitogen-activated protein kinase activation (12, 14). Rho mediates nearly all types of inflammatory response (1), and ICAM-1- the assembly of stress fibers and associated focal contacts in many deficient mice have impaired inflammatory and immune re- cell types, including endothelial cells (12, 15, 16), and induces contractility in HUVECs leading to the formation of intercellular gaps which could aid the transmigration of leukocytes (16). In *Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, and †Department of Biochemistry and Molecular Biology, University Col- addition, Rho is required for monocyte adhesion to HUVECs, and lege London, London, United Kingdom; and ‡Experimental Medicine, GlaxoSmith- mediates clustering of ICAM-1 underneath the monocytes (17). Kline, Addenbrooke’s Center for Clinical Investigation, Addenbrooke’s Hospital, Furthermore, Rho can activate gene expression in coordination Cambridge, United Kingdom with Ras activation, by activating the serum response element Received for publication February 13, 2002. Accepted for publication May 7, 2002. (SRE) promoter sequence (18). In contrast to ICAM-1, a possible The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance signaling function of ICAM-2 in endothelial cells has not been with 18 U.S.C. Section 1734 solely to indicate this fact. investigated. However, in T cells, it has recently been reported that 1 P.W.T. was funded by a Medical Research Council studentship (U.K.). ICAM-2 can activate the serine/threonine kinase Akt/protein 2 Address correspondence and reprint requests to Dr. Anne J. Ridley, Ludwig Institute kinase B (19). for Cancer Research, 91 Riding House Street, London W1W 7PS, U.K. E-mail ad- The cytoplasmic tails of ICAM-1 and ICAM-2 could be respon- dress: [email protected] sible for transducing signals in the cell. Despite their short length 3 Abbreviations used in this paper: DC, dendritic cell; SRE, serum response element; ERM, ezrin, radixin, and moesin; N-ERMAD, N-terminal domain of ERM proteins; (ICAM-1 has 28 aa and ICAM-2 has 26 aa), both ICAM-1 and F-actin, filamentous actin; CPERM, C-terminally phosphorylated ERM protein; ICAM-2 cytoplasmic tails can interact with ezrin in vitro (20). TRITC, tetramethylrhodamine isothiocyanate; EGM, endothelial growth medium; TRBD, rhotekin Rho-binding domain; GST-TRBD, TRBD bound to GST; SHP-2, Src Ezrin is a member of the ERM (ezrin, radixin, and moesin) family, homology 2 domain-containing tyrosine phosphatase-2. which can function as a linker between the plasma membrane and Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 1008 ICAM-1, ICAM-2, AND RHO the actin cytoskeleton (21), and play an important role in lamelli- with human fibronectin until confluent. To obtain quiescent-starved cells, podium extension (22), cell-cell adhesion (23), microvillus assem- the culture medium was replaced by medium containing 10% FCS, but no bly, and cytokinesis (23). Ab-cross-linked ICAM-1 coclusters with heparin or other growth factors. Cells were incubated in this medium for no longer than 24 h. Cells grown in EGM-2 were starved in 1% FCS. ERM proteins in endothelial cells (17). The N-terminal domain of No differences between HUVEC responses were observed between cells ERM proteins (N-ERMAD) can interact with both phosphoino- grown in different media. sitides and a number of proteins, while the C-terminal domain of ERM proteins binds to filamentous actin (F-actin). There is Receptor clustering and immunofluorescence evidence that Rho activation occurs downstream of ERMs as N-ERMADs can interact with RhoGDI, which normally holds Rho To induce receptor clustering, either mouse monoclonal anti-ICAM-2 Ab in an inactive GDP-bound complex in the cytoplasm. N-ERMAD was added to starved cells at a final concentration of 10 ␮g/ml for 60 min, or mouse monoclonal anti-ICAM-1/ICAM-2 were added to cells that had binding to RhoGDI facilitates exchange of GDP for GTP on Rho, been stimulated for 24 h with 10 ng/ml TNF-␣. After incubation with presumably because Rho is released from RhoGDI (24). Also, primary Abs, TNF-␣ and the primary Abs were removed from the cell more recently, the ICAM-1 cytosolic tail has been shown to inter- medium and 10 ␮g/ml of Alexa 488-labeled goat anti-mouse Ab was added act with the Src homology 2 domain containing tyrosine phospha- to the cells for between 15 and 60 min. Cells were then washed three times tase-2 (SHP-2) in a phosphotyrosine-dependent manner (25). in TBS (10 mM Tris, pH 7.5, 150 mM NaCl) containing 0.25% BSA, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized for In this study, we compare the effects of ICAM-1 and -2 cross- 6 min with 0.2% Triton X-100, and then incubated with 1 ␮g/ml TRITC- linking in primary human umbilical cord endothelial cells upon phalloidin for 45 min to stain actin filaments, or for 45 min with rabbit cytoskeletal arrangements and gene expression.