Our present findings clearly demonstrate that JAM-C regulates both inflammation- and angiogenesis-related endothelial permeability. In particular, JAM-C mediates an increase in endothelial permeability by modulating actomyosin-based endothelial contractility and through regulation of VE-cadherin–mediated cell–cell contacts in a Rap1-dependent manner. Consistently, disruption of JAM-C function by soluble JAM-C in vivo decreased vascular permeability in the skin during delayed-type hypersensitivity responses and in the retina during hypoxia-driven retinal neovascularization, and reduced retina angiogenesis.
To date, transmembrane molecules of the tight junctions were thought to function as gatekeepers by interacting in a homotypic fashion (13
); thus, our findings were somewhat surprising. To our knowledge, JAM-C was defined here as the first junctional molecule that can mediate an increase in permeability. JAM-C knockdown mediated a rescue of endothelial barrier function under basal conditions and prevented VEGF- and histamine-mediated disintegration of endothelial junctions.
Interestingly, the subcellular localization of JAM-C in microvascular endothelial cells was found to be divergent from macrovascular endothelial cells. Consistent with our previous reports and those by others (16
), JAM-C in macrovascular endothelial cells was localized in the interendothelial junctions. In contrast, microvascular endothelial JAM-C was mainly intracellular and redistributed rapidly and transiently to the cell–cell contacts upon stimulation with VEGF or histamine. Our findings are in line with the diversity among endothelial cells from different vascular beds especially with respect to the regulation of paracellular permeability or leukocyte transendothelial migration (29
). By immunogold electron microscopy we found intracellular JAM-C in HDMECs to be associated with structures some, but not all, of which resembled Weibel-Palade bodies (unpublished data). The complete subcellular localization of JAM-C must be investigated in a future study. The regulated localization of JAM-C and its recruitment to the interendothelial contacts correlate and coincide with the role of JAM-C in regulating VEGF- and histamine-induced permeability. However, although the redistribution of JAM-C to the junctions may be relevant, it is clearly not essential for the effects of JAM-C on endothelial barrier function, as basal nonstimulated endothelial permeability was also decreased by JAM-C knockdown. In fact, intracellular JAM-C clearly regulates signaling, such as the Rap1 pathway, and participates in the regulation of actin stress fiber formation. This may be attributed to the propensity of JAM-C to be associated with AF-6 or other scaffolding molecules that may provide a link between intracellular JAM-C and these signaling pathways. Further studies addressing the detailed molecular mechanism for the intracellular trafficking of JAM-C and the importance of JAM-C localization for endothelial barrier function in microvascular versus macrovascular endothelial cells are required.
JAM-C knockdown in microvascular endothelial cells resulted in a decrease in F-actin and prevented stress fiber formation. In line with these observations JAM-C knockdown decreased the phosphorylation of MLCs. Of note, the changes in actomyosin caused by JAM-C knockdown were considerably higher than the decrease in basal endothelial permeability, indicating that changes in stress fiber formation may only loosely correspond to permeability changes, although these two processes may be functionally linked. By regulating the status of actomyosin, JAM-C may modulate actomyosin-driven contractility, although endothelial contractility was not directly assessed in the present study. Furthermore, knockdown of JAM-C led to the up-regulation of Rap1 activity, whereas the activity of other GTPases remained unaffected. Rap1 is involved in the formation and maintenance of cadherin-mediated cell–cell contacts and adherens junctions in endothelial and epithelial cells (7
), thereby improving barrier function. The homophilic ligation of E-cadherin in epithelial cells induces Rap1 activation, and vice versa, the affinity and avidity of cadherin-mediated contacts may be modulated by Rap1-triggered inside-out signaling (31
). Consistently, JAM-C knockdown strengthened VE-cadherin–mediated cell–cell contacts. This was a Rap1-dependent process, as down-regulation of Rap1 activity largely prevented the rescue of endothelial barrier function and the increase in VE-cadherin–mediated contacts caused by JAM-C knockdown. In contrast, the effects of JAM-C down-regulation were not altered by inhibition of the activity of PKA, another known regulator of endothelial barrier function (27
Interestingly, JAM-A, which functions to decrease paracellular permeability in endothelial and epithelial cells (13
), was recently found to regulate Rap1 activity in an opposite way to JAM-C, as JAM-A knockdown decreased Rap1 activity in epithelial cells (14
). Although, these authors did not study cadherin-mediated cell–cell contacts, their findings, together with our present data, enable a new mechanism for the regulation of vascular permeability by JAMs to be envisioned: JAM-A acts as a gatekeeper of the endothelial contacts, as its homophilic interaction may participate in the tight junction–mediated adherence, and through stimulating Rap1 it may promote the integrity of adherens junctions. The counter-player, JAM-C, down-regulates Rap1 activity and shifts the equilibrium toward the disruption of VE-cadherin–mediated intercellular contacts. In this intriguing scenario, which needs further experimental proof, it is striking that two structurally very similar molecules, JAM-A and JAM-C, act in an opposite manner to regulate the balance of active versus nonactive Rap1.
The herein reported destabilization of adherens junctions by JAM-C may also be operative in the function of JAM-C to promote transendothelial migration. Besides its interaction with leukocyte Mac-1 during transmigration (16
), junctional JAM-C may disrupt adherens junctions and increase the influx of leukocytes. This is consistent with recent findings from transgenic JAM-C–overexpressing mice that revealed increased accumulation of inflammatory cells (35
). Moreover, we have previously shown that soluble JAM-C blocks neutrophil transendothelial migration in vitro and in vivo (16
). Although soluble JAM-C acts as an inhibitor of Mac-1, it cannot be overlooked that the inhibition of transmigration by soluble JAM-C in vivo may also be attributed to decreased endothelial permeability.
Our findings that JAM-C regulates actin polymerization and actomyosin contractility and VE-cadherin–dependent cell–cell contacts become even more evident in light of two very recent publications deconstructing the cadherin–catenin–actin complex (36
). In particular, α-catenin assembled into the cadherin–catenin complex cannot bind actin. Instead, the recruitment of α-catenin to the adherens junctions upon cadherin ligation functions as a molecular switch to ultimately inhibit actin polymerization. This molecular mechanism would explain our present finding that the increase in VE-cadherin adhesiveness caused by JAM-C knockdown is accompanied by a decrease in F-actin; however, direct experimental evidence needs to be generated for this hypothesis. Furthermore, the findings of these two recent publications (36
) highlight the emergence of alternate mechanisms that regulate adherens junctions such as the one described in the present study. In epithelial cells, nectins, through their homophilic interaction, regulate the stability of cadherin-based cell–cell contacts both via AF-6 and by modulating the activity of small GTPases, particularly Rap1 (38
). JAMs may take over a similar function in regulating the maintenance of adherens junctions in endothelial cells, thereby fine tuning vascular permeability. In addition, the inability of the cadherin–catenin complex to interact with actin (36
) highlights the importance of alternate links between these two components. AF-6 may operate as a potential link between the actin cytoskeleton, adherens junctions, and Rap1 (38
) and may interact with the COOH-terminus of nectins and JAMs (reference 41
and unpublished data). This hypothesis requires further investigation. Nevertheless, our present work uncovers the first functional link between JAMs and VE-cadherin and a novel crosstalk between tight and adherens junctions that may open new directions for studies of endothelial barrier function.