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Endothelial Cell-to-Cell Junctions: Molecular Organization and Role in Vascular Homeostasis

Istituto di Ricerche Farmacologiche Mario Negri, FIRC Institute of Molecular Oncology, Department of Genetics and Biology of Microorganisms, Facolta' di Scienze Matematiche Fisiche e Naturali, Universita' Statale di Milano, Milan, Italy

ABSTRACT

I. GENERAL FEATURES OF ENDOTHELIAL JUNCTIONS

II. ADHERENS JUNCTIONS

A. Molecular Components of AJ

1. The cadherin-catenin complex

A) CADHERINS.

B) CATENINS AND OTHER INTRACELLULAR PARTNERS OF CADHERINS.

2. The nectin-afadin complex

B. Outside AJ

C. Intracellular Signaling Through AJ

1. Transcriptional activity of catenins

2. Other signaling pathways at the junctions

D. Modulation of AJ Organization and Vascular Permeability

E. Conclusions

III. TIGHT JUNCTIONS

A. Introduction to TJ

1. Intercellular strands as morphological and functional units of tight junctions

B. Molecular Components of TJ

1. Integral membrane proteins

A) OCCLUDIN.

B) CLAUDINS.

C) JUNCTIONAL ADHESION MOLECULE-A AND RELATED MOLECULES.

2. Intracellular proteins

A) ZO PROTEINS.

B) OTHER INTRACELLULAR PROTEINS.

C. Molecular Interactions at the TJ

1. Molecular interactions among TJ components

A) INTERACTIONS OF OCCLUDIN.

B) INTERACTIONS OF CLAUDINS.

C) INTERACTIONS OF JAM-A.

D) INTERACTIONS OF INTRACELLULAR TJ PROTEINS.

2. Interactions of TJ components with signaling molecules

A) INTERACTIONS WITH G PROTEINS.

B) INTERACTIONS WITH PROTEIN KINASES.

C) PHOSPHORYLATION OF TJ MOLECULES.

3. Interactions of TJ components with regulators of cell proliferation

D. TJ Assembly

1. General principles of TJ assembly

2. Methods for the study of TJ assembly

E. TJ Function (I): Paracellular Permeability

1. General principles: TJ as barriers or channels?

2. Methods for the measurement of paracellular permeability

3. Regulation of permeability by extracellular agents

F. TJ Function (II): Cell Polarity

1. General principles of cell polarity: TJ as cause or effect of polarity?

G. TJ Function (III): In Vivo Studies

1. Genetic deletion of TJ genes in mouse

2. Genetic diseases in humans associated with mutations of TJ genes

H. Conclusions

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. GENERAL FEATURES OF ENDOTHELIAL JUNCTIONS

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Endothelial cells adhere to one another through junctional structures formed by transmembrane adhesive proteins that are responsible for homophilic cell-to-cell adhesion. In turn, the transmembrane proteins are linked to specific intracellular partners, which mediate anchorage to the actin cytoskeleton and, as a consequence, stabilize junctions (for a review, see Ref. 76). Junctions in endothelial and epithelial cells share common features. In addition, in both cell types, two major types of junctions have been described and named adherens junctions (AJ) and tight junctions (TJ). However, epithelial cells also form desmosomes, which are absent in the endothelium (for a review, see Refs. 30, 76, 179, 273, 290). In addition to cell-to-cell adhesion, another type of junction, the gap junction, mediates cell-to-cell communication. This structure is formed by connexins (Cx), and three of these proteins (i.e., Cx43, Cx40, and Cx37) are expressed in the endothelium. In turn, connexins are organized in connexons, which act as channels for the intercellular passage of ions and small-molecular-weight molecules (292).

In epithelial cells, junctions are better organized, with TJ and AJ following a well-defined spatial distribution along the intercellular cleft. TJ (or zonula occludens) are concentrated at the apical side of the rim, while AJ (or zonula adherens) are located below the TJ. In contrast, in endothelial cells, the junctional architecture is less defined and, along the cleft, AJ are intermingled with TJ (290).

As to the function, junctions in the endothelium control different features of vascular homeostasis. For instance, permeability to plasma solutes is controlled, to a considerable extent, by junction permeation. In addition, leukocyte extravasation and infiltration into inflamed areas require finely regulated opening and closing of cell-to-cell contacts (159, 221). Notably, junctional proteins can also transfer intracellular signals, which modulate endothelial cell growth and apoptosis (179). We may expect that the transduced information communicates to the cells their own position, mediates contact-dependent inhibition of growth, and establishes cell polarity.

The organization of endothelial junctions varies along the vascular tree in function of organ-specific requirements (290). For instance, in the brain, where a strict control of permeability between blood and the nervous system is required, junctions are well developed and rich in TJ (274, 352). In contrast, postcapillary venules, which allow dynamic trafficking of circulating cells and plasma proteins, display poorly organized TJ. These morphological features may also account for the high sensitivity of postcapillary venules to permeability-increasing agents, such as histamine and bradikinin. In contrast, the endothelium of large arteries, which tightly controls permeability, has a well-developed system of TJ. Finally, lymphatic endothelium displays specific junctional structures, the complexus adhaerentes (283, 284), which are likely to be specialized in controlling the passage of lymphocytes from and to different compartments.

The development of fully mature endothelial junctions is reached only late in development and, in general, junctions are not completely differentiated in the embryo. A typical example is the blood-brain barrier, which reaches full functional differentiation only after birth (274, 304). This conclusion is supported by data obtained in the in vitro system of embryonic stem cells' differentiation to endothelial cells, where junctional proteins (i.e., VE-cadherin and PECAM) become detectable after a few days of differentiation (187, 336).

In general, junctions are dynamic structures. During the organization of an endothelial monolayer, cell-to-cell contacts follow different steps of maturation. Evidence in epithelial cells indicates that adhesive membrane proteins of AJ and TJ first form adhesion complexes at sites of cell-to-cell contacts and then tend to organize in zipperlike structures by lateral adhesion along the cell border (57, 230, 361, 362). The intracellular partners of transmembrane adhesive proteins also vary during junction maturation and stabilization (18, 177, 179). In addition, even after contacts have been stably formed, adhesion proteins are still in a dynamic equilibrium and recycle continuously between plasma membrane and intracellular compartments.

In this review, we focus on AJ and TJ, as well as on their molecular organization and functional changes. In some cases, considering the high homology of the two systems and the paucity of information on endothelial cells, we will draw a parallelism between epithelial and endothelial cells, taking into account cell-specific differences, when they are present. We also focus on the role of junctions in the organization of vessels and in the control of permeability to plasma solutes. We consider only marginally the role of junctions in leukocyte extravasation, since several excellent and recent reviews on this specific topic are already available in the literature (see, for instance, Refs. 159, 221).

	II. ADHERENS JUNCTIONS

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A. Molecular Components of AJ

1. The cadherin-catenin complex

A) CADHERINS.  AJ are ubiquitously distributed along the vascular tree and are expressed in both blood and lymphatic vessels. These structures are formed by transmembrane adhesion proteins of the cadherin family, which mediate homophilic adhesion and are able to organize in multimeric complexes at the cell borders (1, 10, 11, 122, 358). Endothelial cells express a specific cadherin called vascular endothelial (VE)-cadherin (77). This protein cannot be found in any other cell type, including blood cells or hemopoietic stem cells (178), and, like a signature for the endothelium, is expressed during development, when cells become committed to the endothelial lineage (46). VE-cadherin is present in all endothelial cells of all types of vessels.

Extrapolation of data obtained from the crystal structure of other cadherins (10) suggests that VE-cadherin dimerizes laterally in-cis and makes head-to-head contacts in-trans, via the most amino-terminal repeats, thus promoting cell-to-cell adhesion. Other data, however (54, 70), point to deep interdigitations among cadherins on two contiguous cells and strongly suggest that there are multiple binding domains along the extracellular domain. The transmembrane and intracellular domains of cadherins may also play a role in dimerization and possibly in multimerization (141).

The remarkable cell specificity of expression prompted investigators to study the VE-cadherin promoter (113). The promoter contains three domains important for transcriptional regulation: a proximal domain, which promotes transcription in a cell type-independent manner, and two negative control regions, which abolish transcription in nonendothelial cells. Such regulation, with positively acting proximal domains and cell type-specific silencing domains, is a rather unique feature among endothelial promoters but is reminiscent of promoters that confer tissue-specific expression. Transgenics expressing chloramphenicol acetyltransferase (CAT) under the VE-cadherin promoter confirmed the endothelial specificity of promoter expression in vivo (113).

The tissue specificity of VE-cadherin has an exception, which is the cytotrophoblast. During the establishment of human placenta, cytotrophoblasts invade the uterine interstitium and vasculature, thus anchoring the fetus to the mother and establishing blood flow to the placenta (369). Cytotrophoblasts invading spiral arterioles replace the maternal endothelium and express adhesion molecules that are typical of endothelial cells, such as PECAM, _v3-integrin, and VE-cadherin (369).

Even if VE-cadherin is the most prominent cadherin at the AJ, it is not the only cadherin expressed in endothelial cells. N-cadherin can be found at comparable levels in most of the endothelial cells examined so far (228). P-cadherin expression was noticed by PCR analysis but could not be seen by antibody staining (273). T-cadherin was also found in the vasculature in tissue sections (154), but its biological function in this cell type has not been clarified yet.

Despite high expression levels, in the presence of VE-cadherin, N-cadherin does not localize to AJ and remains diffusely distributed on the cell membrane (228). This peculiar behavior is not cell specific but is related to functional or structural features of these two cadherins. Cotransfection of VE- and N-cadherin in Chinese hamster ovary (CHO) cells prevented N-cadherin localization at the junctions. A similar result was obtained upon transfection of a VE-cadherin mutant (truncated in the cytoplasmic domain), suggesting that the extracellular region of VE-cadherin (or the residual cytoplasmic tail) may be responsible for N-cadherin exclusion from intercellular junctions. Regardless of the mechanism, these observations suggest that VE- and N-cadherin may exert different functions in endothelial cells. An interesting possibility is that N-cadherin promotes endothelial cell adhesion and communication with mesenchymal cells expressing N-cadherin, such as pericytes, smooth muscle cells, and astrocytes. Direct contacts among endothelial cells and the underlying smooth muscle cells, the myoendothelial junctions, have been described by electron microscopy, suggesting that they may be required for a coordinated response of the vessel wall to stimuli (290). N-cadherin is indeed clustered at the basal side of endothelial cells in contact with pericytes and astrocytes in the brain (194), suggesting that this interaction may be important in the elongation of vascular sprouts and in the protection of endothelial cells from apoptosis. Finally, Williams et al. (343) identified a region in the N-cadherin extracellular domain that is responsible for the activation of fibroblast growth factor receptors. This receptor is present in endothelial cells and is able to induce cell growth. It is possible that N-cadherin modulates angiogenesis in this way.

In the endothelium, another cadherin-like protein was described and named VE-cadherin-2 (314). This protein displays homology with other members of the cadherin family, but has a completely unrelated cytoplasmic tail. For its structural characteristics, it was included in the protocadherin family (98).

B) CATENINS AND OTHER INTRACELLULAR PARTNERS OF CADHERINS.  The cytoplasmic tail of VE-cadherin is highly homologous to that of other classic cadherins (10, 313) and, through its carboxy-terminal region, it binds -catenin and plakoglobin (also called -catenin). These two proteins are homologous and contain 10–13 so-called armadillo repeats, which are also present in many other signaling proteins. Both -catenin and plakoglobin link -catenin, which is homologous to vinculin and anchors the complex to actin (38, 337). -Catenin can also bind -actinin (168, 234) and vinculin (340), which may further stabilize AJ anchorage to actin (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1. Molecular organization of adherens junctions (AJ) and other junctional transmembrane proteins. VE-cadherin is a transmembrane protein at AJ. Through its cytoplasmic tail it binds -catenin, plakoglobin, and p120. -Catenin and plakoglobin in turn link -catenin, which binds vinculin, -actinin, and ZO-1. These interactions mediate the anchorage to actin microfilaments. Nectin is a transmembrane protein of the IgG superfamily, which promotes homophilic cell-cell adhesion. At the intracellular level, nectin binds afadin, which binds ponsin, which in turn associates to -catenin and vinculin and through them to actin. Outside AJ, PECAM, another member of the IgG family, induces intercellular adhesion and binds the phosphatase SHP-2 and -catenin. S-endo-1, also called CD146 and Muc 18, promotes homophilic adhesion, but its intracellular signaling and cytoskeletal partners are unknown.

An additional VE-cadherin partner is p120, which is a src substrate and is homologous to -catenin and plakoglobin (4, 5). However, p120 binds to a membrane-proximal domain of VE-cadherin (316) and does not associate to -catenin or other actin binding proteins. The functional role of p120 is complex. In some cell types, p120 increases cadherin-based adhesion, but in others it has an inhibitory role (13, 244, 316, 362). Tyrosine phosphorylation of VE-cadherin correlates with higher association to p120 (177). N-cadherin in endothelial cells presents lower phosphorylation in tyrosine and lower binding to p120 (227). Whether this may explain some of the functional differences of these two cadherins in the endothelium is unknown. It is intriguing that classic cadherins bind essentially the same molecules at their cytoplasmic domain but may exert different biological activities (166, 233). There should be additional and specific intracellular partners that are able to transfer cadherin- or cell-specific signals.

VE-cadherin interacts with endothelial-specific signaling proteins. For instance, it can bind to vascular endothelial growth factor (VEGF) receptor 2 (49, 286, 367) and modulate signaling through phosphatidylinositol (PI) 3-kinase. In addition, it was recently found that VE-cadherin interacts with an endothelial-specific receptor-protein tyrosine phosphatase called VE-PTP. This interaction is unusual, as it is mediated by the extracellular domains of the two proteins. VE-PTP reduces the VEGF-induced phosphorylation of VE-cadherin and strengthens cell-to-cell adhesion (229).

A specific feature of VE-cadherin is that it may behave as a desmosomal-like cadherin. As mentioned above, endothelial cells do not have classic desmosomes and do not express desmosomal cadherins (111). However, in lymphatics and in some blood endothelial cells, VE-cadherin, through plakoglobin or p0071 (48), can recruit the desmosomal proteins desmoplakin and vimentin at the membrane (172, 324). This novel desmosomal-like structure (complexus adhaerentes; Refs. 283, 284) is specific for endothelial cells, since, in epithelial cells, plakoglobin and demoplakin are mostly concentrated in desmosomes in association with desmocollins and desmoglins, which are absent in the endothelium.

Interestingly, the in vivo inactivation of the desmoplakin gene leads to a complex phenotype, which includes vascular defects, such as a reduced number of capillaries and the presence of discontinuities among endothelial cells in these types of vessels (109). These data suggest that the lack of a correct organization of complexus adhaerentes may be the reason for these abnormalities.

2. The nectin-afadin complex

This complex has been described in epithelial cells and, although its components are present in the endothelium, little is known about its functional role in this cell type. The complex consists of at least three components, i.e., nectin, afadin, and ponsin. The nectin family of calcium-independent cell adhesion molecules comprises four members. Through the cytoplasmic tails, nectins bind to the PDZ-containing protein afadin (also called AF-6), which in turn connects nectins to the actin cytoskeleton (310, 312). Ponsin binds afadin, vinculin, and -catenin. Nectins and afadin colocalize with cadherins and can reciprocally interact through -catenin. Therefore, it has been suggested that nectin/afadin may be involved in AJ organization. However, afadin was also found in association with TJ proteins, such as JAM-A (88), and nectins can interact with ZO-1. So, the specific localization of these proteins at AJ may depend on the cell type and/or the complex may play a more general role in the organization of different junctional structures.

B. Outside AJ

Endothelial cells express other adhesive proteins, which are concentrated to the intercellular clefts but are not specifically confined to the AJ and TJ. Among these molecules, PECAM is one of the most extensively studied. It is a transmembrane immunoglobulin concentrated at intercellular contacts in the endothelium and is also expressed in leukocytes and platelets (for a review, see Refs. 79, 159, 221, 231, 232). PECAM promotes either homophilic or heterophilic adhesion (221). Heterophilic ligands include the _v3-integrin (259). Using blocking antibodies, PECAM was found to participate in vascular angiogenesis in the adult (78), even if the null mutation of the PECAM gene did not cause detectable changes in vascular development in the embryo (86, 315). Several in vitro and in vivo data indicate that PECAM may modulate leukocyte infiltration (for a review, see Refs. 159, 221, 231, 334). Inactivation of the PECAM gene in mouse increases vascular permeability and sensitivity to experimental autoimmune encephalomyelitis (117). In other models of inflammation, however, the role of PECAM was less apparent, thus suggesting compensation with other adhesive proteins (86, 315). It has been recently reported that neutrophils of PECAM-null mice can traverse endothelial cells but remain entrapped between endothelial monolayer and basement membrane (315). This effect seems to be due to defective expression of ₆₁ on the neutrophil membrane upon activation (75). More recent data show those leukocytes need to interact sequentially with PECAM and another junctional protein, i.e., CD99, for efficient transmigration (282).

The way through which PECAM transfers intracellular signals is not yet fully clear. It can bind intracellular partners mainly through phosphorylation-related kinases (for review, see Ref. 145). The tyrosine residues are located in an immunoreceptor tyrosine based activation motif (ITAM) (198). Among the intracellular partners of PECAM, the best studied are SHP-2 and -catenin (144). SHP2 may play a role in the Ras-mitogen-activated protein kinase (MAPK) activation cascade (for review, see Ref. 145). Tyrosine phosphorylated -catenin associates with PECAM, even if PECAM itself does not need to be phosphorylated. PECAM may exert similar biological activities like the cadherins through its binding to -catenin (see below). In addition, it has been suggested that PECAM can modulate cadherin-mediated cell-cell interactions through the interaction with different intracellular proteins and activation of the MAPK pathway (287). Finally, PECAM can modulate cell adhesion and migration possibly through an interaction with matrix adhesion receptors, such as integrins (156, 306, 308).

Another endothelial junctional protein is S-endo 1, which is also called CD146 and Muc 18. This immunoglobulin-like protein was originally described in melanoma, but is also expressed in smooth muscle and endothelial cells and not in hemopoietic stem cells and blood cells. S-endo 1 is located at endothelial junctions (27) and induces homophilic cell adhesion.

Endoglin is expressed at high levels on vascular endothelial cells and is concentrated at interendothelial junctions. Its role as an accessory component of the receptor system of members of the transforming growth factor- superfamily has been investigated in detail (256). Mutations of the endoglin gene in humans determine a vascular disorder called hereditary hemorrhagic telangiectasia type 1 that is characterized by arterovenous malformations. In mice, inactivation of the endoglin gene leads to a vascular phenotype that is reminiscent of VE-cadherin-null animals (42, 212). Whether the junctional localization of endoglin has functional consequences for its interaction with transforming growth factor- receptor and/or for cell interaction and vascular remodeling is still an open issue.

C. Intracellular Signaling Through AJ

1. Transcriptional activity of catenins

A prominent feature of -catenin, plakoglobin, and p120 is the ability to translocate to the nucleus and, in association with other transcription factors, to modulate gene expression. -Catenin is a key member of the Wnt signaling pathway (38, 39, 258). This family of growth and differentiation factors plays a crucial role in cell specification during embryonic development. Many members of the Wnt family act through -catenin transcriptional activity (for a review, see Ref. 39). When -catenin is released in the cytosol, it can be quickly inactivated through phosphorylation in amino-terminal serine and threonine by the action of casein I and glycogen synthase kinase-3 (GSK-3) in complex with the tumor suppressor axin and adenomatous polyposis coli (APC) (39, 196). Upon phosphorylation, -catenin is ubiquitinated and degraded in proteasomes. If -catenin phosphorylation is inhibited (as by Wnt signaling), it can translocate to the nucleus and bind the Tcf/LEF transcription factors complex and influence gene transcription. Mutations of either -catenin or members of its degrading machinery have been associated with malignant cell transformation (see for a review Ref. 261). In general, overexpression of -catenin in different cell systems leads to increased cell proliferation and reduced sensitivity to apoptosis, so that -catenin stabilization or permanent signaling may facilitate tumor progression. There is a long list of -catenin target genes, which will likely increase in the future and includes genes important in cell division and apoptosis (38, 39, 261).

When -catenin is bound to cadherins, it is stabilized and retained at the cell membrane. Therefore, absence or mutation of cadherins may increase the pool of free -catenin in the cytosol and its signaling activity (115, 297). An extensive literature is available showing an inverse correlation between cadherin expression and tumor progression (for a review, see Ref. 313).

An important question is how -catenin may detach from the cadherin tail. Tyrosine phosphorylation of -catenin and, in particular, phosphorylation of residue Tyr-654, causes a sixfold reduction in affinity of -catenin for the cytoplasmic tail of E-cadherin (139, 272). The reduction in affinity likely increases -catenin release in the cytosol and eventually promotes its nuclear translocation. In contrast, serine phosphorylation of specific residues in the cadherin tail leads to specific phosphoserine interactions with -catenin (139). Both casein kinase II and GSK-3 can induce phosphorylation of these sites and strongly increase -catenin binding to cadherins, thus reducing -catenin signaling and increasing the strength of cell-to-cell adhesion (192).

Little is known about the transcriptional role of -catenin in endothelial cells. As other cadherins in other cell types (297), VE-cadherin may contribute to contact inhibition of growth (50) by limiting -catenin transcriptional activity (333). Interestingly, similarly to other malignant tumors, angiosarcomas (while expressing several endothelial markers) are negative for VE-cadherin staining (204), thus suggesting that the absence of the cadherin might increase the levels of free -catenin and contribute to endothelial cell growth and transformation.

Plakoglobin was also shown to bind to Tcf/LEF, although to a site different than -catenin (215), and influence cell transcription in both a positive and a negative way (for review, see Ref. 38). Similarly to -catenin, overexpression of plakoglobin in different tissues induced cell proliferation and transformation (128, 370). p120 also binds to specific transcriptional partners, including Kaiso (5), and can transcriptionally upregulate different genes, including E-cadherin (147).

A general consideration emerging from this complex picture is that several cytoplasmic members of AJ (but also TJ; see below), in addition to promoting anchorage of junctional transmembrane proteins to actin, may also translocate to the nucleus and influence transcription. This finding, together with the observation that junctions are frequently altered during tumor transformation, supports the concept that these structures may have a more complex role, and that, in addition to simply promoting cell-to-cell adhesion, they may also modulate cell growth and differentiation.

2. Other signaling pathways at the junctions

In addition to maintaining -catenin at the membrane and preventing its nuclear translocation, cadherins may also signal through other pathways. In endothelial cells, null mutation of VE-cadherin has lethal effects, and embryos die in utero at early stages of development (49). Although endothelial cells are able to form the primitive vascular plexus, vascular remodeling is missing and, already at embryonic day 8.75–9.00, endothelial cells appear disconnected from each other, detach from the underlying basement membrane, and lay scattered inside the vascular lumen. In the heart, the endocardium is highly disorganized, trabeculation is impaired, and the myocardium is loosely assembled.

To gain a mechanistic view of the defects linked to the absence of VE-cadherin, we cultured endothelial cells from VE-cadherin-null animals. By comparing isogenic endothelial cell lines that differ for VE-cadherin expression, we found that several vascular responses are severely impaired by the absence of the protein.

First, VE-cadherin –/– cells are more prone to apoptosis and are unable to respond to VEGF protective signals (49). We found that, similarly to other cadherins (360), VE-cadherin clustering at the junctions can activate PI 3-kinase and Akt phosphorylation. Upon activation with VEGF, the VEGFR2 and PI 3-kinase subunits associate to the VE-cadherin/catenin complex, leading to more efficient activation of Akt (49). Mutants of VE-cadherin missing -catenin or plakoglobin binding domain cannot form this multiprotein complex (49). In contrast, mutants lacking the p120 binding domain can still associate to VEGFR2, albeit less efficiently.

Dismantling VE-cadherin from cell-to-cell contacts with blocking antibodies reduced both Akt phosphorylation by VEGF and its protective effect on apoptosis (49). This finding suggests that not only VE-cadherin expression but also its clustering at junctions is required for a correct interaction with intracellular partners. Others have found that cadherins may interact with growth factor receptors, such as epidermal growth factor (EGF) receptor (85, 256).

Both N- and VE-cadherins can directly bind to shc, an adaptor protein that participates in the Ras signaling pathway (357, 367). We found that shc binding to VE-cadherin requires cell activation by VEGF and tyrosine phosphorylation of VE-cadherin cytoplasmic tail. The interaction decreased shc phosphorylation after VEGF and likely reduced the persistence of the mitogenic stimulus (367).

Similarly to E-cadherin (224, 242), VE-cadherin expression and clustering at the junctions influence the activation of small GTPases by inducing Rac and reducing RhoA activation (181). The Rac guanosine exchange factor Tiam (123, 201) codistributes with VE-cadherin at AJ, but remains diffuse on the cell membrane in VE-cadherin negative cells (181). It is likely that VE cadherin activates and recruits Tiam through the induction of the PI 3-kinase pathway and that Tiam, in turn, activates Rac (Fig. 2). The biological consequences of inducing this pathway are multiple. First, Rac induces actin redistribution in endothelial cells and acquisition of the typical cobblestone morphology (181). In addition, Rac may be also involved in cell migration, growth, and differentiation (265).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Schematic representation of VE-cadherin signaling through phosphatidylinositol 3-kinase (PI3K). VE-cadherin can associate to vascular endothelial growth factor receptor 2 (VEGFR2) and induce PI3K activation. The enzyme inhibits cell apoptosis through phosphorylation of Akt. In addition, PI3K activation brings Tiam at the membrane. Tiam is a guanosine exchange factor, which activates Rac and, as a consequence, induces actin rearrangement. VE-cadherin can bind shc when cells are stimulated by vascular endothelial growth factor (VEGF), but the functional consequences of this interaction are still unknown.

Also p120 may influence small GTPases. When it is released in the cytosol, it binds and inhibits Rho with complex consequences for cell adhesion and motility (3, 120, 239).

Several phosphatases (PTP-µ, PTP-K, SHP-1, SHP-2, PTP-LAR, and PTP-B) have been found to associate with the cadherin/catenin complex (25, 43, 99, 176, 317, 323) and are likely to modulate phosphorylation of the complex and/or its intracellular partners. As mentioned above, VE-PTP is an endothelial-specific phosphatase, which associates with VE-cadherin (229). Another interesting phosphatase is DEP1 or CD148 (250), which, although not endothelial specific, is upregulated by cell confluency and localizes at intercellular junctions in confluent endothelial cells (311). In other cells, it can bind p120 and -catenin (135). In a recent paper, we have reported that VE-cadherin is important in contact inhibition of cell growth. More specifically, VE-cadherin binds VEGFR2 and induces its dephosphorylation through the action of DEP-1 (182).

Other unexpected activities of VE cadherin have been reported. It was found that VE-cadherin may act as a transducer of shear stress signals acting in concert with VEGFR2 (286). When endothelial cells are exposed to shear, VEGFR2 associates to the VE-cadherin--catenin complex and translocates to the nucleus. This phenomenon does not occur in absence of VE-cadherin and, consistently, VE-cadherin expression is required for induction of shear-responsive promoters. Similarly, also PECAM was reported to transduce shear-dependent cell activation (248). In endothelial cells exposed to shear, PECAM is phosphorylated in tyrosine, associates with SHP-2, and regulates extracellular signal-regulated kinase (ERK). It is of interest that two endothelial-specific junctional proteins, such as PECAM and VE-cadherin, may act as "mechanosensors" for shear changes.

VE-cadherin may also act as fibrin receptor, and this interaction induces endothelial cells to form capillary-like structures in three-dimensional gels (18). The intracellular signals transferred by fibrin through VE-cadherin are still unknown but suggest a "nonjunctional" role of this protein.

From all these observations, it appears that cadherins, similarly to integrins, may promote the formation of multiprotein complexes, by binding different effectors and facilitating their reciprocal interaction. Cadherin ability to cluster at junctions may further amplify this process by creating multimeric complexes, where protein-protein interaction is optimized.

Cadherins may trigger rapid and short-lasting responses (257), such as PI 3-kinase or MAPK activation at early moments of contact formation, which may be important for quick signaling of cell position. However, cadherins and, in particular, VE-cadherin may also transfer lasting signals, which contribute to the maintenance of cell homeostasis. Comparing long-term confluent cultures of VE-cadherin +/+ and –/– cells, we found that this protein protects endothelial cells from apoptotic stimuli and attenuates cell growth. These are lasting effects, which require constitutive expression and clustering of VE-cadherin at junctions and likely continuous intracellular signaling.

D. Modulation of AJ Organization and Vascular Permeability

Endothelial cells control the passage of plasma proteins and circulating cells from blood to tissues. This function is finely regulated by the so-called transcellular and paracellular pathways (87, 97, 214, 268, 290, 298). The transcellular pathway defines the passage of plasma components through the endothelial cytoplasm by the action of vesicular systems and fenestrae. The paracellular pathway is regulated by opening of cell-to-cell junctions and/or by rearrangement of their architecture. Although both TJ and AJ can play an important role in the control of endothelial permeability, TJ have been always considered as the key regulators of this function (see sect. III).

A clear role for VE-cadherin in maintaining permeability was demonstrated by experiments in vivo, where anti-VE-cadherin antibodies were injected in mice (69). This treatment induced a marked increase in vascular permeability within a few hours. In vitro and in vivo staining of VE-cadherin showed that the antibodies were selective, as they were able to disrupt VE-cadherin clustering at the junctions, while leaving the distribution of other junctional components unchanged. Notably, the effect of VE-cadherin inhibition was strong in lungs and heart but undetectable in other organs, such as brain, muscles, and skin. This observation suggests that the relevance of VE-cadherin in controlling vascular permeability is different along the vascular tree and is likely dependent on the type and organization of other junctional structures.

AJ organization displays different characteristics in growing vessels compared with resting vasculature. It was found that tumor microvasculature is particularly sensitive to VE-cadherin blocking antibodies. Some monoclonals were able to dismantle VE-cadherin junctions in tumors without affecting permeability of constitutive vessels (70, 191). In addition, VE-cadherin presents epitopes, which are only exposed in tumor vasculature and not in systemic vessels (191).

These in vivo data are supported by in vitro observations. AJ organization is different at different stages of cell confluency. In sparse cells, VE-cadherin is highly phosphorylated in tyrosine and preferentially binds -catenin and p120 (177). In contrast, in long confluent cells, the amount of plakoglobin linked to VE-cadherin is higher (178). These changes correlate with a lower control of permeability in subconfluent cells, compared with stabilized long-confluent cultures (9).

It is conceivable that the response to permeability-increasing agents is influenced by the degree of junction organization and strength. For instance, the effect of histamine is more marked in loosely confluent cells, where this agent induces cell retraction and gap formation. This was accompanied by stronger increase in VE-cadherin tyrosine phosphorylation compared with long-confluent cultures (9). Hence, as suggested by in vivo data, agents able to disrupt junctions in subconfluent and growing endothelium may be inactive in stabilized long-confluent cells.

An important issue is how permeability-increasing agents modify junctional architecture and eventually create intercellular gaps. Cell retraction is certainly an intuitive mechanism, which has been observed in cultured cells upon exposure to thrombin, histamine, plasmin, and other permeability-increasing agents (9, 68, 84, 92, 130, 288, 298, 338) or in vivo in inflamed vessels (26). More subtle changes, such as phosphorylation of junctional proteins and their dissociation from the actin cytoskeleton, may result in increased permeability without frank appearance of intercellular gaps (9). VE-cadherin –/– endothelial cells present increase in permeability but no intercellular gaps (70, 227). This may explain why in vivo conditions of apparent increase in permeability are not always accompanied by morphological evidence of junction disruption. In general, however, it is likely that the combination of both cell retraction and junctional changes leads to marked increase in permeability. Endothelial cell contraction has many characteristics in common with smooth muscle cell retraction and depends on calcium/calmodulin-dependent myosin light-chain kinase (for a review, see Refs. 84, 298).

Small GTPases may regulate the organization of AJ and TJ (44, 351). In keratinocytes, the activity of Rho and Rac is required for successful assembly of cadherins and AJ formation (44). This, however, is not the case for endothelial cells, where VE-cadherin remains unperturbed, even in the presence of dominant negative mutants of these small GTPases (45). The lack of response is related to the cell-specific context, since the function of VE-cadherin in transfected CHO cells was susceptible to Rho and Rac inhibition. It is possible that different targets of the small GTPases are expressed in different cell types and that endothelial and epithelial cells regulate permeability in different and cell-specific ways.

Although apparently dispensable for the formation and maintenance of VE-cadherin at AJ in the endothelium, small GTPases play an important role in the action of permeability-increasing agents. Thrombin strongly increases RhoA and Rac activation (329, 330, 338, 351), and this causes endothelial cell rounding and retraction, as well as increase in permeability (92, 130, 338). However, Rho activation alone is not sufficient to increase endothelial permeability. When the Escherichia coli cytotoxic necrotizing factor-1 was added to endothelial cells, it caused strong induction of Rho and stress fibers reorganization (339). VE-cadherin, however, remained at junctions, and no sign of cell retraction was reported. These observations underline that both cytoskeletal rearrangements and junctional protein modifications are needed for clear appearance of intercellular gaps and eventually increase in permeability.

Phosphorylation in tyrosine of junctional components has been described after thrombin (323), VEGF (91), or histamine (9). Thrombin would increase phosphorylation by inducing dissociation of the phosphatase SHP2 from the VE-cadherin complex. Thrombin may also change VE-cadherin-catenin organization at junctions and reduce the amount of plakoglobin associated with the complex (262). Inhibitors of protein kinase C (PKC) inhibit junction reorganization and the increase in permeability induced by thrombin.

VEGF induces a rapid phosphorylation of VE-cadherin and catenins, which is followed by increase in junction permeation (91) and in same cases VE-cadherin redistribution from intercellular junctions (355). This effect may be inhibited by the phosphatase VE-PTP (see above) (229).

Overall, these data support the concept that tyrosine phosphorylation of VE-cadherin and/or its intracellular partners is involved in regulating the strength of cell-to-cell contacts. In general, however, it cannot be excluded that cadherin and catenin phosphorylation may represent a general reaction to stressful agents, which results in increased permeability but may also trigger more complex cell responses and interactions.

Association of the cadherin-catenin complex to actin microfilaments stabilizes junctions. Truncated VE-cadherin, which is unable to bind -catenin and to link actin, exerts a lower control of paracellular permeability. Mobilization of VE-cadherin and catenin in the detergent-soluble fraction, which is considered an indirect measure of dissociation from the cytoskeleton, is always increased in conditions of high permeability (9, 177). Similar findings were obtained using E-cadherin-expressing epithelial cells (175, 331). Therefore, part of the mechanism of action of permeability-increasing agents may consist of dissociating VE-cadherin from the actin cytoskeleton.

Activation of Rho and increase in intracellular Ca²⁺ levels may act in this direction. More specifically, it has been found that IQGAP1 (100), an effector of small GTPases, such as Rho, Cdc42 and Rac1, negatively regulates cadherin-mediated cell-to-cell adhesion by displacing -catenin from its binding to -catenin. This would in turn cause cadherin-catenin complex dissociation from the actin cytoskeleton and cell-cell dissociation (134). These effects have been described in epithelial cells, but IQGAP is also expressed in endothelial cells and thus may modulate permeability in this cell system as well.

Other ways for rapid cadherin regulation is recycling (185) and digestion by proteases (68, 148, 203, 238, 267). Classic cadherin and, in particular, VE-cadherin extracellular domain is highly susceptible to lysis (77). Induction of metalloproteases may induce cleavage and secretion of the extracellular domain of E-cadherin (58, 238) and promote junction disruption. However, cadherins may be cleaved also at the membrane-cytoplasm interface by proteases activated during apoptosis or Ca²⁺ imbalance (129). It was found that this process might be mediated by the presenilin 1/-secretase system, which dissociates E-cadherin and -catenin from the cytoskeleton (203). Furthermore, the cleavage induces the release of E-cadherin cytoplasmic domain and increases the levels of cytosolic -catenin and -catenin. This system works also for VE-cadherin, thus suggesting that it may promote disassembly of junctions and increase in permeability. Finally, ADAM15, which belongs to a family of proteins containing both a disintegrin and a protease domain, codistributes and associates with VE-cadherin at AJ. The biological role of this interaction is not yet clear, but it is possible that ADAM15 contributes to the local cleavage of VE-cadherin (124).

Finally, other proteins outside junctions may contribute to the control of permeability. For instance, PECAM can be phosphorylated by permeability-increasing agents, such as VEGF (91), shear stress, or can be displaced and internalized upon activation with inflammatory cytokines (266). In PECAM-null animals, the absence of this protein leads to increase in vascular permeability, but only in pathological conditions (117).

E. Conclusions

AJ in endothelial cells are key structures for the maintenance of cell-specific properties, such as permeability to solutes or inflammatory cells, contact inhibition of cell growth, and apoptosis. The molecular organization of AJ, although not fully elucidated yet, is complex and includes both adhesive and signaling molecules. It is likely that additional membrane and cytoplasmic junctional proteins will be found in the future, which may interact, modulate AJ organization, and promote the transfer of intracellular signals. Manipulation of AJ and other junctional adhesive proteins may be important in the therapy of vascular inflammatory reactions, edema, and angiogenesis.

	III. TIGHT JUNCTIONS

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A. Introduction to TJ

1. Intercellular strands as morphological and functional units of tight junctions

Forty years ago, in 1963, TJ were defined by electron microscopy as a specialization of the plasma membrane and were named zonula occludens. In thin sections, TJ appear like a sequence of fusions (or "kisses"), which are formed between two adjacent cells by the outer leaflets of the plasma membrane. At higher magnification, however, it becomes clear that the membranes are not fused, but in tight contact to each other. In freeze-fracture preparations, TJ look like anastomosis of fibrillar strands within the plasma membrane. In turn, the strands are composed of particles that, with some exceptions, are preferentially localized to the internal leaflet (P-face). In correspondence of the strands, mostly on the external leaflet (E-face), there are complementary grooves (96). TJ are the most apical component of the junctional complex. Together with AJ, desmosomes, and gap junctions, TJ mediate adhesion and communication between adjoining cells. Specifically, TJ are responsible for regulating paracellular permeability and maintaining cell polarity, which are often referred to as "barrier" and "fence" function, respectively.

The appearance of TJ as fusions between adjacent membranes, right at the boundary between apical and basolateral domains, suggested that TJ might be instrumental in occluding the lateral intercellular space (LIS). In addition, it was hypothesized that the function of TJ might consist of restricting both the diffusion of solutes across intercellular spaces (barrier function) and the movement of membrane molecules between the apical and basolateral domains of the plasma membrane (fence function). Subsequent studies lent further support to this hypothesis, by correlating number and complexity of the TJ strands with the ability to restrict fluxes of electrolytes. In this conceptual framework, it was proposed that cohesive interactions between TJ molecules localized on apposing strands might provide the mechanistic basis for the barrier and fence function of TJ. However, at present, demonstration that strands are indeed the morphological and functional units of TJ is far from being proven (320). In the past, even the biochemical nature of the strands has been debated. Some researchers proposed that the strands were made of lipids forming cylindrical micelles within the membrane bilayer (161). Now, it is commonly accepted that the strands are primarily (but perhaps not exclusively, Ref. 118) composed of proteins. However, important questions have not been exhaustively answered yet. Specifically, which proteins are the essential building blocks of TJ? How do they mutually interact during TJ assembly? Finally, how do these proteins mediate the barrier and fence functions?

As in some other areas of cell biology, appreciation of the functional role of TJ predated description of their molecular architecture. Only in the past decade, a powerful combination of biochemical, immunochemical, and recombinant techniques has allowed the discovery and characterization of several TJ proteins. In parallel, researchers have been realizing that these proteins are capable of multiple interactions, both among themselves and with other molecules, including signaling mediators and transcription factors. We provide here a description of the individual TJ molecules (see sect. IIIB) and an overview of their complex network of interactions (see sect. IIIC). The challenging task to understand how interactions among TJ components bring about TJ assembly and function is just at the beginning. In this review, we emphasize the molecular basis of TJ function and regulation, with reference to TJ assembly (see sect. IIID), as well as TJ-mediated regulation of paracellular permeability (see sect. IIIE) and cell polarity (see sect. IIIF). Finally, we survey in vivo studies on the physiological role of TJ molecules (see sect. IIIG).

TJ are expressed in both epithelial and endothelial cells. In columnar epithelial cells, TJ clearly subdivide the plasma membrane into apical and basolateral domains (which face lumen and connective tissue, respectively). In addition, some epithelial cell lines have been widely used as in vitro systems for the study of TJ structure, function, and regulation. Hence, research on TJ has focused on epithelial cells, and most information reviewed here of necessity refers to epithelial biology. At variance, the role of TJ in endothelial cells has been analyzed less extensively. Yet, in these cells too, TJ restrict permeability and separate the membrane into apical and basolateral regions (which face blood and perivascular spaces, respectively). Hence, several conclusions obtained from epithelial systems can be reasonably applied to endothelial cells. However, some important caveats should be taken into account when making such extrapolations. First, in most endothelial cells, the precise localization of TJ and their separation from other junctional organelles is not so clear-cut as in epithelial cells. Second, even if endothelial and epithelial cells share numerous TJ components, the same molecules might be differentially assembled and regulated in the two cell types. Third, there is considerable variability among different segments of the vascular tree. Specifically, in large vessels, TJ are well developed in arteries and less elaborate in veins. Similarly, in small vessels, TJ are well organized in arterioles, but loosely organized (even with some gaps) in postcapillary venules, which are a preferential site for the extravasation of plasma proteins and circulating leukocytes. Finally, TJ are well developed in brain vessels, where they contribute to the blood-brain barrier, and less organized in other organs, which are characterized by high rate trafficking (291). When available, we highlight information that specifically relates to endothelial TJ and their unique role in vascular function.

B. Molecular Components of TJ

Like other junctional organelles, TJ are composed of both transmembrane and intracellular molecules (Fig. 3). In 1986, zonula occludens-1 (ZO-1) was discovered as the first intracellular component of TJ. In subsequent years, extensive effort aimed to identify integral membrane proteins that might act as partners for ZO-1. Occludin, the first transmembrane protein identified at the TJ, was discovered 7 years later, in 1993. The last few years have been witnessing the identification of novel TJ components at an ever-increasing pace. The long list of TJ proteins in itself speaks for the complexity of the molecular architecture of TJ. Our aim in this section consists of providing a brief survey of individual TJ molecules as an introduction to subsequent sections on molecular interactions and the molecular basis for TJ function. For a more detailed description, the reader is referred to other reviews (51, 216, 303).

View larger version (40K): [in this window] [in a new window]   FIG. 3. Schematic representation of tight junctions (TJ). The apparent fusions of adjoining plasma membranes are represented here as the prefential site for TJ strands. Occludin, claudins, and small junctional immunoglobulins (sJI; e.g., JAM-A) are transmembrane proteins at the TJ. Through their cytoplasmic tails, they bind a vast array of cytoplasmic and cytoskeletal proteins, which in turn are involved in functions as diverse as cell signaling, cell polarity, transcriptional regulation, and membrane trafficking. A prominent role is represented by the structural assembly of protein complexes, which link the transmembrane proteins to cortical actin and to actin microfilaments.

A) OCCLUDIN.  Occludin was identified using monoclonal antibodies raised against the junctional fraction of chick liver. Occludin, which has a molecular mass of 65 kDa, predictably contains two extracellular loops and four membrane-spanning regions. Both the amino and carboxy termini are localized in the cytoplasm (104). Transcripts of occludin from different vertebrate species only display a high degree of identity in the first extracellular loop (which contains several glycine and tyrosine residues) and in the carboxy-terminal tail (8). The functional importance of these domains in cell adhesion and molecular associations is discussed below. Occludin-1B is an alternatively spliced variant that contains an insertion of 56 amino acids. Its subcellular localization and tissue distribution are indistinguishable from those of occludin (223). Occludin is exclusively localized at the TJ of epithelial and endothelial cells. Interestingly, expression of occludin in the endothelium correlates with the permeability of different segments in the vascular tree. Occludin is expressed at high levels (with a continuous distribution) in brain endothelial cells and at much lower levels (with a discontinuous pattern) in endothelial cells of nonneural tissues (131).

Like other transmembrane components of TJ, occludin might contribute to intercellular adhesion. Upon expression in fibroblasts, exogenous occludin localizes to points of cell-cell contact (in confluent cells) and induces aggregation (in cells kept in suspension). Surface expression and adhesion, however, are not intrinsic properties of occludin, but require that endogenous ZO-1 be organized at the membrane, probably at the AJ. Adhesion is likely dependent on the conserved first extracellular loop of occludin, as synthetic peptides corresponding to this domain inhibit adhesion (326). At variance, a peptide that encompasses the second loop removes occludin from the TJ of Xenopus kidney epithelial cells and affects transepithelial electrical resistance (TER; Ref. 354). In Madin-Darby canine kidney (MDCK) cells, expression of a deletion mutant of occludin lacking the cytoplasmic tail removes endogenous occludin from TJ and affects both the flux of nonelectrolyte solutes and the fence function of TJ, even if it leaves TER unaffected (24). Finally, occludin may also play a role in the transepithelial migration of leukocytes (140). Thus, in addition to contributing to intercellular adhesion, occludin is involved in multiple TJ functions.

B) CLAUDINS.  The family of claudins comprises more than 20 members. Like occludin, claudins have four membrane-spanning regions, two extracellular loops, and two cytoplasmic termini. However, they are smaller (with a molecular mass of 22 kDa) and display no sequence similarity to occludin (218). Claudins-1 and -2 were discovered in the same fraction where occludin had been identified. Upon expression in L fibroblasts, claudins-1 and -2 concentrate at sites of cell contact, reconstitute TJ-like strands, and recruit occludin to the strands. Interestingly, claudin-1-based strands are continuous and associated with the P-face, whereas claudin-2-based strands are fragmented and associated with the E-face (106), thus suggesting that claudin-2 forms more leaky TJ than claudin-1. In addition, claudin-2 is differentially expressed in two MDCK strains that are characterized by different TER. Specifically, claudin-2 is absent in MDCK I but present in MDCK II (the strain with lower TER), and transfection of claudin-2 in MDCK I decreases TER to the levels observed in MDCK II (102). In vivo, claudin-2 is expressed in the proximal tubules of the kidney, the site of passive resorption of sodium and water (90).

Some claudins have a limited distribution. For instance, claudin-5 is restricted to endothelial cells. Upon transfection in L fibroblasts, claudin-5 forms TJ-like strands that associate with the E-face (220), as it is commonly found in most endothelial cells. In contrast, strands associate with the P face in endothelial cells of the blood-brain barrier, which express both claudins-1 and -5. Interestingly, small vessels of glioblastoma do not express claudin-1, and the remaining claudin-5-based TJ strands are only detectable on the E-face (193). These molecular alterations might contribute to the formation of edema, which is a clinical feature of brain tumors. The involvement of claudins in paracellular permeability is discussed in detail in section IIIE1.

C) JUNCTIONAL ADHESION MOLECULE-A AND RELATED MOLECULES.  Junctional adhesion molecule-A (JAM-A, which was previously called JAM, JAM-1, and F11R) is a 32-kDa glycoprotein that is composed of an extracellular region, a transmembrane segment, and a short cytoplasmic tail. Upon transfection in CHO cells, JAM-A localizes to sites of cell contact and reduces paracellular fluxes, possibly by favoring intercellular adhesion. The molecule was named after its subcellular localization and predicted function in adhesion (205) (see for review, Refs. 29, 31, 159, 221). In addition, a blocking antibody and a recombinant fragment (190, 197) inhibit the establishment of TER, thus further supporting a role for JAM-A in TJ function. Finally, JAM-A contributes to the transendothelial migration of leukocytes, as determined using in vitro assays and in vivo models of inflammation in mice, such as accumulation of leukocytes in subcutaneous spaces (205) and in cerebrospinal fluid (80).

The extracellular segment of JAM-A comprises two Ig-like domains, an amino-terminal (V_H-type) and a carboxy-terminal (C₂-type) fold, respectively. In solution, a recombinant soluble protein, which corresponds to the whole extracellular domain of JAM-A, binds in a homophilic manner, thus suggesting that JAM-A may mediate homotypic cell adhesion (32). In addition, JAM-A forms parallel and noncovalent homodimers, which might expose at the cell surface an adhesive interface for the homophilic interactions (171). Interestingly, JAM-A may also mediate heterophilic adhesion to ligands as diverse as the leukocyte _L2-integrin (249) and the attachment protein sigma1 of Reovirus (28).

More recently, independent groups identified two molecules that are homologous to JAM-A, namely, JAM-C and JAM-B. Similarly to JAM-A, these novel members of an emerging family of small junctional proteins all display an amino-terminal V_H-type and a carboxy-terminal C₂-type Ig-like fold, a single transmembrane region, and a short cytoplasmic tail ending with a putative PDZ-binding motif. JAM-C, which has been described in mouse as JAM-2 (17) and in humans as JAM-3 (14), has been localized in high endothelial venules and lymphatic vessels of lymphoid organs, as well as in vascular structures of the kidney. Ectopically expressed JAM-C localizes closely to the TJ of transfected epithelial cells and increases paracellular permeability (16). Similarly, lymphocyte migration is increased in JAM-C-transfected endothelial cells (158). Hence, it is intriguing to speculate that different JAMs at the TJ may differentially regulate junction patency. JAM-B has also been described in mouse and humans and was originally named VE-JAM (253) and JAM-2 (73). It has a broad tissue distribution and is capable of both homophilic and heterophilic interactions with JAM-C. However, albeit localized to intercellular contacts, its precise localization to the TJ remains unclear.

Finally, two additional small junctional Ig-like proteins with a similar V_H-C₂ tandem arrangement have been described at the TJ. The former is ESAM, an acronym for endothelial cell-selective adhesion molecule (132). The localization of ESAM at the TJ is supported by its colocalization with ZO-1 in brain and muscle capillaries (226). The latter molecule is CAR (Coxsackie- and adeno-virus receptor), which also participates in TJ assembly and in the regulation of paracellular permeability (65).

2. Intracellular proteins

Among intracellular TJ components, ZO-1 (together with the related proteins ZO-2 and ZO-3) is perhaps the most extensively studied molecule (Fig. 4). The ZO proteins belong to the family of membrane-associated guanylate kinases (MAGUK), which possess a distinct modular organization, associate peripherally with the membrane, and assemble molecular complexes at junctional structures, such as TJ and synapses (for a review, see Ref. 93). In addition to the ZO proteins, TJ comprise other MAGUKs, as well as a heterogeneous class of cytoplasmic molecules that mediate functions as diverse as anchorage to actin, establishment of cell polarity, membrane trafficking, cell signaling, and control of gene expression.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Molecular complexes at the TJ. Modular structure of the three ZO proteins. The binding regions for most of the ZO-interacting proteins are indicated.

ZO-1 contains three PDZ, an SH3, and a guanylate kinase domain, as well as an acidic and a proline-rich region. Between these two regions, alternative splicing inserts a stretch of 80 amino acids, which is termed motif (345). The ZO-1-⁺ isoform is expressed in most epithelia, while the ^– isoform is only detectable in endothelial cells, Sertoli cells, and slit diaphragms of kidney glomeruli. As these cells have dynamic junctions, absence of the motif might correlate with TJ plasticity (20, 173). As to ZO-1 regulation, calcium-dependent intercellular adhesion is required for ZO-1 localization to the TJ (7, 289). Once localized to the TJ, ZO-1 becomes insoluble in nonionic detergents, suggesting strong association with cortical actin (6).

ZO-2 and ZO-3 share sequence homology and domain organization with ZO-1 (157). ZO-2 has a molecular mass of 160 kDa and may contain the motif , an alternatively spliced segment of 32 amino acids (34). Although initially regarded as a TJ-specific molecule (121), ZO-2 can also localize to spotlike AJ in nonepithelial cells (150). ZO-3 has a molecular mass of 130 kDa. At variance with ZO-1 and -2, the proline-rich motif is located between the second and third PDZ domain (127). Expression of the amino-terminal half of ZO-3 perturbs junction assembly (348) and, as more recently reported, produces global effects on the actin cytoskeleton, with decreased formation of stress fibers and focal adhesions, which is likely related to reduced RhoA activity (349). Finally, in addition to ZO proteins, other MAGUK (i.e., MAGI and CASK) have been reported at the TJ (see sect. IIIC1).

B) OTHER INTRACELLULAR PROTEINS.  The remaining class of intracellular proteins comprises three non-MAGUK that contain PDZ domains (i.e., AF-6/Afadin, PAR-3/ASIP, and MUPP-1). AF-6 contains a Ras-binding, a PDZ, and a myosin V-like domain. The subcellular distribution of AF6 is still controversial, as it has been reported either at the TJ (359) or at the AJ (202) (see also above). PAR-3/ASIP has been localized at the TJ of MDCK cells and enterocytes (155). The analogy with its Caenorhabditis elegans homolog, which controls axis formation during early embryonic development, suggests that PAR-3 might play a role during the establishment of cell polarity in vertebrate cells (245). Its involvement in cell polarity is discussed in section IIIF. Finally, multi-PDZ domain protein-1 (MUPP-1) contains 13 PDZ domains and has been detected exclusively at the TJ of epithelial cells (125).

Additional molecules that lack PDZ domains localize at the TJ. Cingulin is a 140- to 160-kDa component of endothelial and epithelial TJ (60). It contains a globular head, an -helical rod domain, and a carboxy-terminal tail. The central rod domain mediates the formation of coiled-coil parallel dimers, which can further aggregate (71). Albeit colocalized with ZO-1, cingulin is more distant from the TJ than ZO-1, as evaluated by electron microscopy (302). Symplekin is a widely distributed 125-kDa protein. In epithelial and Sertoli cells, it localizes both at the TJ and in the nucleus. In other cell types, symplekin is only detectable in the nucleus, which suggests a possible role in the regulation of gene expression. It is not expressed in endothelial cells (165). Finally, 7H6 is a 155-kDa antigen localized at the TJ of hepatocytes and other polarized cells (368). It likely plays a role in the barrier function of TJ in epithelial and endothelial cells (281).

PILT and JEAP have been identified recently at the TJ. PILT (protein incorporated later into TJ) is a 61-kDa protein containing a proline-rich domain. As the acronym indicates, the molecule is only recruited to TJ following the formation of claudin-based strands, even if it does not directly interact with claudin (164). JEAP (junction-enriched and -associated protein) is a 98-kDa protein that contains a polyglutamic acid repeat, a coiled-coil domain, and a carboxy-terminal consensus motif for binding PDZ domains. It is specifically expressed in epithelia of exocrine glands, such as pancreas, lachrymal, and salivary glands (235). An intriguing finding is the localization at the TJ of the serine-threonine kinase WNK-4, whose mutations cause a form of secondary hypertension in humans (346). Finally, the monomeric G proteins Rab3b and Rab13 (341, 366), as well as VAP-33 and the Sec6/Sec8 complex (119, 183), localize to the TJ. The role of these proteins in membrane and vesicle trafficking suggests that TJ may act as target sites for vesicular targeting and dockin