Here we have used small interfering RNA to investigate the effect of the loss of JAM1 expression on epithelial cell function. Consistent with our previous study, knockdown of JAM1 was observed to increase paracellular permeability in epithelial monolayers. Interestingly, knockdown of JAM1 also produced dramatic changes in cell morphology, and a similar effect was observed with expression of a JAM1 mutant lacking the putative homodimer interface. Further studies revealed that JAM1 knockdown decreased cell-matrix adhesion and spreading on matrix proteins that are ligands of ␤1 integrins. These changes were characterized by a decrease in ␤1 integrin protein levels and loss of ␤1 integrin staining at the cell surface. Immunolabeling of cells for the small GTPase Rap1, a known activator of ␤1 integrins, revealed colocalization of Rap1 with JAM1 at intercellular junctions, and knockdown of JAM1 resulted in decreased Rap1 activity. Lastly, knockdown of Rap1b resulted in diminished ␤1 integrin expression and altered cell morphology analogous to that observed with knockdown of JAM1. Together, these results suggest that JAM1 regulates epithelial cell morphology and ␤1 integrin expression by modulating activity of the small GTPase Rap1.

No abstract

Junctional adhesion molecule-1 (JAM1) is a tight junction-associated immunoglobulin superfamily protein implicated in the regulation of tight junctions and leukocyte transmigration. The structural basis for the function of JAM1 has yet to be determined. Here we provide evidence that JAM1 homodimer formation is important for its function in epithelial cells. Experiments were conducted to determine the effects of a panel of JAM1 monoclonal antibodies on epithelial barrier recovery after transient disruption by calcium switch. Two monoclonal antibodies were observed to inhibit barrier recovery in contrast to another monoclonal antibody that had no effect. Epitope mapping by phage display revealed that both inhibitory antibodies bind to a region of JAM1 located within the N-terminal Ig-like loop (residues 111-123). Competition experiments with synthetic peptides and site-directed mutagenesis confirmed the location of this epitope. Analysis of the crystal structure of JAM1 revealed that this epitope includes residues within the putative homodimer interface, and one of the two inhibitory antibodies was then shown to block JAM1 homodimer formation in vitro. Finally, mutations within the homodimer interface were shown to prevent enrichment of JAM1 at points of cell contact, presumably by interference with homophilic interactions. These findings suggest that homodimer formation may be important for localization of JAM1 at tight junctions and for regulation of epithelial barrier function.A crucial function of epithelial cells is in the maintenance of a selective barrier separating the external from internal environments. It is well known that tight junctions (TJs) 1 play an important role in the maintenance and regulation of these barriers. Located at the apical-most part of the lateral epithelial cell membrane, TJs regulate paracellular diffusion of ions and small molecules across epithelial barriers. In addition, TJs serve as a fence between the apical and basolateral domains of polarized cells and facilitate bi-directional signal transduction between the intracellular and extracellular environments. TJs are composed of both transmembrane and cytoplasmic structural elements. Transmembrane protein members include occludins, claudins, coxsackie adenovirus receptors, and junctional adhesion molecules (JAMs). Knowledge of how these various proteins contribute to TJ function is continually evolving.JAM1 is an immunoglobulin superfamily protein that localizes to TJs of epithelial and endothelial cells (1) as well as to the surface of leukocytes (2). Evidence suggests a role for JAM1 in TJ assembly (3). Exogenous expression of JAM1 in CHO cells has been shown to promote localization of ZO-1 and occludin at points of cell contact (4). Monoclonal antibodies to JAM1 have been shown to inhibit reassembly of TJs and block recovery of transepithelial resistance to ion flux (3). Similar effects on epithelial barrier function were observed with recombinant soluble JAM1 consisting of the extracellular domain fused to IgG Fc (5). In add...

Junctional adhesion molecule-A (JAM-A

Epithelial cell migration is a critical event in gastrointestinal mucosal wound healing and is dependent on actin cytoskeletal reorganization. We observed increased expression of an actin regulatory protein, annexin 2, in migrating intestinal epithelial cells. Small interfering RNA (siRNA)-mediated knockdown of annexin 2 expression in Caco-2 epithelial cells resulted in significant reductions in cell spreading and wound closure associated with decreased formation of filamentous actin bundles along the base of migrating cells. Because annexin 2 has been shown to influences actin cytoskeletal remodeling through targeting signaling molecules to membrane domains, we examined the membrane association and activation status of Rho GTPases after annexin 2 knockdown. We observed Rho dissociation from membranes and decreased Rho activity following annexin 2 siRNA transfection. Inhibition of cell spreading and wound closure in annexin 2 siRNA-transfected cells was prevented by expression of constitutively active RhoA. Rho colocalized with annexin 2 in lamellipodia and along the cytoplasmic face of the plasma membrane. In addition, annexin 2 was observed to co-immunoprecipitate with endogenous Rho and constitutively active RhoA. These findings suggest that annexin 2 plays a role in targeting Rho to cellular membranes, thereby modulating Rhorelated signaling events regulating cytoskeletal reorganization during epithelial cell migration. (Am J Pathol