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Role of p27^Kip1 in cAMP- and TGF-ß2–Mediated Antiproliferation in Rabbit Corneal Endothelial Cells

¹ From the Doheny Eye Institute and ³ Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles; ² Department of Ophthalmology, Kon Yang University, Seoul, Korea.

PURPOSE. To determine whether p27^Kip1 plays a role in antiproliferation mediated by antimitogens (cAMP and TGF-ß2) in rabbit corneal endothelial cells (CECs).

METHODS. Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of cell cycle–regulatory proteins was determined by immunofluorescent staining, and expression of the proteins was analyzed by immunoblot analysis.

RESULTS. When cells were treated with cAMP or TGF-ß2, serum-mediated cell proliferation was inhibited in a dose-dependent manner. Simultaneous treatment of the two antimitogens did not show a synergistic effect on inhibition of cell growth. Expression of cell cycle–regulatory proteins, such as cyclin-D1, cyclin-E, cdk2, cdk4, p21^Cip1, and p27^Kip1 was determined using immunofluorescent staining. A strong nuclear staining was observed for p27^Kip1. The other proteins were not stained or were only very faintly stained. Treatment of cells with either cAMP or TGF-ß2 did not change the staining potential of any proteins other than p27^Kip1, but all cells were positive for nuclear p27^Kip1 when treated with either TGF-ß2 or cAMP. In contrast, mitogen (FGF-2)-containing medium decreased the number of p27^Kip1-positive cells. When the expression level of p27^Kip1 was determined using immunoblot analysis in the cells treated either with FGF-2 alone or with a concomitant treatment with FGF-2 and cAMP for 24 hours, FGF-2 markedly decreased the p27^Kip1 level, and cAMP prevented the decrease in p27^Kip1 level induced by FGF-2. No such phenomenon occurred during a short-term exposure of cells to either FGF-2 or cAMP or to a combination of the two. When cells were stained for phosphorylated p27^Kip1, FGF-2 markedly enhanced the staining of phosphorylated p27^Kip1 in nuclei, whereas both cAMP and TGF-ß2 prevented the phosphorylation of p27^Kip1.

CONCLUSIONS. These findings suggest that both antimitogens upregulate the expression of p27^Kip1 as they prevent the decrease of the p27^Kip1 level mediated by mitogen. Furthermore, cAMP and TGF-ß2 may inhibit the G₁-to-S transition by blocking phosphorylation of p27^Kip1, which is a prerequisite for nuclear export of the inhibitor molecule for degradation.

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