While the importance of JAM-A in endothelial and epithelial barrier function is appreciated,8,7,5,1
downstream pathways linking JAM-A to paracellular permeability are unknown. As mentioned above, signaling pathways regulating JAM-A dependent cell migration have been described.4
From these studies, it is reasonable to postulate that similar pathway(s) may regulate barrier function. In migration studies, it was found that JAM-A associates with the scaffold protein afadin and the guanine nucleotide exchange factor PDZ-GEF2 resulting in the activation of the small GTPase Rap1a, stabilization of β1 integrins and enhanced cell migration. While these effector molecules have not been reported to directly affect barrier in epithelia, afadin, PDZ-GEFs, and Rap1 have been widely implicated in regulation of endothelial barrier, as will be discussed below.
Afadin, a large PDZ-containing scaffold protein shown to associate with JAM-A,4
has been strongly implicated in the regulation of barrier function. Mice with intestinal epithelial-targeted loss of afadin have increased intestinal permeability26
and a phenotype similar to that observed with JAM-A KO mice. JAM-A KO mice have normal intestinal mucosal architecture but a leaky colonic epithelium, increased mucosal lymphoid follicles and enhanced susceptibility to acute injury-induced colitis.2
While complete genomic deletion of afadin is lethal, mice with intestinal epithelial targeted loss of afadin (cKO) are viable, have a similar increase in gut permeability, and exhibit seemingly normal intestinal morphology. Similarly, afadin cKO mice show enhanced susceptibility to acute injury-induced colitis. Although the above parallels between JA KO and afadin cKO animals are consistent with afadin regulation of barrier function downstream of JAM-A, afadin has also been regarded as a cadherin-associated scaffold that mediates outside–in signaling after nectin-driven nascent junctions are initiated.27
While the latter observation might implicate afadin in controlling barrier downstream of nectin, mice lacking nectins-2 and -3 in the intestinal epithelium have no increase in intestinal permeability compared to wild type counterparts.26
Furthermore, the intestinal epithelium of such nectin-deficient mice has normal localization of afadin. These findings suggest that intestinal barrier function and junctional localization of afadin can occur by nectin-independent mechanism(s).
In addition to afadin, the guanine nucleotide exchange factor PDZ-GEF2 has been reported to associate with JAM-A and mediate β1 integrin dependent epithelial cell migration,4
presumably through activation of the small GTPase Rap1a. Despite this observation, the role for PDZ-GEF2 or the closely related PDZ-GEF1 in regulating epithelial barrier is not understood. Loss of PDZ-GEF1/2 in epithelial and endothelial cells has been shown to affect the composition and architecture of the AJ so that its morphology resembles nascent puncta,28,29
suggesting that PDZ-GEF1/2 may be important in the maturation of initial puncta into functionally developed AJCs. On the other hand, the differences in AJ architecture described in these reports were only detectable when cells were recultured at low confluence, suggesting that the role of PDZ-GEFs in barrier function may be limited to early events in junction formation. Additionally, loss of PDZ-GEF2 in epithelial cells did not affect the localization of the TJ proteins occludin or ZO-1,28
supporting the idea that altered AJ morphology does not necessarily translate to defects in TJ composition. In endothelial cells, Pannekoek et al.
reported that PDZ-GEF1/2 depletion resulted in decreased transendothelial impedance, a proxy measure of barrier function,29
but no analogous functional observations were reported for epithelial cells. Such observations support the concept of distinct regulatory mechanisms governing endothelial and epithelial barrier, which may be explained in part by the fact that the AJC in endothelial cells differs from that of epithelial cells by having a less defined separation between AJs and TJs.30
It is therefore important to confirm the functional importance of PDZ-GEFs in epithelial barrier function so as to determine whether they may act downstream of JAM-A to affect epithelial permeability.
As is the case for PDZ-GEFs, the small GTPase Rap1 has been implicated in downstream signaling events from JAM-A mediated regulation of cell migration. Interestingly, Rap1a/b have been widely implicated in regulation of endothelial barrier function31,29,32
but much less is known regarding the role of Rap1 as an effector of barrier in epithelial cells. In epithelial cells, Rap1a has been implicated in mediating trans-dimerization of E-cadherin32
and in organization of E-cadherin along cell–cell contacts.33,28
However, inactivation of Rap1 by the guanine nucleotide activating protein RapGAP does not affect the localization of ZO-1 to cell–cell contacts,33
suggesting that TJ formation does not require Rap1 in epithelial cells. Furthermore an in vivo
study on the role of the oxidized phospholipid OXPAPC in lung epithelial permeability reported no changes in transepithelial resistance after downregulation of Rap1.34
Additionally, Rap1 null c. elegans
display normal epithelial architecture of the epidermis and gut.35
Notably, most studies claiming a role for Rap1 in regulating endothelial and epithelial barrier are in fact describing functions of proteins known to alter the activation of Rap1, such as EPAC, RapGAP, and PDZ-GEF1/2.29,33
Since these mediators lack specificity for Rap1,36–39
the possibility of involvement of other small GTPases has not been excluded. The paucity of data directly relating Rap1 to functional measures of epithelial permeability leaves more questions than answers regarding a link between Rap1 and JAM-A dependent regulation of barrier function.
ZO-1 is an important TJ-associated scaffold protein and one of three zonula-occludens proteins. ZO-1 has three PDZ domains (PDZ1–3), an Src homology 3 domain (SH3), and a guanylate kinase homology domain (GUK) and has been reported to associate with JAM-A.40
A recent crystallography study identified PDZ3 as the putative binding pocket in ZO-1 responsible for JAM-A association, and that this association required the presence of the SH3 domain.41
Interestingly, mice lacking ZO-1 or ZO-2 do not survive, suggesting that both proteins are required for embryonic development.42,43
ZO-3 null mice, however, have little or very subtle phenotypic differences compared to their wild-type counterparts, suggesting a redundant role of ZO-3 in epithelial function.41
Cell culture studies have further defined barrier-inducing roles of ZO proteins through simultaneous silencing of ZO-1, 2 and 3 followed by their replacement one at a time.44,45
Epithelial cells lacking all three ZO proteins have no TJs, highlighting the importance of these scaffold proteins to barrier formation. Interestingly, addition of either ZO-1 or ZO-2 to ZO-null cells is sufficient for establishing TJs. Additionally, it was found that protein levels of JAM-A, claudins, and occludin are unchanged in ZO-depleted cells, suggesting that ZO-1 or 2 are likely necessary and sufficient for recruitment of TJ components to the AJC during barrier formation, but that synthesis of TJ proteins occurs independently of ZO-1/2 expression.46
ZO-1 has also been shown to associate with claudins via PDZ1, occludin through its GUK domain, afadin via its SH3 domain, and with other ZO proteins via PDZ2, allowing it to cluster several scaffold proteins, potentially leading to TJ maturation. It is possible that ZO-1, afadin, PDZ-GEFs, and other PDZ-containing scaffold proteins associate with transmembrane PDZ motif-containing proteins, such as JAM-A, to direct maturation and maintenance of barrier function. Ebnet and others have reported co-association between JAM-A and ZO-1 from cell lysates,47,40
and Nomme et al.
have shown in vitro
direct association of these proteins via crystallography studies.40
However, there are no reports showing a direct association of JAM-A and ZO-1 in cells. Additionally, there is limited data on whether ZO-2 can associate with JAM-A or participate in the same signaling module that ZO-1 and JAM-A are reported to share. While the role of ZO-1 and -2 proteins in barrier formation are well appreciated, the mechanisms linking ZO-1 and -2 to JAM-A mediated regulation of barrier function require further elucidation.
The GEFS and scaffold proteins discussed above are attractive candidate mediators regulating JAM-A dependent barrier function. However, potential distal signaling elements downstream of JAM-A, which are intimately associated with regulation of epithelial permeability, merit careful consideration. In particular, cytoskeletal dynamics and claudin composition of TJs directly affect paracellular permeability ().48–49
The tetraspan TJ forming claudins are classified as either leaky or tight and dimerize across the apical intercellular space to form channels that control the permeability of monolayers.47,51,49
It was previously reported that JAM-A null mice and JAM-A-depleted intestinal epithelial cells demonstrate enhanced levels of the leaky claudins 10 and 15,2
but not of claudin 2 or occludin, suggesting that JAM-A affects the claudin composition of TJs. It is not known how JAM-A does this, however previous studies indicate that JAM-A affects levels of β1 integrin by maintaining its stability at the cell surface.52
Given the likely overlap in function of some of the signaling elements discussed above, it is reasonable to hypothesize that JAM-A may regulate barrier function through effects on the stability of certain claudin family members at the TJ. Clearly, further studies should help to answer this important question.
Figure 2 Possible downstream mechanisms linking JAM-A dimerization to barrier function. While PDZ-GEF2, Afadin, and Rap1 have been reported to associate with JAM-A, their potential roles in regulation of epithelial barrier require further elucidation. Other key (more ...)
JAM-A associated AJC scaffold proteins, such as afadin and ZO molecules, have actin binding domains that allow for communication between the AJC and the apically positioned actin-myosin ring, a critical component of barrier integrity.25
The association of the AJC with cytoskeletal components is necessary for maintaining AJC structure.53,54
An attractive potential mechanism for JAM-A regulation of barrier would involve signaling through effectors such as afadin to induce cytoskeletal changes that control paracellular flux and epithelial permeability. JAM-A modulates epithelial cell migration, a process dependent on dynamic restructuring of actin, and induces activation of Rap GTPases, which have important cytoskeletal regulatory properties.11,55
Analogous JAM-A–dependent pathways that regulate barriers are therefore easily envisioned and require further exploration.