Interendothelial junctions: structure, signalling and functional roles

674

Interendothelial junctions: structure, signalling and functional roles Maria Grazia Lampugnani and Elisabetta Dejana Endothelial cell-cell adhesive junctions are formed by transmembrane adhesive proteins linked to a complex cytoskeletal network. These structures are important not only for maintaining adhesion between endothelial cells and, as a consequence, for the control of vascular permeability, but also for intracellular signalling properties. The establishment of intercellular junctions might affect the endothelial functional phenotype by the downregulation or upregulation of endothelial-specific activities.

Address Vascular Biology Laboratory, Mario Negri Institute for Pharmacological Research, 20157 Milan, Italy Current Opinion in Cell Biology 1997, 9:674-682

http:llbiomednet.com/elecref10955067400900674 © Current Biology Ltd ISSN 0955-0674 Abbreviations AJ adherens junction adenomatous polyposis coli APC epithelial E epidermal growth factor EGF membrane-associated guanylate kinase MAGUK PDZ/DHR PSD-95, discs large, ZO-1/discs-large homology region platelet/endothelial cell adhesion molecule PECAM protein tyrosine phosphatase PTP tight junction TJ vascular endothelial VE zonula occludens ZO

Introduction Endothelial cells constitute the main barrier between blood and tissues. T h e integrity of the endothelial cell layer is required to control the flow of plasma components and circulating cells between the blood and subendothelial compartments. Endothelial permeability mostly comprises the passage of solutes through paracellular junctions (paracellular route) [1] and a vesicle-mediated uptake of components and their active transport from the apical to the basal side of the cell (transcellular route) [2]. In contrast to the vesicular system, it is likely that the passage of plasma components through endothelial (paracellular) junctions does not require specific receptors but is regulated by diffusion and by the dynamic opening and closing of interendothelial gaps. T h e molecular organization of intercellular junctions in the endothelium has been partly elucidated during the last few years [1,3°°]. At endothelial contacts, it is possible to distinguish at least two types of junctional organelle, tight junctions (TJs) and adherens junctions

(AJs). These structures share the common characteristic of being formed by specific transmembrane proteins which, through their extracellular domains, promote homotypic adhesion between cells. Through their cytoplasmic regions the transmembrane proteins are anchored to a complex network of cytoskeletal proteins and actin microfilaments [4,5°°,6°°,7]. In addition to these junctional structures, the endothelium presents a series of transmembrane proteins, such as platelet/endothelial cell adhesion molecule (PECAM), which can concentrate at intercellular contacts and display homotypic adhesive properties [8]. T h e endothelium is functionally and structurally heterogeneous along the vascular tree and this diversity is also reflected by the organization of intercellular junctions. For instance, in the brain, where permeability needs to be under strict control, the intercellular junctions are particularly well organized and form a complex and intersected seal. In the postcapillary veins where the interchanges between blood and tissues need to be highly dynamic, intercellular junctions are very simple; frequently TJs are absent or present in only a primitive organization [9]. T h e functional state of interendothelial junctions can also be changed by the state of growth and activation of the endothelial cells. For instance, inflammatory cytokines or growth factors change the expression or phosphorylation state of the junctional proteins [10"']. In addition to their role in promoting homotypic cell adhesion, increasing amounts of evidence are accumulating to suggest that intercellular junctions can transfer cell-cell signals. This probably occurs after the engagement and clustering of the transmembrane adhesive proteins at junctions and the association of their cytoplasmic tails with signalling molecules. T h e intracellular molecules that associate with AJs are different from those that associate with TJs and from those that link other junctional adhesion molecules, suggesting that a certain specificity in the signalling pathways should exist. In this review, we attempt to describe both the molecular organization and the signalling properties of interendothelial junctions. We also summarize the endothelial activities that are modified by the state of confluency of the cells. We suggest that these functional changes may be, at least in part, determined by the signals transferred by interendothelial junctions.

InterendothelialjunctionsLampugnani and Dejana

Adherens junctions Structural organization AJs are formed by transmembrane adhesive proteins belonging to the cadherin family [4,5°°,6°°]. Cadherins are single-chain transmembrane polypeptides that undergo, in the majority of the cases, homophilic-type binding (Figure 1). T h e major and specific transmembrane component of endothelial AJs is vascular endothelial (VE)-cadherin [1,10°°]. This protein belongs to the type II cadherin subgroup [5°°] and in mouse is encoded by a gene that resides in a cluster with the epithelial (E), placental (P) and muscle (M) cadherin genes on chromosome 8 [11]. T h e short cytoplasmic domain of VE-cadherin is linked to cytoplasmic proteins called catenins, in particular to 13-catenin, plakoglobin and p120, which belong to the so-called Armadillo family [12°°,13,14]. T h e region of VE-cadherin that mediates binding to ~-catenin and plakoglobin resides in the last 80 amino acids of the cytoplasmic tail, while p120 associates with a different domain closer to the transmembrane region [15]. Only ~-catenin and plakoglobin, but not p120, can associate with (x-catenin [16]. a-catenin, which is homologous to vinculin, can in turn bind to the actin cytoskeleton and promote junction stabilization [5°°]. As p120 does not bind to (x-catenin and actin, cadherin association with p120 would lead to a junctional complex that is more mobile and dynamic than those that contain only [3-catenin or plakoglobin. In ras-transformed cells [17], which lack stable inter~ltular junctions, most of the E-cadherin in the cells was bound to p120. Consistent with this observation, we found that in recently confuent endothelial cells, where intercellular junctions are still weak and immature, VE-cadherin is bound to p120 [15]. When junctions stabilize after longer periods of confluency, p120 dissociates from VE-cadherin [15]. Signal transduction via adherens junction components AJs are considered to be communication centers that are necessary for transducing signals between neighboring cells. Different receptor-like phosphatases have been found at AJs. These include leukocyte antigen-related protein (LAR) [18], protein tyrosine phosphatase (PTP)IB [19], and hPTP~c [20]. Kinases such as src, lyn and yes and receptor kinases such as the epidermal growth factor (EGF) receptor and c-erb-B2 are concentrated at intercellular contacts and, in the case of the EGF receptor and c-erb-B2, are directly associated with the cadherin-catenin complex [21,22]. [3-catenin and plakoglobin, like other members of the Armadillo family, can act as signalling molecules. [3-catenin directly participates in the Wnt growth factor signalling cascade. In particular, the binding of Wnt to its receptor leads to inactivation of glycogen synthase kinase-3 (GSK-3), which in its active form is responsible for the

675

phosphorylation and consequent rapid inactivation, via a ubiquitin-proteasome pathway, of [3-catenin. This process is regulated by the adenomatous polyposis coli (APC) protein which can bind to GSK-3 and facilitate further ~-catenin phosphorylation and degradation [23°°]. Wnt signalling therefore stabilizes ~-catenin in the cytoplasm. Cytoplasmic ~-catenin or its Drosophila homolog can then bind to at least three high mobility group (HMG) transcription factors (lymphoid enhancer-binding factor-l, Xenopus T cell specific transcription factor-3 and pangolin) and translocate to the nucleus [24°°-26°°]. This process may regulate the expression of a series of homeobox genes [23 °° ] which are probably involved in celt growth and differentiation. Cytoplasmic free ~-catenin can also affect cell behavior through other pathways. For instance, it can directly bind to the actin fasciculation protein fascin [27], and overexpression of a stabilized form of [3-catenin leads to its co-distribution with the APC protein in tubulin-containing cellular puncta [28]. These observations suggest that cytoplasmic free [3-catenin can affect the cellular cytoskeleton in a cadherin-independent way. Role of adherens junctions in signalling

How the signalling activities of free cytoplasmic catenin are linked and controlled by AJs is still unclear. AJs can indirectly modify cellular responses by binding of cadherins to ~-catenin and thus decreasing the free cytoplasmic pool of ~-catenin. However, this does not seem to be relevant in all cases. In some conditions, the overall levels of I]-catenins are high and therefore the association with cadherins does not significantly affect the amount of cytoplasmic [~-catenin [29°]. In the endothelium, VE-cadherin mediates specific intracellular signalling. VE-cadherin limits cell migration and participates in contact inhibition of cell growth in the endothelium [30]. This last effect requires binding of VE-cadherin to catenins [30]. In addition, a null mutation of the VE-cadherin gene leads to altered vascular morphogenesis in vitro [31]. In this system, the lack of VE-cadherin did not alter endothelial cell differentiation but instead abolished the capacity of the cells to organize themselves into vascular-like structures. It is not yet known how VE-cadherin elicits intracellular signalling. However, the VE-cadherin-catenin complex is very dynamic and its composition rapidly changes according to the functional state of the cells. At early stages of junction assembly or when the cells are released from tight confluency and migrate, VE-cadherin is heavily tyrosine phosphorylated and mostly linked to p120 and [3-catenin. Only a small amount of the complex is bound to the actin cytoskeleton. When the junctions stabilize, tyrosine residues in VE-cadherin are dephosphorylated, and p120 and 13-catenin tend to detach from the complex

676

Cell-to-cell contact and extracellular matrix

Figure 1

Figure 1 Schematic representation of the molecular complexes localized to interendothelial junctions. The general organization of each of these complexes includes transmembrane proteins acting as receptors and cytoplasmic partners transducing signals to inside the cell and, very often, modulating the activity of the transmembrane component too. Connections with cytoskeletal elements are also present. Transmembrane and cytoplasmic components are generally specific for each type of junction, with the exception of VE-cadherin. Adherens junctions are formed by VE-cadherin dimers which are connected inside the cell to a cluster of catenins (c~- and I]-catenins, plakoglobin and p120) and actin microfilaments. Tight junctions consist of the transmembrane protein occludin and a group of cytoplasmic proteins (ZO-1, ZO-2, cingulin and 7H6) as well as actin microfilaments. The complexus adherens presents VE-cadherin as the transmembrane component and possibly plakoglobin, desmoplakin and vimentin inside the cell. The question mark indicates the fact that although desmoplakin co-localizes with VE-cadherin [62], no direct connection between the VE-cadherin-plakoglobin complex and desmoplakin has been reported yet. Outside these junctional complexes, PECAM is also present at endothelial cell-cell contacts where, as represented in the figure, it would express homophilic binding properties [8]. N-cadherin (top), although bound to each of the known catenins (~t- and I~-catenin, plakoglobin and p120), and thereby to actin, is mostly nonjunctional. Only the proteins with reported endothelial expression are represented; however, some molecular relationships among cytoplasmic components are also inferred from data obtained in other cell types rather than endothelial cells. This is the case for the association of the protein tyrosine phosphatase SHP-2 with PECAM that has been reported during platelet aggregation as a result of phosphorylation of Tyr663 of PECAM [8]. This interaction might regulate PECAM tyrosine dephosphorylation which takes place during endothelial migration [66]. Noteworthy in other cell types as a new possible signalling system at/Us, tyrosine phosphorylation of N-cadherin has recently been shown to recruit the adaptor protein Shc (not shown) [67]. Assembly of TJs can be regulated by serine phosphorylation of the transmembrane component, occludin, the highly phosphorylated form being concentrated at TJs [68]. ~t, c~-catenin; 13, I~-catenin; p, plakoglobin/7-catenin; VE-cad, VE-cadherin; N-cad, N-cadherin; SHP-2, Src homology 2 domain containing PTP-2.

Occludin junction

/

/\

Actin

t

\

/l~JJtm lunctlon

and are substituted by plakoglobin [15]. These changes are accompanied by a stronger association of the VE-cadherin complex with actin.

PECAM

I V'~nentin

Desmoplakin

© 1997 Current Opinion in Cell Biology

How these changes could be related to intracellular signalling is still a matter of speculation. It might be that, when detached from AJs, p120 and [3-catenin become available for signalling. Indeed, p120 is a substrate for src and might participate in an src-dependent signalling cascade [16]. It is also possible that the binding of actin to AJs is responsible for intracellular signalling, c~-catenin is indeed required for the E-cadherin-mediated retardation of growth in a tumor cell line [32]. Another way in which AJs can participate in signalling is by clustering signalling molecules and growth factor receptors and in this way facilitating their reciprocal interaction. In addition to kinases and phosphatases, components of the ras signalling pathway and small GTP-binding proteins can also concentrate at cell-cell junctions. Recent data indicate that the small GTP-binding proteins Rho and Rac

Interendothelial junctions Lampugnaniand Dejana

participate in organization of AJs, and inhibition of Rho and Rac leads to AJ disassembly [33°°].

Tight junctions Structural organization AJs represent the basic and ubiquitous type of organized structure at interendothelial contacts. TJs or occludens junctions, on the other hand, are particularly well developed in those endothelia that strictly control exchanges between blood and tissues (typically at the blood-brain barrier and in the large arteries [7]). At the electron microscopic level, TJs (see Figure 1) present an apparent fusion of the outer leaflets of the plasma membranes of two contiguous cells. This suggests that they literally seal the intercellular space, giving rise to a true adhesive belt located towards the apical surface of the cell layer. Morphologically, endothelial TJs display an organization that is very similar to that of TJs in epithelia. In contrast to epithelial TJs, possibly as a result of the far more flat aspect of the endothelium, endothelial TJs can be less strictly localized to the apical cell membrane and can be found to be spatially intermingled with AJs [7,34]. More recently, several molecular components of TJs have been defined which are common to endothelium and epithelium. However, subtle but significant differences may exist between the two cell types; these differences still await experimental analysis. In endothelial cells, the complexity of TJ organization may be influenced by the microenvironment. T h e best example of this is the brain microvasculature where TJs are uniquely complex. This characteristic is not predetermined but is induced by the neural microenvironment and, in particular, by still undefined factors released by astroglial cells [35]. In brain tumors, the neovasculature formed by angiogenesis does not have the same barrier properties of the rest of brain vessels even if it originates from them [35]. This is probably due to the interaction with tumor-derived factors which strongly modify the original properties of the vessels. Oceludin

T h e only transmembrane constituent of TJs described thus far is occludin [36]. This is a 65 kDa protein with four putative membrane-spanning domains that ensure that both the amino- and the carboxy-terminal regions of the protein are found in the cytoplasm. Occludin might bind an identical molecule present on an adjacent cell, possibly through the second extracellular domain [37°]. In parallel to the high concentration of TJs in brain endothelium, occludin staining was found to be particularly intense in brain capillaries [36]. It was also present, but stained more weakly, in heart, muscle and intestinal endothelia [36].

677

One of the mediators of such an interaction could be the cytoplasmic protein zonula occludens (ZO)-I, a protein of 195-200kDa [39,40], which binds to the 250 amino acid carboxy-terminal region of occludin [37°]. Binding of occludin to ZO-1 is necessary, but apparently not sufficient, for targetting occludin to TJs. Some deletion mutants of occludin bind ZO-1, but are not concentrated at TJs [38]. Therefore, other factors must be required for the correct junctional distribution of the molecule. ZO-1 might connect occludin to the actin cytoskeleton through its association with spectrin [41] and may also bind actin directly using its carboxy-terminal domain [42]. Occludin binds ZO-I via the amino-terminal region of ZO-1 [42]. ZO-1 in turn interacts via one of its three PSD-95, discs large, ZO-1 (PDZ) domains (PDZ2; see below) with ZO-2 (a protein of 160kDa [43]) and both proteins form part of the cytoplasmic undercoat of TJs. Both ZO-1 and ZO-2 are members of the membrane-associated guanylate kinase (MAGUK) family, as they bear a domain homologous to the catalytic domain of guanylate kinase, although they are devoid of enzymatic activity [44]. T h e cytoplasmic proteins of the MAGUK family in general express organizing and targetting activity towards cell membrane proteins [44]. Beyond the guanylate kinase homologous region, ZO-1 and ZO-2 contain multiple copies of a 80 amino acid repeat (as mentioned above), the P D Z / D H R (discs-large homology region) domain. These domains are involved in protein-protein interactions. ZO-2 is exclusively found at TJs in both endothelia and epithelia, while ZO-1 shows a far less specific distribution [43]. Indeed, it is located at cell-cell contacts independently of TJs and can be also found in cell types that never develop TJs such as fibroblasts or cardiac muscle cells [41,45]. Targetting of ZO-1 to the plasma membrane can be regulated by its binding to cytoplasmic components of AJs, typically catenins (or- and 13-catenin and plakoglobin [46]). In cells that do not develop TJs, ZO-1 is found associated with AJs [41]. However, when TJs form, ZO-1 is segregated with them. Therefore, ZO-1 may represent a cross-talking element between AJs and TJs. Interestingly, endothelial cells express a specific ZO-1 isotype, called ZO-loc [34]. It derives from alternative RNA splicing, and it lacks a 80 amino acid region (in the proline-rich carboxy-terminal half of the molecule). No alteration of the molecular relationships of this isoform with other TJ components (for example ZO-2) has been reported, although it has been suggested that ZO-lot characterizes more dynamic junctions [34].

Zonula occludens-1 and -2

Other components of tight junctions

It has been suggested that occludin needs association with cytoskeletal proteins to be localized at TJs [38].

Two other cytoplasmic components of TJs that are common to epithelia and endothelia are cingulin (molecular

678

Cell-to-cellcontact and extracellular matrix

weight 108-140kDa [47]) and 7H6 antigen (molecular weight 155-175 kDa [48]). T h e expression of 7H6 antigen is restricted to brain capillaries in vivo [48]. Cingulin is located more peripherally to the plasma membrane than ZO-1 and ZO-2, and its molecular interaction with the other constituents of TJs remains to be defined. Epithelial cells also express molecules at TJs that are absent in endothelial cells, such as symplekin [49°]; this suggests that besides the strong similarities a certain degree of cell specificity exists in the TJ organization.

Functions of tight junctions T h e functional effects of TJs have been classically designated as the control of paracellular permeability and cell polarity, functions that most epithelia and many endothelia must display [7]. Increasing evidence indicates that occludin directly contributes to paracellular barrier function (or restriction of permeability) [50]. Occludin may achieve its barrier function by homophilic recognition of another molecule of an adjoining cell. Interestingly, when occludin is displaced from its junctional localization, the cytoplasmic components of TJs (ZO-1, ZO-2 and cingulin) remain in place at the plasma membrane even if both permeability and electrical resistance are severely affected [37°]. As expected, cultured endothelial cells from the brain microvasculature express higher levels of occludin in comparison with aortic endothelium, and this higher expression parallels their higher capacity to maintain effective barrier functions [51]. Of the cytoplasmic components of TJs, the most studied in terms of functional effects is ZO-1. In cultured endothelial cells the amount of ZO-1 is upregulated by cell confluency [52] and is downregulated in response to a vasoactive agent such as histamine [53]. Tyrosine phosphorylation of ZO-1 correlates with increased paracellular permeability in both epithelial and endothelial cells [54]. Induction of transcription of another cytoplasmic component of TJs, 7H6, with dbcAMP (dibutyryl cyclic AMP, a cell-permeable synthetic analog of cyclic AMP) or retinoic acid enhances the barrier function of endothelial cells in culture [55]. Signalling via tight junctions As in AJs, there is indirect evidence that TJs can act as cell-cell signalling organelles. Non-classical signalling pathways (those that result in nuclear localization of components of cell-cell junctions) are being delineated for components of TJs. In epithelial cells, ZO-1 shows a nuclear localization, which is inversely related to the maturity of cell-cell contacts [56°]. Symplekin was also found to be concentrated in the nucleus, where it might modify gene expression [49°]. Many of the possible novel signalling routes in epithelial cells make use of the muhidomain nature of the MAGUK proteins [44]. ZO-1 and ZO-2 contain the same P D Z / D H R sequence that is present in hdgl, the human homolog of

the Drosophila discs large (dig) protein, another MAGUK family member. T h e P D Z / D H R sequences are recognized by the tumor suppressor protein APC [57°°]. Hdlg, using its P D Z / D H R sequences, binds protein 4.1 (at the 30 kDa amino-terminal domain of protein 4.1) and ezrin (at a similar conserved region of ezrin) [58°]. At its Src homology 3 domain ZO-1 binds a serine protein kinase that can phosphorylate ZO-1 itself [59]. Although the functional consequences of these molecular associations are largely unknown at the moment, Drosophila dlg, which is a component of septate junctions, is involved in multiple functions from the control of epithelial proliferation to maintenance of apicobasal polarity [60°°]. Interestingly, another MAGUK family member in Caenorhabditis elegans, lyn 2, acts in the pathway that is homologous to the E G F / E G F receptor signalling system of mammals [44]. In addition, E G F can induce tyrosine phosphorylation of ZO-1 and ZO-2 in human epidermal cells [61]. Whether or not similar interactions can be expressed by ZO-1 and ZO-2 (and possibly other members of the MAGUK family) at endothelial TJs is, at the present, totally ignored. The exploration of this aspect could help us to understand the molecular basis of endothelial morphogenesis and its alteration in pathological conditions. O t h e r s t r u c t u r e s at i n t e r e n d o t h e l i a l j u n c t i o n s In contrast to epithelial cells, endothelial cells do not possess classic desmosomes. However, they might express similar but possibly simplified desmosomal structures. Endothelial cells synthesize desmoplakin, which is a specific component of desmosomes. As viewed by confocal microscopy, desmoplakin co-distributes with VE-cadherin, plakoglobin and vimentin [62]. It is possible that the association of these molecules (see Figure 1) constitutes a desmosomal-like structure (also called the complexus adherens) [63]. Endothelial cells express high amounts of N-cadherin, but this molecule is preferentially localized in a diffuse pattern on the cell membrane (Figure 1) [64]. This suggests that it can play a role in the anchorage of endothelial cells to other N-cadherin-expressing cells such as smooth muscle cells, astrocytes or pericytes. Other adhesive molecules, not directly associated with AJs or TJs, have been found to be concentrated at endothelial cell-cell contacts. These include PECAM, S-endo-1/Muc 18, CD34, and endoglin (for a review, see [10"°]). PECAM is certainly the best studied of the proteins at interendothelial contacts (for a review, see [8]). It can transfer and receive signals, possibly through binding to a tyrosine phosphatase, SHP-2 (Src homology 2 containing PTP-2), which may participate in the signalling cascades of different growth factors. T h e function of the redundancy of adhesive structures at interendothelial contacts is not clear. It is difficult to believe that it is simply due to the need of maintaining

Interendothelial junctions Lampugnani and Dejana

Table 1 Differences in amounts of protein or enzymatic activity in confluent endothelial cells versus subconfluent/migrating cells.

Protein/activity

Increase/

References

decrease*

Growth factors and morphogens Insulin-like GF-1 binding protein 3 Non-PDGF mitogens bFG F TGF-113 Production Responsiveness to

1" 1` ,L

[69] [70] [71 ]

$ ~,

[?3,74]

Follistatin

J,

[75]

SPARC

$

[76]

$ $

[77] [78]

1" 1"

[79,80] [80]

,1, 1"

[81 ] [82]

Nuclear proteins Cyclin A Pigpen Enzymes PTPs (p., ~) PC5 (subtilisin/kexin) endoprotease PLA2 Activity Localization: cytoplasmic : nuclear uPA uPAr PAl-1 Inflammation/vascular tone PGI2 MCP1 Endothelin TNF (affinity for/

[79]

$

[82]

$ $ $

[83] [83] [84]

1" $ $ J,

[85] [86] [87] [88]

1" 1" 1` 1` 1` .1. $

[89] [59] [62] [90] [91] [15,89] [15]

$ $

[92] [93]

responsiveness to)

Structural and cell-cell junction associated proteins

Plakoglobin ZO-i Desmoplakin Vimentin Non-muscle filamin (ABP-280) 13-catenin p120 Other proteins/activities Hyaluronic acid binding 25 kDa, 48 kDa surface glycoproteins

*Upward arrows indicate increased amounts of protein or enzymatic activity, and downward arrows indicate decreased amounts of protein or enzymatic activity, unless otherwise specified, in confluent epithelial monolayers as compared with subconfluent/migrating endothelial cells. Please note that subconfluent and migrating situtations are taken as one entity for the sake of clarity. GF-1, growth factor-I; PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor; TGF-113, transforming growth factor-l~; SPARC, secreted protein acidic and rich in cysteine; PC5, proprotein convertase 5; PLA2, phospholipase A2; uPA, urokinase-type plasminogen activator; uPAr, uPA receptor; PAl-l, plasminogen activator inhibitor-I; PGI2, prostaglandin 12/prostacyclin; MCP1, monocyte chemoattractant protein 1 ; TNF, tumor necrosis factor.

intercellular adhesion, as, in the majority of cases, each adhesive molecule plays roles in both adhesion and signalling. It is more attractive to consider that these

679

structures have specific biological activities and possibly initiate intracellular signalling pathways.

Cell-cell contacts and the modulation of endothelial functions A relevant question is whether the establishment of intercellular junctions could modify the cellular phenotype. Several reports describe changes in endothelial functional behavior as a function of the state of confluency (which affects the establishment of intercellular junctions) of the cells. Table 1 attempts to summarize this information. T h e endothelium in the adult, under most physiological situations, is actually a confluent monolayer. T h e release from this state occurs in conditions of neoangiogenesis or wound repair. Many of the functions that are upregulated when the junctions are disrupted are relevant for cell motility and proliferation. T h e opposite occurs when the cells reach confluency: activities important for vascular homeostasis, such as the expression of cytoskeletal proteins or of antithrombotic proteins like prostacyclin, are induced. Interestingly, when the endothelial cells are migrating, such as during repair of a vascular lesion, they release growth factors (basic fibroblast growth factor or transforming growth factor-13; see Table 1) that are important in the process of wounding but which may also stimulate the proliferation of the vascular media and contribute to the development of an atherosclerotic lesion. In addition, leukocyte adhesion proteins or the release of inflammatory agents are upregulated in migrating endothelial cells, suggesting that disruption of the endothelial integrity might facilitate inflammatory reactions. While it is reasonable that signals coming from cell-cell contacts could directly regulate specific endothelial activities, whether and how this actually takes place are largely unknown at present. It is, however, attractive to speculate that the same signals from AJs, TJs or other structures that can regulate complex differentiation phenotypes [65] could also operate in changing endothelial cell responses.

Conclusions We are now beginning to understand the structural features of endothelial junctions. Some molecular components of endothelial junctions are identical to those described in epithelial cells, but others are selective for this cell type and might regulate cell-specific activities. We still know little about the pathological consequences of alterations in the functional behavior or synthesis of endothelial junctional proteins. It is possible that diseases linked to altered endothelial permeability or vascular morphogenesis are associated with alterations in the intercellular junction composition. In addition, many functional changes that occur in proliferating endothelial cells as compared with in resting confluent endothelium might be

680

Cell-to-cell contact and extracellular matrix

influenced by the signals transferred by the establishment or rupture of intercellular junctional structures.

18.

Kypta RM, Su H, Reichardt LF: Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J Cell Biol 1996, 134:1519-1529.

Acknowledgements

19.

Balsamo J, Leung TC, Ernst H, Zanin MKB, Hoffman S, Lilien J: Regulated binding of a PTP 1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of 13-catenin. J Cell Biol 1996, 134:801-813.

20.

Fuchs M, Muller T, Larch MM, UIIrich A: Association of human protein-tyrosine phosphatase K with members of the Armadillo family. J Biol Chem 1996, 271:16712-16719.

21.

Hoschuetzky H, Aberle H, Kemler R: Beta-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J Cell Biol 1994, 127:1375-1380.

22.

KanaiH, Ochiai A, Shibata T, Oyama S, Ushijima S, Akimoto S, Hiroashi S: c-erb-B2 gene product directly associates with 13catenin and plakoglobin. Biochem Biophys Res Commun 1995,

This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro and the European Community (projects C T 960036, BMH4 CT960669 and BMH4 C T 950875) and by the Human Frontier Science Program.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • .o

of special interest of outstanding interest

208:1067-1072.

1.

Dejana E, Corada M, Lampugnani MG: Endothelial cell-to-cell junctions. FASEB J 1995, 9:910-918.

2.

Schnitzer JE: Update on the cellular and molecular basis of capillary permeability. Cardiovasc Med 1993, 3:124-130.

3. Staddon JM, Rubin LL: Cell adhesion, cell junctions and the •° blood brain barrier. Curr Opin Neurobio/1996, 6:622-627. Describes endothelial cell junction organization in the blood brain barrier. 4.

Takeichi M: Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Bio11993, 5:806-811.

5. =°

Aberle H, Schwartz H, Kemler R: Cadherin-catenin complex: protein interactions and their implications for cadherin functions. J Cell Biochem 1996, 61:514-523. A review on the structural biology of cadherins and catenins. The authors discuss the role of cadherin-catenin complexes in promoting cell adhesion and tissue morphoganesis during embryonic development. 6. An

Gumbiner BM: Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 1996, 84:345-357. extensive review on cell adhesion mechanisms and their role in signal transduction, cell growth and differentiation.

23. Nusse R: A versatile transcriptional effector of wingless ** signalling. Cell 1997, 89:321-323. A short, excellent review summarizing the latest progress in the study of signal transduction via wingless/Writ and the role of catenin/Armadillo proteins. 24. •-

Beherans J, yon Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W: Functional interaction of J~-catenin with the transcription factor LEF-I. Nature 1996, 382:638-642. This paper, together with [25°',26"'], gives experimental support to the concept that a component of adherens junctions such as ~-catenin can directly intervene in the regulation of gene transcription. 25. •°

Molenaar M, van de Wetering M, Oosterwegel M, PetersonMaduro J, Godsave S, Korinek V, Roose J, Destree O, Clevers H: XTcf-3 transcription factor mediates ~-catenin-induced axis formation in Xenopus embryos. Cell 1996, 86:391-399. See annotation [24"]. 26. •°

Brunner E, Peter O, Schweizer L, Basler K: Pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. Nature 1997, 385:829-833. See annotation [24"*].

Anderson JM, Van Itallie CM: Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 1995, 269:G467-G475.

27.

Tao YS, Edwards RA, Tubb B, Wang S, Bryan J, Mc Crea PD: ~-catenin associates with the actin-bundling protein fascin in a noncadherin complex. J Cell Biol 1996, 134:1271-1281.

8.

NewmanPJ: The biology of PECAM-1. J Clin Invest 1997, 99:3-8.

28.

9.

Simionescu N, Simionescu M: Endothelial transport macromolecules: transcytosis and endocytosis. Cell B/o/Rev 1991, 25:5-80.

Barth AIM, Pollack AL, Altschuler Y, Mostov KE, Nelson WJ: N H 2 terminal deletion of ~-catenin results in stable colocalization of

7.

10. ••

Dejana E: Endothelial cell-cell adherens junctions: implications in the control of vascular permeability and angiogenesis. J Clin Invest 1996, 98:1949-1953. A review which makes an attempt to discuss the specific role of adherens junctions in the endothelium. 11.

Huber P, Dalmon J, Engiles J, Breviario F, Gory S, Buchberg AM, Dejana E: Genomic structure and chromosomal mapping of the mouse VE-cadherin gene (Cdh5). Genomics 1996, 32:21-28.

Miller JR, Moon RT: Signal transduction through 13-catenin and specification of cell fate during embryogenesis. Genes Dev 1996, 10:2527-2539. An update on recent progress in the study of the role of Armadillo proteins in signal transduction and embryonic development.

mutant p-catenin with adenomatous polyposis coil protein and altered MDCK cell adhesion. J Cell Biol 1997, 136:693-706. 29.

Orsulic S, Peifer M: An in vivo structure-function study of Armadillo, the ~-catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for Wingless signaling. J Cell Biol 1996, 134:1283-1300. This paper reports direct evidence for different roles of Armadillo in the wingless signalling pathway and in cell-cell adhesion. •

30.

Caveda L, Martin-Padura I, Navarro P, Breviario F, Corada M, Gulino D, Lampugnani MG, Dejana E: Inhibition of cultured cell growth by vascular endothelial cadherin (cadherin-5/VEcadherin). J C/in Invest 1995, 98:885-893.

31.

Vittet D, Buchou T, Schweitzer A, Dejana E, Huber P: Targeted null-mutation in the VE-cadherin gene impairs the organization of vascular-like structures in embryoid bodies. Proc Nat/Acad Sci USA 1997, 94:6273-6278,

32.

Watabe M, Nagafuohi A, Tsukita S, Takeichi M: Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J Cell Biol 1994, 127:247-256.

12. ••

13.

Hulsken J, Behrens .I, Birchmeier W: Tumor-suppressor gene products in cell contacts: the cadherin-APC-armadillo connection. Curt Opin Cell Biol 1994, 6:711-716.

14.

Peifer M: Cell adhesion and signal transduction: the Armadillo connection. Trends Cell Bio/1995, 5:224-229.

15.

Lampugnani MG, Corada M, Andriopoulou P, Esser S, Risau W, Dejana E: Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells. J Cell Sci 1997, in press.

16.

Daniel JM, Reynolds AB: The tyrosine kinase substrata p120 cas binds directly to E-cadherin but not to the adenomatous polyposis coil protein or (x-catenin. Mol Cell Biol 1995, 15:4819-4824.

17.

Kinch MS, Clark GJ, Der C J, Burridge K: Tyrosine phosphorylation regulates the adhesion of ras-transformed breast epithelia. J Cell Biol 1995, 130:461-471.

33. •*

Braga VMM, Machesky L, Hall A, Hotchin NA: The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J Cell Biol 1997, 137:1421-1431. This paper provides an underlying molecular mechanism to the 'classical' concept of the regulation of adherens junctions by the actin cytoskeletal undercoat. See also annotation [57°']. 34.

Balda MS, Anderson JM: Two classes of tight junctions are revealed by Z0-1 isoforms. Am J Phys 1993, 264:C918-C924.

35.

Breier G, Risau W: Angiogenesis in the developing brain and in brain tumours. Trends Exp Med 1996, 6:362-376.

Interendothelial junctions Lampugnani and Dejana

36.

Fumse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita Sh: Oocludin: a novel integral membrane protein localizing at tight junctions. J Cell Bio/1993, 123:1777-1788.

37. •

Wong V, Gumbiner BM: A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barder. J Cell Biol 199?, 136:399-409. The functional activity of occludin in terms of control of junctional permeability is demonstrated and mapped to specific regions of the molecule. 38.

Furuse M, Itoh M; Hirase T, Nagafuchi A, Yonemura S, Tsukita Sa, Tsukita Sh: Direct association of occludin with Z0-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 1994, 127:1617-1626.

39.

Stevenson BR, Siciliano JD, Mooseker MS, Goodenough DA: Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Bio11986, 103:755-766.

40.

Anderson JM, Stevenson BR, Jesaitis LA, Goodenough DA, Mooseker MS: Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J Cell Bio11988, 106:1141-1149.

41.

Itoh M, Yonemura S, Nagafuchi A, Tsukita Sa, Tsukita Sh: A 220KD undercoat-constitutive protein: its specific localization at cedherin-based cell-cell adhesion sites. J Cell Biol 1991, 115:1449-1462.

42.

Fanning A, Jameson BT, Anderson JM: Molecular interactions among the tight junction proteins ZO-1, ZOo2 and occludin [abstract]. Mol Biol Cell 1996, 7:3530.

43.

Gumbiner B, Lowenkopf T, Apatira D: Identification of a 160-kDa polypoptide that binds to the tight junction protein ZO-1. Proc Natl Acad Sci USA 1991, 88:3460-3464.

44.

Kim SK: Tight junctions, membrane-associated guanylate kinases and cell signalling. Curr Opin Cell Bio11995, 7:641649.

45.

Howarth AG, Hughes MR, Stevenson BR: Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 1992, 262:CA61-C469.

46.

Rajasekaran AK, Hojo M, Huima T, Rodriguez-Boulan E: Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J Cell Biol t 996, 132:451-463.

681

cell-cell contacts. Proc Nat/Acad Sci USA 1996, 93:1077910784. In this paper and [49"], a novel nuclear localization for the cell-cell junction components zonula occludens-1 and symplekin is reported. This IocaJization suggests potential new signalling pathways from cell-cell junctions directly to the nucleus for components of tight junctions as well as for components of adherens junctions (see also [22,23"'-26"']). 57. ••

Matsumine A, Ogai A, Senda T, Okumura N, Satoh K, Baeg G-H, Kawahara T, Kobayashi S, Okada M, Tohyoshima K, Akiyama T: Binding of APC to the human homolog of the drosophila discs large tumor suppressor protein. Science 1996, 272:1020-1023. Hdlg, which belongs to the membrane-associated guanylate kinase (MAGUK) family, is reported to be a novel partner for adenomatous polyposis coil (APC) protein. The paper also envisages a potential association of both zonula occludens-1 and zonula occludens-2, members of the MAGUK family and components of tight junctions, with the APE; protein through their conserved PDZ/DHR repeats. 58. •

Lue RA, Brandin E, Chan EP, Branton D: Two independent domains of hDIg are sufficient for subcellular targeting: the PDZl-2 conformational unit and an alternatively spliced domain. J Ceil Biol 1996, 135:1125-1137. These authors mapped the domains of a membrane-associated guanylate kinase (MAGUK) member that are involved in its binding to protein 4.1 and ezrin. This paper, together with [33"'], offers a molecular basis to the regulation of cell-cell junctions by the cytoskeletal/cytoplasmic undercoat of the plasma membrane. 59.

Balda MS, Anderson JM, Matter K: The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain. FEBS Lett 1996, 399:326-332.

60. t•

Woods DF, Hough C, Peel D, Callain G, Bryant PJ: Dig protein is required for junction structure, cell polarity and proliferation control in Drosophila epithelia. J Cell Biol 1996, 134:14691482. This paper demonstrates the effect of a junctional molecule on very important cell functions such as cell proliferation and polarity. 61.

Van Itallie CM, Balda MS, Anderson JM: Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells. J Cell Sci 1995, 108:1735-1742.

62.

Valiron O, Chewier V, Usson Y, Breviario F, Job D, Dejana E: Desmoplakin expression and organization at human umbilical vein endothelial cell-to-cell junctions. J Cell Sci 1996, 109:2141-2149.

47.

Citi S, Sabanay H, Jakas R, Geiger B, Kendrick-Jones J: Cingulin, a new peripheral component of tight junctions. Nature 1988, 333:272-276.

63.

48.

Zhong Y, Saitoh T, Minase T, Sawada N, Enomoto K, Mori M: Monodonal antibody 7H6 reacts with a novel tight junctionassociated protein distinct from ZO-1, cingulin, and ZO-2. J Cell Bio11993, 120:477-483.

Schmalz M, Franke WW: Complexus adhaerens, a new group of desmoplakin-containing junctions in endothelial cells: the syndesmos connecting retothellal cells in lymph nodes. Eur J Cell Biol t 993, 61:274-289.

64.

Salomon D, Ayalon O, PateI-King L, Hynes RO, Geiger B: Extrajunctional distribution of N-cedherin in cultured human endothelial cells. J Cell Sci 1992, 102:1-11.

65.

Huber O, Bierkamp C, Kemler R: Cadherins and cetenins in development. Curt Opin Cell Biol 1996, 8:685-691.

49. •

Keon BA, Schafer S, Kuhn C, Grund C, Franke WW: Symplekin, a novel type of tight junction plaque protein. J Cell Biol 1996, 134:1003-1018. See annotation [56"]. 50.

McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, Schneeberger EE: Occludin is a functional component of the tight junction. J Cell Sci 1996, 109:22872298.

66.

Lu "l'Y, Yan LG, Madri JA: Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1. Proc Nat/Acad Sci USA 1996, 93:1180811813.

51.

Staddon JM, Saitou M, Furuse M, Tsukita Sh, Rubin LL: Occludin in endothelial cells [abstract]. Mol Biol Cell 1996, 7:3520.

67.

52.

Li C, Poznansky MJ: Characterization of the ZO-1 protein in endothelial and other cell lines, J Cell Sci 1990, 97:231-237.

Xu Y, Guo D, Davidson M, Inagami T, Carpenter G: Interaction of the adaptor protein Shc and the adhesive molecule cadherin. J Biol Chem 1997, 272:13463-13466.

68.

53.

Gardner "I'W, Lasher T, Khin S, Vu C, Barber AJ, Brennan WA: Histamine reduces ZO-1 tight-junction protein expresssion in cultured retinal microvascular endothelial cells. Biochem J 1996, 320:717-721.

Sakakibara A, Furuse M, Ando-Akatsuka Y, Tsukita S: Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Bio/1997, 137:1393-1401.

69.

Delafontaine P, Ku L, Anwar A, Hayzer D: Insulin-like growth factor 1 binding protein 3 synthesis by aortic endothelial cells is a function of cell density. Biochem Biophys Res Commun 1996, 222:478-482.

70.

Gajdusek CM: Release of endothelial cell-derived growth factor (ECDGF) by heparin. J Cell Physiol 1984, 121:12-21.

71.

Singh RK, Llansa N, Bucana CD, Sanchez R, Koura A, Fidlar IJ: Cell density-dependent regulation of basic fibroblast growth factor expression in human renal cell carcinoma cell. Cell Growth Diff 1996, 7:397-404.

72.

Merrilees MJ, Sodek J: Synthesis of TGF-beta 1 by vascular endothelial cells is correlated with cell spreading. J Vasc Res 1992, 29:376-384.

54.

Staddon JM, Herrenknecht K, Smales C, Rubin LL: Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci 1995, 108:609-619.

55.

Satoh H, Zhoug Y, Isomura H, Saitoh M, Enomoto K, Sawada M, Mori M: Localization of 7H6 tight junction-associated antigen along the cell border of vascular endothelial cells correlates with paracellular barrier function against ions, large molecules and cancer cells. Exp Cell Res 1996, 222:269-274.

56. *

Gottardi C J, Arpin M, Fanning AS, Louvard D: The junctionassociated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of

682

Cell-to-cell contact and extracellular matrix

73.

Gamble JR, Khew-Goodall Y, Vadas MA: Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells. J Immunol 1993, 150:4494-4503.

84.

Pepper MS, Sappino AP, Montesano R, Orci L, Vassalli JD: Plasminogen activator inhibitor-1 is induced in migrating endothelial cells. J Cell Phys 1992, 153:129-139.

74.

Flaumenhaft R, Rifkin DB: Cell density dependent effects of TGF-~ demonstrated by a plasminogen activator-based assay for TGF-13. J Cell Physio/1992, 152:48-55.

85.

Eldor A, Vlodavsky I, Hy-Am E, Atzmon R, Weksler BB, Raz A, Fuks Z: Cultured endothelial cells increase their capacity to synthesize prostacyclin. J Cell Phys 1983, 114:179-183.

75.

Kozian DH, Augustin HG: Rapid identification of differentially expressed endothelial cell genes by RNA display. Biochem Biophys Res Commun 1995, 209:1068-1075.

86.

76.

Iruela-Arispe ML, Hasselaar P, Sage H: Differential expression of extracellular proteins is correlated with angiogenesis in vitro. Lab Invest 1991,64:174-186.

Wempe F, Lindner V, Augustin HG: Basic fibroblast growth factor (bFGF) regulates the expression of the CC chemokine monocyte chemoattractant protein-1 (MCP-1) in autocrine activated endothelial cells. Arther Throm Vasc Bio/1997, in press.

87.

77.

Yoshizumi M, Hsieh C-M, Zhou F, Tsai J-C, Patterson C, Perrella MA, Lee M-E: The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells. Mol Cell Biol 1995, 15:3266-3272.

Zaragoza R, Budzik GP, Dillon TP, Opgenorth TJ: Effect of cell density on endothelin release from endothelial cells and phosphoremidon dependent inhibition. Biochem Pharmacol 1992, 44:851-856.

78.

Alliegro MC, Alligro MA: A nuclear protein regulated during the transition from active to quiescent phenotype in cultured endothelial cells. Dev Biol 1996, 174:288-297

88.

Gerlach H, Lieberman H, Bach R, Godman G, Brett J, Stern D: Enhanced responsiveness of endothelium in the growing/motile state to tumor necrosis factor/cachectin. J Exp Med 1989, 170:913-931.

79.

Gaits F, LI RY, Ragab A, Ragab-Thomas JMF, Chap H: Increase in receptor-like protein tyrosine phosphatase activity and expression level on density-dependent growth arrest of endothelial cells. Biochem J 1995, 311:97-103. Campan M, Yoshizumi M, Seidah NG, Lee ME, Bianchi C, Haber E: Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family. Biochemistry 1996, 35:3797-3802.

89.

Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E: The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, ~catenin and (x-catenin with vascular endothelial (VE)-cadherin. J Cell Bio/1995, 129:203-217

90.

Savion N, Vlodavsky I, Greenburg G, Gospodarowicz D: Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and organization. J Cell Physio/1982, 110:129-141.

91.

Wang Q, Patton WF, Chiang ET, Hechtman HB, Shepro D: Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases. J Cell Biochem 1996, 62:383-396.

92.

Antonelli A, D'Amore PA: Density-dependent expression of hyaluronic acid binding to vascular cells in vitro. Microvasc Res 1991,41:239-251.

93.

Augustin-Voss HG, Pauli BU: Migrating endothelial cells are distinctly hyperglycosylated and express specific migrationasssociated cell surface glycoproteins. J Cell Biol 1992, 119:483-491.

80.

81.

Whatley RE, Satoh K, Zimrnerman GA, Mclntyre TM, Prescott SM: Proliferation-dependent changes in release of arachidonic acid from endothelial cells. J C/in Invest 1994, 94:1889-1900.

82.

Sierra-Honigmann MR, Bradley JR, Pober JS: 'Cytosolic' phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation. Lab Invest 1996, 74:684-695. Pepper MS, Sappino AP, Stocklin R, Montesano R, Orci L, Vassalli JD: Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Bio11993, 122:673-684.

83.