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Abstract
PURPOSE: To study isoform expression and cellular distribution of CD44, a cell surface glycoprotein thought to be an adhesion molecule in cell-cell and cell-substratum interactions, during corneal epithelial wound healing. METHODS: Reverse transcription-polymerase chain reaction was performed to determine alternatively spliced rat CD44 isoforms. In situ hybridization was carried out on frozen sections of the rat corneas obtained at different time points after epithelial debridement. 35S-Labeled sense and antisense cRNA that recognizes rat CD44 standard form was used as a probe. Immunofluorescence was used to assess expression and localization of CD44 in the rat corneas during reepithelialization. RESULTS: Corneal epithelia contained several alternatively spliced CD44 variants. Four large CD44 variants with inserts V1 through V10, V2 through V10, V3 through V10, and V4 through V10 were differentially expressed in migratory epithelia. The silver grains, indicating CD44 transcripts, started to increase in the epithelial cells surrounding the wound margin 3 hours after wounding and peaked at 18 hours in the basal epithelial cell layers, at which time the epithelia were actively migrating. As the cells began proliferation after wounding, the density of CD44 mRNA label declined but was still significantly higher than that in control specimens. The label returned to basal level as epithelial cells reverted to their normal phenotype. The location of CD44 on cell surfaces during corneal reepithelialization was consistent with the pattern of mRNA production. In the corneas at 18 hours after wounding, CD44 immunoreactivity was elevated in the entire epithelium, from the leading edge to the limbal-corneal border. As happened for the mRNA, the cell surface CD44 declined as cells differentiated to reestablish the multilayered epithelium. CONCLUSIONS: The expression of CD44 correlates with corneal reepithelialization, suggesting that CD44 may be involved in cell-cell interactions that provide adhesive strength for the much-stressed epithelial sheet and in the cell-substratum interactions that mediate cell migration during reepithelialization.