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

It has been demonstrated that cells migrating to cover an epithelial débridement wound exit the cell cycle and that the cell-cycle inhibitor p15(INK4b) is upregulated in these cells. TGF-beta signaling has been implicated in both of these processes, and this study was conducted to determine whether the expression and localization of TGF-beta receptor (TbetaR)-I and -II are altered during corneal epithelial wound repair. Three-millimeter superficial keratectomy wounds and 3-mm débridement wounds were made in central rat cornea and allowed to heal in vivo for 1 to 48 hours. Immunofluorescence microscopy and Western blot analysis were used to determine the localization and expression of TbetaR-I and -II. Unwounded rat corneas served as control samples. To determine the effect of epidermal growth factor (EGF) and TGF-beta1 on p15(INK4b) and TbetaR-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-beta1 (2 ng/ml) were added for 6 hours. Cells were harvested and p15(INK4b) and TBR-I and -II levels were assayed by using Western blot analysis. In unwounded corneas, TbetaR-I and TbetaR-II were present at low levels across the cornea, with higher levels in limbal epithelium. Both TbetaR-I and -II were upregulated after wounding. However, levels of TbetaR-II appeared to increase in the epithelial cells that had migrated to cover the wound area, whereas TbetaR-I was upregulated in the entire corneal epithelium. Western blot analysis indicated that both TbetaR-I and -II were upregulated threefold after wounding. In cultured cells, EGF and TGF-beta1 stimulated TbetaR-II; however, neither one stimulated TbetaR-I expression. TGF-beta1 stimulated p15(INK4b) protein levels threefold. After wounding, TbetaR-I and TbetaR-II were both expressed at 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 inhibitor p15(INK4b). In addition, although both TbetaR-I and TbetaR-II are upregulated during wound repair, they appear to be differentially regulated.