TGF-beta receptor types I and II are differentially expressed during corneal epithelial wound repair

PURPOSE. It has been demonstrated that cells migrating to cover an epitheli al debridement wound exit the cell cycle and that the cell-cycle inhibitor p15(INK4b) is upregulated in these cells. TGF-beta signaling has been impli cated in both of these processes, and this study was conducted to determine whether the expression and localization of TGF-beta receptor (T betaR)-I a nd -II are altered during corneal epithelial wound repair. METHODS. Three-millimeter superficial keratectomy wounds and 3-mm debrideme nt wounds were made in central rat cornea and allowed to heal in vivo for 1 to 48 hours. Immunofluorescence microscopy and Western blot analysis mere used to determine the localization and expression of T betaR-I and -II. Unw ounded rat corneas served as control samples. To determine the effect of ep idermal growth factor (EGF) and TGF-beta1 on p15(INK4b) and T betaR-I and - II expression, human corneal epithelial cells were grown in culture to 50% to 60% confluence, and EGF (5 ng/ml) and/or TGF-betaI (2 ng/ml) were added for G hours. Cells were harvested and p15(INK4b) and T betaR-I. and -II lev els were assayed by using Western blot analysis. RESULTS. In unwounded corneas, T betaR-I and T betaR-II were present at low levels across the cornea, with higher levels in limbal epithelium. Both T betaR-I and -II were upregulated after wounding. However, levels of T betaR -II appeared to increase in the epithelial cells that had migrated to cover the wound area, whereas T betaR-I was upregulated in the entire corneal ep ithelium. Western blot analysis indicated that both T betaR-I and -II were upregulated threefold after wounding. In cultured cells, EGF and TGF-beta1 stimulated T betaR-II; however, neither one stimulated T betaR-I expression . TGF-beta1 stimulated p15(INK4b) protein levels threefold. CONCLUSIONS. After wounding, T betaR-I and T betaR-II were both expressed a t high levels in cells migrating to cover a corneal wound, suggesting that TGF-beta signaling is involved in blocking migrating cells from progressing through the cell cycle. This blockage, at least in part, involves the inhi bitor p15(INK4b). In addition, although both T betaR-I and T betaR-II. are upregulated during wound repair, they appear to be differentially regulated .