Perspectives Series: Cell Adhesion in Vascular Biology The Biology of PECAM-1 Peter J. Newman Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233-2121 Introduction Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T cell subsets. It is also a major constituent of the endothelial cell intercellular junction (1–3), where up to 106 PECAM-1 molecules (4) are concentrated. With a few minor exceptions, PECAM-1 is not present on fibroblasts, epithelium, muscle, or other nonvascular cells. Since its cloning nearly 10 yr ago (5, 6), much has been learned about the structure of this cell adhesion receptor and its function in vascular cells. The purpose of this brief Perspective is to review progress in the field of PECAM-1 biology, and to bring the reader up to date on current concepts about (a) the function of PECAM-1 in the different vascular cells in which it is expressed; (b) the molecular mechanisms by which PECAM-1 mediates cell–cell interactions; and (c) its role in bidirectional transmembrane signal transduction. In keeping with the intent of this series to discuss issues of cell adhesion in the context of human biology and pathophysiology, the potential clinical relevance of PECAM-1–mediated cellular interactions to thrombotic, inflammatory, and immunological diseases will be underscored at relevant points throughout the review. Structure of the PECAM-1 gene and protein PECAM-1 is encoded by a 75-kb gene that resides at the end of the long arm of chromosome 17 (7). The earliest reports of PECAM-1 in the literature described it variously as a myeloid differentiation antigen (8, 9) or as a homologue of platelet GPIIa (the integrin b1 subunit) present within the plasma membrane of endothelial cells (1). After the determination of its primary structure in 1990 (6, 10, 11), however, PECAM-1 was assigned definitively to the growing family of type I transmembrane cell adhesion molecules that are members of the Ig superfamily (Ig-CAMs). The 574 amino acids that comprise the extracellular portion of this molecule are organized into six Ig-like homology domains, and typical of other members of the Address correspondence to Peter J. Newman, Blood Research Institute, The Blood Center of Southeastern Wisconsin, 638 N. 18th Street, Milwaukee, WI 53233-2121. Phone: 414-937-6237; FAX: 414937-6284; E-mail: pjn@bcsew.edu Received for publication 11 November 1996. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/97/01/0003/06 $2.00 Volume 99, Number 1, January 1997, 3–8 Ig superfamily, each of these is encoded by a single exon (12). After a short, single-pass transmembrane domain, PECAM-1 contains a 118–amino acid cytoplasmic domain that is both structurally and functionally complex, being encoded by eight different exons that can be alternatively spliced to yield PECAM-1 isoforms that variously contain or lack residues that serve as sites for palmitoylation, phosphorylation, and assembly of cytosolic signaling molecules (see cover of this issue for a schematic diagram of the PECAM-1 protein). Functions of PECAM-1 in vascular cells There is good evidence to suggest that PECAM-1 is a key participant in the adhesion cascade leading to extravasation of leukocytes during the inflammatory process. Muller et al. (13) were the first to show that pretreating monocytes or neutrophils with antibodies specific for PECAM-1 inhibited their emigration across an endothelial cell monolayer in a quantitative in vitro assay of transendothelial migration. Blocking endothelial cell junctional PECAM-1 also effectively inhibited leukocyte transmigration, indicating that PECAM-1 molecules on both the endothelial cell as well as the leukocyte side contributed to the transmigration process. The requirement for PECAM-1 in leukocyte recruitment appears to be operative in vivo as well, since antibodies to PECAM-1 have been shown to block the accumulation of neutrophils into the peritoneal cavity, the alveolar compartment, and human skin grafts in three different intact rat models of inflammation (14). An mAb to murine PECAM-1 has also been shown to be effective in reducing leukocyte emigration into the peritoneal cavity in a mouse model of acute peritonitis (15). In studies of direct potential relevance to human disease, two different groups have shown that antibodies to PECAM-1 reduce myocardial infarct size in both rat (16, 17) and feline models of ischemia-reperfusion injury (18). Together, these studies have combined to make agents that activate or antagonize the adhesive and/or signaling properties of PECAM-1 (see below) attractive potential therapeutics for the treatment of acute and chronic inflammatory conditions. Functions mediated by PECAM-1 in other cells of blood or vascular origin have been less well worked out. Ohto et al. (8) reported that certain mAbs to PECAM-1 inhibit neutrophil and monocyte chemotaxis, but Muller et al. (13) observed no effect of the blocking anti–PECAM-1 mAb, hec7, on leukocyte migration into collagen gels in response to chemotactic stimuli. There is one report that transfection of PECAM-1 into NIH/3T3 cells actually diminishes the rate of cell migration (19), probably by virtue of its ability to stabilize intercellular contacts. PECAM-1 has also been implicated in T-lymphocyte function during the development of an alloimmune response, The Biology of PECAM-1 3 as Zehnder et al. (20) reported that an mAb directed against Ig-domain 6 of PECAM-1 inhibited T cell activation in a mixed lymphocyte reaction, while Behar et al. (21) noted a tendency for bone marrow transplant recipients unmatched for a Leu98Val polymorphism in PECAM-1 to develop acute graftversus-host disease. Finally, despite the name platelet endothelial cell adhesion molecule-1, virtually nothing is known about the role that PECAM-1 plays in platelet function, though recent studies suggest that it may serve as an agonist receptor to modulate b3 integrin function (to be discussed in greater detail below). Mechanisms of PECAM-1–mediated adhesion The first indication that PECAM-1 actually possessed adhesive activity was provided by the studies of Albelda et al. (22), who showed that although PECAM-1 was not present at intercellular junctions formed between PECAM-1–positive and PECAM-1–negative cells, it became strongly localized to cell– cell borders when adjacently transfected cells contacted one another. These findings led to the hypothesis that PECAM-1/ PECAM-1 homophilic interactions are responsible for concentrating this molecule at endothelial cell intracellular junctions. Further support for a homophilic mechanism derived from the observation that murine L cell fibroblasts transfected with PECAM-1 acquire the ability to interact with one another in an aggregation assay. Finally, Sun et al. (23) have shown recently that purified, full-length PECAM-1 reconstituted into artificial phospholipid membranes interacts directly with other PECAM-1 molecules in a divalent cation-independent manner, a process that is completely inhibited by small Fab frag- ments of mAbs that bind to Ig-homology domains 1 and 2 of PECAM-1. That this amino-terminal region of the extracellular domain is physiologically relevant to PECAM-1–mediated cellular interactions is further supported by the finding that anti–PECAM-1 mAbs that block leukocyte transendothelial migration almost without exception epitope map to Ig-domains 1 or 2 (24, 25). In addition to homophilic interactions, several studies have suggested that PECAM-1 may also be capable of interacting heterophilically with other components of the cell surface, as PECAM-1–transfected cells have been found to interact not only with each other, but also with nontransfected, PECAM-1– negative L cells in a heterotypic process that is dependent upon the presence of divalent cations (22, 26) and involves cell surface glycosaminoglycans (27). Two additional studies have implicated the integrin avb3 as another potential counterreceptor for PECAM-1 (28, 29). However, recent experimental observations cast doubt on there being physiologically important receptors for PECAM-1 other than PECAM-1 itself. First, neither heparin, heparin-sulfate, nor EDTA inhibit the interaction of purified PECAM-1, presented either as a full-length adhesion receptor incorporated into phospholipid membranes or as a high-affinity chimeric PECAM-1/IgG immunoadhesin (23), with human umbilical vein endothelial cells or PECAM-1– transfected L cell fibroblasts, both of which express abundant levels of glycosaminoglycans, including chondroitin-6-sulfate and heparan sulfate, on their surface. Second, despite having a near-consensus heparin-binding motif within Ig-domain 2 (22), neither cellular nor recombinant PECAM-1 binds glycosaminoglycans (30). Finally, blocking antibodies to avb3 prebound Figure 1. Dual functional roles for PECAM-1 in leukocyte–endothelial cell interactions. Two non-mutually exclusive receptor functions are depicted. On the left, homophilic engagement of PECAM-1 molecules between circulating leukocytes and the underlying endothelial cell monolayer (1) initiates intracellular signal transduction events that result in the upregulation of leukocyte integrin affinity, facilitating subsequent interactions with endothelial cell counterreceptors (2). Integrin-mediated cellular interactions (3), in turn, amplify cytosolic signal transduction pathways, some of which result in the phosphorylation of PECAM-1 and the binding of additional signaling molecules, some of which modulate the affinity of PECAM-1 and enable transendothelial migration (4). While good experimental evidence exists for many of these events, others are tentative assumptions, based on paradigms already established for other adhesion receptor signal transduction pathways, which remain to be proven. The depicted ability of PECAM-1 homophilic interactions to mediate leukocyte transendothelial migration, while at the same time maintaining the permeability barrier established by the monolayer of endothelial cells, is adapted from a model originally proposed by Muller et al. (13). 4 P.J. Newman Figure 2. Proposed model for transmembrane signaling through PECAM-1. Antibody-induced dimerization of PECAM-1 has been shown recently to affect the phosphorylation state of its cytoplasmic domain, creating a docking site for one or more signaling molecules. At the same time, engagement or dimerization of PECAM-1 on the surface of lymphocytes, endothelial cells, or platelets results in the upregulation of integrin function (outside-in signaling from PECAM-1 to integrins). PECAM-1 not only modulates integrin function, but is itself affected by integrins, as integrin-mediated cellular contact leads directly to tyrosine phosphorylation of the PECAM-1 cytoplasmic domain (outside-in signaling from integrins to PECAM-1). The signaling molecules that mediate bidirectional cross-talk between integrins and PECAM-1 are just now beginning to be identified and may involve elements of the pathway shown schematically in this figure. to cells that abundantly express this integrin have no effect on the subsequent binding of PECAM-1 proteoliposomes or PECAM-1/IgG, while an mAb to PECAM-1 Ig-domain 1 bound to these same cells completely abolishes their binding (23). Together, these studies argue that the mechanism by which introduction of PECAM-1 into cells promotes interactions with PECAM-1–negative cells must be an indirect one. As will be discussed below, there is now increasing evidence that, at least in some cell types, PECAM-1 functions as an agonist receptor, and that its activation initiates specific signal transduction pathways that result in secondary adhesion events mediated by non–PECAM-1 receptors. PECAM-1 transmits signals into and receives signals from the cell interior The first experimental evidence that PECAM-1 might be involved in outside-in signal transduction came from the studies of Tanaka et al. (31), who showed that antibody-induced dimerization of PECAM-1 on the surface of T cells resulted in their increased adherence to the b1 integrin substrates VCAM-1 (via a4b1) and fibronectin (via a5b1). Monovalent Fab fragments of PECAM-1 mAbs were ineffective, suggesting that dimerization of PECAM-1 on the cell surface might be responsible for the observed upregulation of b1 integrin function. Since that time, affinity modulation of b1 integrins induced by cross-linking PECAM-1 has been reproduced in CD341 hematopoietic progenitor cells (32), and b2 integrin function has been shown to be modulated by PECAM-1 dimerization in lymphokine-activated killer cells (33), monocytes and neutrophils (34), and natural killer cells (35). The ability of PECAM-1 to mediate outside-in signal transduction has been extended recently to b3 integrins on platelets by the studies of Varon and co-workers, who showed that the binding of F(ab9)2 fragments specific for PECAM-1 Ig-domain 6 results in the transformation of the resting aIIbb3 complex into an activated conformational state and augments platelet adhesion and aggregation (Varon, D., D.E. Jackson, B. Shenkman, and P.J. Newman, manuscript in preparation). Finally, DeLisser and colleagues (36, 37) have shown that L cells transfected with PECAM-1 isoforms missing all or parts of their cytoplasmic domain are unable to associate with nontransfected L cells (i.e., they lose heterophilic binding ability), consistent with the hypothesis that PECAM-1–mediated signal transduction, which presumably requires the cytoplasmic domain to relay signals from the extracellular domain into the cytosol, is required for heterotypic L cell aggregation. Taken together, it seems likely that PECAM-1 dimerization or engagement may be capable of transducing signals into the cell, a process that may mimic hoThe Biology of PECAM-1 5 mophilic PECAM-1/PECAM-1 interactions that are thought to occur between leukocytes and endothelial cells during the process of transendothelial migration. The dual functional roles proposed for PECAM-1, serving as both an agonist receptor as well as an adhesion receptor in vascular cells, are depicted in Fig. 1. What might be the mechanism by which PECAM-1 engagement leads to downstream signal transduction events that result in integrin activation and heterotypic cellular activation? Like many protein-tyrosine kinase receptors (including those for PDGF, FGF, VEGF, NGF, and M-CSF), the extracellular domain of PECAM-1 is comprised of Ig-like domains. However, the PECAM-1 cytoplasmic domain does not contain a catalytic kinase domain and therefore is unable to become activated by receptor autophosphorylation after ligand (i.e., PECAM-1) binding has occurred. However, previous studies have shown that PECAM-1 can become tyrosine phosphorylated in both human platelets (38) and in cultured endothelial cells (39), and we have found recently that, when phosphorylated, Tyr663 within the cytoplasmic domain of PECAM-1 becomes a specific docking site for the Src-homology 2 (SH2) domains of the protein-tyrosine phosphatase, SHP-2 (40). SHP-2 is a ubiquitously expressed protein-tyrosine phosphatase that contains two SH2 domains at the amino terminus of the protein, followed by a catalytic phosphatase domain, and a carboxyl-terminal region that can itself become tyrosine phosphorylated (41–44). SHP-2 has been found previously to associate with several autophosphorylated receptor-tyrosine kinases, including the platelet-derived growth factor receptor (43–45) and the epidermal growth factor receptor (43, 44, 46). Though the precise way in which the association of SHP-2 with the cytoplasmic domain of PECAM-1 might lead to downstream signaling in vascular cells has not been worked out yet, SHP-2 in other cell types has been implicated as a multifunctional signaling molecule, acting both as a phosphatase to activate nearby Src family kinases and/or as an upstream mediator of p21ras activation via its ability to bind the Grb2/Sos complex (for reviews on the role of SHP-2 in signaling see references 47–49). In addition to its role in activating integrins via outside-in signal transduction, PECAM-1 also appears to be able to respond to integrin-mediated cell–cell and cell–matrix interactions. Lu et al. (39) have shown recently that engagement of integrins on cultured endothelial cells results in dephosphorylation of PECAM-1 (39), whereas Jackson et al. (40) have shown that integrin-mediated interactions result in an increase in tyrosine phosphorylation of PECAM-1 in aggregating human platelets. While the differences in observed phosphorylation state of PECAM-1 in these two cell types in response to integrin engagement are not well understood, it is likely that the relative balance of kinase and phosphatase activity controls the phosphorylation state not only of PECAM-1, but of other cellular receptors as well. A schematic diagram of the proposed involvement of PECAM-1 in signal transduction in blood and vascular cells is shown in Fig. 2. Conclusions Recent studies on the adhesive and signaling properties of PECAM-1 have impacted our understanding of its role in vascular cell biology in important and exciting ways. Definition of the specific regions of the extracellular domain that mediate PECAM-1 homophilic interactions as well as the elucidation 6 P.J. Newman of the specific molecular events that take place during signal transduction events involving PECAM-1 remain important avenues of future investigation. As our knowledge of the basic cellular and molecular mechanisms by which PECAM-1 exerts its adhesive and cell modulatory effects improves, so should our understanding of the relative role that this novel cell adhesion receptor plays in thrombosis, hemostasis, immunity, and the inflammatory response. Acknowledgments I am indebted to both past and present members of my laboratory who have made significant contributions to many of the studies reviewed herein, including Richard Gumina, Denise Jackson, Nancy Kirschbaum, Chao-Yan Liu, Cathy Paddock, Kim Piotrowski, QiHong Sun, Ronggang Wang, and Christopher Ward. I am also grateful to Drs. Steven Albelda, Horace DeLisser, and William Muller for many years of fruitful and enjoyable collaboration, and for their constant stream of insights and suggestions. This work was supported in part by grants HL-40926 and HL44612 from the National Institutes of Health. Dr. Newman is the recipient of an Established Investigator Award from the American Heart Association. References 1. van Mourik, J.A., O.C. Leeksma, J.H. Reinders, P.G. de Groot, and J. Zandbergen-Spaargaren. 1985. Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from platelet membrane glycoprotein IIa. J. Biol. Chem. 260:11300–11306. 2. 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Newman Adhesion and signaling in vascular cell–cell interactions ........................................Guy Zimmerman, Tom McIntyre, and .................................................................................................................................Stephen Prescott Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis ................................................................................Elisabetta Dejana Genetic manipulation of vascular adhesion molecules in mice ................................. Richard O. Hynes and Denisa D. Wagner The extracellular matrix as a cell cycle control element in atherosclerosis and restenosis ................................................................................Richard K. Assoian and .................................................................................................................................Eugene E. Marcantonio Effects of fluid dynamic forces on vascular cell adhesion .......................................Konstantinos Konstantopoulos and .................................................................................................................................Larry V. McIntire The biology of PECAM-1 .........................................................................................Peter J. Newman Selectin ligands: will the real ones please stand up? ..............................................Ajit Varki Cell adhesion and angiogenesis..............................................................................Joyce Bischoff and Judah Folkman New advances in von Willebrand Factor biology.....................................................Zaverio Ruggeri Therapeutic inhibition of carbohydrate-protein interactions in vivo..........................John Lowe and Peter Ward Proteoglycans and proteoglycan-binding proteins in vascular biology....................Robert Rosenberg Platelet GPIIb/IIIa antagonists: the first anti-integrin receptor therapeutics.............Barry Coller Importance of shear stress in endothelial adhesion molecule expression ..............Michael Gimbrone Integrins and vascular matrix assembly ..................................................................Erkki Ruoslahti New insights into integrin-ligand interaction ............................................................Robert Liddington and Joseph Loftus Adhesive interactions of Sickle erythrocytes with endothelium ...............................Robert Hebbel Cell migration in vascular biology ............................................................................Stephen Schwartz Integrin signaling in vascular biology.......................................................................Sanford Shattil and Mark Ginsberg Multi-step mechanisms of leukocyte homing...........................................................Eugene Butcher Role of PSGL-1 binding to selectins in leukocyte recruitment.................................Rodger McEver and Richard Cummings