Expression of JAM-A in the human corneal endothelium and retinal pigment epithelium: localization and evidence for role in barrier function

Abstract

Purpose: Junctional adhesion molecules (JAMs) are a family of adhesion proteins found in intercellular junctions. Evidence suggests that JAM-A is important for the regulation of tight junction assembly and epithelial barrier function. The authors recently reported that JAM-A is expressed in rabbit corneal endothelium and that antibody to JAM-A produces corneal swelling. In the present study, they investigate JAM-A expression in the human corneal endothelium and retinal pigment epithelium (RPE) and examine the effect of a function-blocking antibody to JAM-A on the permeability of cultured RPE cell monolayers.

Methods: Expression of JAM-A in human corneal endothelium, human RPE tissue, and cultured ARPE-19 monolayers was assessed by immunofluorescence confocal microscopy. Localization of JAM-A was compared with the tight junction-associated protein zonula occludens-1 (ZO-1). To investigate JAM-A function in ARPE-19 cells, ARPE-19 monolayers were subjected to a calcium switch protocol to disrupt cell junctions and treated with a function-blocking antibody to JAM-A or an isotype-matched control. Dextran flux assays were performed to assess the effect of JAM-A antibody on ARPE-19 monolayer permeability.

Results: Expression of JAM-A was observed in human corneal endothelium, and its distribution correlated with the tight junction-associated protein ZO-1. In addition, expression of JAM-A was observed in human RPE and in intercellular junctions of ARPE-19 monolayers. The localization pattern of JAM-A in the RPE and ARPE-19 monolayers was similar to that of ZO-1. ARPE-19 monolayers treated with antibody to JAM-A demonstrated a 33% increase in permeability to 10,000 MWt dextran compared with monolayers treated with control antibody.

Conclusions: Results of this study provide new information about JAM-A expression in tight junctions of the human corneal endothelium and human RPE. The observation that antibodies to JAM-A increase ARPE-19 monolayer permeability is consistent with previous findings of JAM-A function in epithelial tight junctions and suggests JAM-A may have a role in the regulation of RPE barrier function.

Figure 1

Expression of JAM-A in the human corneal endothelium. (A) Immunofluorescence confocal micrographs demonstrate the expression of JAM-A (green) and ZO-1 (red) in human corneal endothelial junctions. Below each en face image is a corresponding Z-section. Note that JAM-A and ZO-1 were observed to colocalize in the junctional complexes (yellow). Scale bar, 10 μm. (B) Western blot analysis of JAM-A expression in human corneal endothelial cells. ZO-1 is shown as a control.

Figure 2

Localization of JAM-A in intercellular junctions of the human RPE. Immunofluorescence confocal microscopy was performed to investigate expression of JAM-A (green) in human RPE flat mounts. The junction-associated protein ZO-1 is shown for comparison (red). Note that JAM-A and ZO-1 were observed to colocalize in junctional complexes (yellow). Below each en face image is a corresponding Z-section. Scale bar, 10 μm.

Figure 3

Expression of JAM-A in cultured ARPE-19 cells. (A) Immunofluorescence confocal micrographs of cultured ARPE-19 monolayers illustrate JAM-A (green) and ZO-1 (red) expression in intercellular junctions. Colocalization of JAM-A and ZO-1 is shown in yellow. Below each en face image is a corresponding Z-section. Scale bar, 10 μm. (B) Western blot analysis of JAM-A in ARPE-19 cells. ZO-1 is shown as a control.

Figure 4

Analysis of actin organization in ARPE-19 monolayers. (A) Immunofluorescence confocal micrographs illustrate the distribution of actin (green) and JAM-A (red) in ARPE-19 cells. Below each en face image is a corresponding confocal Z-section. Scale bar, 10 μm. (B) An enlarged confocal Z-section illustrates the distinct apical and basal actin populations (green) and the perijunctional actin that partially colocalizes with JAM-A (yellow). Note that the vertical scale of this image was increased to allow better discrimination of the apical and basal actin.

Figure 5

Markers of tight junction maturity in ARPE-19 cells. Immunofluorescence confocal microscopy was performed to assess the expression of the tight junction markers in ARPE-19 cell monolayers. (A) Detection of the canonical tight junction proteins occludin (Occ) and claudin-1 (CL-1). (B) Detection of JAM-A–related proteins JAM-C (JAM-C) and CAR (CAR), two IgSF proteins also commonly expressed in epithelial tight junctions.

Figure 6

Effect of JAM-A antibody on permeability of cultured ARPE-19 monolayers. ARPE-19 monolayers cultured on a porous substrate were subjected to a “calcium switch” and then incubated with antibody to JAM-A or control IgG. (A) Permeability to 10,000 MWt dextran was measured fluorometrically at various time points thereafter. Dextran flux is reported in AFUs, with error bars representing the SEM at each time point. *Statistically significant differences with P < 0.005. (B) Confocal micrographs demonstrate binding of the JAM-A antibody in intercellular junctions of ARPE-19 cells. No binding to ARPE-19 cells was observed for the IgG control. Scale bar, 10 μm.

Figure 7

JAM-A and its interactions in the tight junction. (A) JAM-A consists of two extracellular immunoglobulinlike loops, a single transmembrane domain, and a short cytoplasmic tail that terminates in a PDZ-binding motif. The N-terminal immunoglobulinlike loop contains a putative dimerization motif. (B) Monoclonal antibodies to JAM-A have been shown to block dimerization of JAM-A and inhibit JAM-A function in epithelial tight junctions. (C) The extracellular domain of JAM-A mediates homophilic interactions, and the cytosolic tail is known to bind to PDZ proteins such as ZO-1, AF-6, and Par-3.–,– These PDZ proteins are linked to the actin cytoskeleton and presumably serve as docking sites for small GTPases such as Rho, Rac and Rap-1, which regulate junction assembly and cell polarity.