TGF-beta 2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells

PURPOSE. Corneal endothelium in vivo is arrested in G1, the phase of the ce ll cycle that prepares cells for DNA synthesis. In many cell types, transfo rming growth factor (TGF)-beta inhibits proliferation by inducing G1-phase arrest. Evidence indicates that-corneal endothelial cells synthesize mRNA f or TGF-beta 1 and are also bathed in aqueous humor that contains TGF-beta 2 (mainly in a latent form), As such, this cytokine may maintain the corneal endothelium in a G1-phase-arrested state in vivo. The purpose of these stu dies was to determine the effect of exogenous TGF-beta 2 and TGF-beta 2 in aqueous humor on DNA synthesis in cultured corneal endothelial cells. METHODS. Rat corneal endothelial cells were grown in explant culture and id entified by morphology and reverse transcription-polymerase chain reaction using primers for specific corneal cell markers. Subconfluent cells were sy nchronized in the GO phase (quiescence) by serum starvation for 24 hours. S erum was then added to the cells in the presence or absence of exogenous TG F-beta 2 or activated rat aqueous hunter. [H-3]Thymidine was added, and rad ioactivity was measured at various time points to detect DNA synthesis, Pre incubation,of exogenous TGF-beta 2 or activated rat aqueous humor with neut ralizing antibody was used to test fur cytokine specificity. RESULTS. A linear increase in [H-3]thymidine incorporation-began approximat ely 16 hours after serum addition, and peak incorporation occurred at appro ximately 24 hours. Exposure of cells to serum plus TGF-beta 2 suppressed [H -3]thymidine incorporation in a dose-dependent manner at concentrations ran ging from 5 pg/ml to 5 ng/ml. [H-3]Thymidine incorporation was also suppres sed in cells exposed to serum plus rat aqueous humor diluted 1:10. Neutrali zing antibody reversed the effects of both exogenous TGF-beta 2 and aqueous humor. CONCLUSIONS. Exogenous TGF-beta 2 and TGF-beta 2 in aqueous humor suppress S-phase entry of rat corneal endothelial cells. These results suggest that this cytokine in aqueous humor could help maintain the corneal endothelium in a G1-phase-arrested state in vivo.