Expression Of Junctional Adhesion Molecule-1 In Human Corneal Epithelium

INTRODUCTIONThe cornea which stands in the outermost part of the eye serves as the frontal barrier of the whole eyeball. It provides a biodefense barrier to the ever-changing external environment, protecting the eye not only from the biological and chemical insults, but also the penetration of tear fluid and its chemical constituents. Four types of intercellular junctions have been identified in the corneal epithelium: gap junctions, desmosomes, adherent junctions, and tight junctions(TJ). TJ functions as the seal between cells, forming the primary barrier to restrict the free diffusion of fluids, electrolytes, molecules and cells through paracellular pathways. It plays a central role in the regulation of paracellular permeability, and also is crucial in the generation and maintenance of cellular polarity in endothelial and epithelial cells. On ultrathin section electron microscopes, TJs appears as a series of kissing points between the abutting cells, at the point intercellular space is completely obliterated.TJ components can be divided into four categories:peripherally associated scaffolding proteins, signaling proteins, proteins regulate membrane vesicle targeting, and transmembrane proteins. Human junctional adhesion molecule-1 (JAM-1), also referred to as JAM-A, is the latest identified transmembrane protein component. It was found to be among the first tight junction-associated proteins appearing at cell-cell contacts during junction formation, and play an important role in recruiting and localizing signaling complex to sites of cell-cell adhesion, promoting the formation of tight junctions. It also involveds in leukocyte migration, platelet activation, angiogenesis and reovirus binding. On human cornea, tight junction was detected in superficial layer of corneal epithelial and corneal endothelium. Many reports revealed that several tight junction related proteins such as occludin, claudin and ZO-1 were detected in human corneal epithelial cells. However, the expression of JAM-1 was remained unreported. The current study was designed to study the expression and distribution of JAM-1 in human cornea, and compared with that of occludin.MATERIALS AND METHODS1. AntibodiesMouse anti-human monoclonal antibody to JAM-1 was obtained from Hycult biotechnology b.v. (Uden, Netherlands), rabbit polyclonal antibody to occludin from Zymed (South San Francisco, CA).2. CorneaHuman cornea obtained from eyebank of Juntendo University, the third hospital of Beijing University and the Shengjing affiliated hospital of China Medical University.3. Cell cultureHuman corneal epithelial cells were purchased from Kurabo. Cells were cultured to subconfluent with Epilife medium (20%FCS). Human corneal keratocytes and endothelial cells were separated from donor corneas. Insolated keratocytes from donor cornea were cultured with 1% or 20% FCS+DMEM. 2 or 3 passaged keratocytes were cultured with 20% FCS+DMEM.4. Reverse transcriptase polymerase chain reactionTotal RNA was isolated from cultured human corneal epithelial cells, keratocytes or endothelial cells using Trizol reagent. Reverse transcription was carried out with the Superscript TM First-Strand Synthesis System. Approximately 0.3μg total RNA was used in each reverse transcription reaction and the final volume was 20μl. Duplex PCR for JAM-1 and occludin was performed using 1μl of reverse transcription reaction in a 20μl reaction using Taq enzyme and buffer. The PCR reaction was carried out as follows: initial denaturation at 94℃for 5 min, followed by denaturation at 94℃for 60 s, annealing at 54～60℃for 30～60 s, and polymerization at 72℃for 60 s. The reaction was carried out over 30 cycles and completed by an extended polymerization at 72℃for 10 min. After completion of the PCR amplification, 8ul of each PCR products were examined by 1.5% agarose gel and stained with ethidium bromide.5. Flow cytometric analysisAfter harvested in PBS, cultured human corneal cells were incubated with the primary antibody for 30 min on ice and washed twice with cold PBS. Then incubated with 10% human serum for 15min,washed with cold PBS twice. After incubation with secondary antibodies for 30 min on ice and washed, flow cytometry was performed.6. ImmunofluorescenceColocalization of JAM-1 and occludin was studied by double immunochemistry. Four-μm-thick cryostat sections of the cornea were initially air-dried in room temperature for 1 hour and then fixed in chilled acetone for 10 minutes followed by a subsequent blocking of nonspecific staining using Protein Block serum-free (Dako Cytomation California, USA) for lOminutes. Sections were then incubated with Mouse anti-JAM-1 antibody (1: 300) for 1 hour at room temperature in a moist chamber, washed, and then exposed to FITC conjugated Goat anti-mouse IgG (1:500, 1h, room temperature). Sections were then exposed to the rabbit polyclonal anti-occludin antibody(1: 300; 1h, room temperature), Cy3 conjugated Goat anti-rabbit IgG(1:500,1h, room temperature) subsequently. After mounting, sections were observed and photographed. Primary antibodies were omitted in negative controls.RESULTS1. Reverse transcriptase polymerase chain reactionWhile checking the amplification reaction after 30 cycles, strong expression of JAM-1 and occludin was detected in cultured epithelial cells, small amount was also detected in the endothelial cells but negligible in primary cultured keratocytes. Decreasing expression of Keratocan, CD34 and increasing expression ofα-SMA was observed with cell passage of the keratocytescultured in DMEM+20%FCS. At the mean time, the expression of JAM-1 and occludin was found to be up-regulated consist with that ofα-SMA.2.FACSThe expression of JAM-1 was detected in cultured human corneal epthelial cells, endothelial cells and the third generation of keratocytes, but not the primary cultured keratocytes.3. ImmunofluorescenceIn normal cornea, the occludin immunoreactivity was found largely limited to the most-supeficial layer. The staining pattern between wing cells was punctate, no staning at the junction between column basal cells was observed. While staining for JAM-1 was detected at cell borders in the entire epithelium, encircling the cells, intense labeling was observed in the superficial layer, coexisting with that of occludin.The immunoreactivity was also observed in the endothelium but almost invisible in the stroma. In recovery corneal ulcer JAM-1 appear at the margin superior to the occludin and in corneal inflamation it was detected in the stroma. In keratoconus the expresion of JAM-1 is similar to normal cornea but redistributed in the gelatinous corneal dystrophy.CONCLUSION1. JAM-1 was expressed by cultured human corneal epithelial cells and endothelial cells.2. JAM-1 was not expressed by primary cultured human keratocytes.3. JAM-1 coexisted with occludin in the superficial layer of the cornea epithelium, suggesting its possible role in the corneal epithelial barrier, but it also present at cell borders between superficial cells, wing cells and basal cells where tight junction is not existed revealed that JAM-1 was not only one of compornents of TJ protein, but also membrane adhesion protein between cornal epithelial cells.4. When keratocytes were subjected to serial passage in media containing 20%FCS, keratocytes became activated and transdiffereciated into fibroblasts, and expressedα-SMA which was considered as a marker for myofibroblast. These gave us the clue that JAM-1 may also in some way participate in wound healing process.5. In pathologic cornea JAM-1 redistributed and was expressed in corneal stroma. In corneal recovery ulcer it appeared in the ulceratic margin prior to occludin.