Transforming Growth Factor-beta Suppresses Proliferation of Rabbit Corneal Endothelial Cells In Vitro

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Abstract
Corneal endothelial cells in vivo appear to be inhibited in G1 phase of the cell cycle. Studies were carried out to determine whether cultured rabbit corneal endothelium expresses transforming growth factor-beta (TGF-beta) receptor types I, II, and III, suggesting they would be sensitive to a TGF-beta-induced signal. In addition, we explored if TGF-beta might mediate this G1 phase inhibition by implementing flow cytometry and 5-bromo-2'-deoxyuridine (BrdU) immunofluorescence. Reverse transcription-polymerase chain reaction (RT-PCR) products of the expected size were obtained for all three TGF-beta receptor types. Flow cytometry revealed a dosedependent suppression in the percentage of S phase cells in cultures treated with TGF- beta1 or TGF-beta2. The lowest percentage of S phase cells was found for 10 ng/ml TGF-beta1 and 0.1 ng/ml TGF-beta2. BrdU, an S phase marker, was immunolocalized, and semiquantitative analysis of stained cells showed a maximum suppression of S phase entry at 18 h for 10 ng/ml of TGF-beta1 and 24 h for 10 ng/ml of TGF-beta2. In rabbit, the corneal endothelium expresses TGF-beta receptor types I, II, and III, permitting a TGF-beta signal to be transduced. Flow cytometry reveals a dose-dependent response to both TGF-beta1 and TGF-beta2, and the cells are more sensitive to TGF-beta2. At optimal TGF-beta concentrations, the percentage of S phase cells is comparable to that of a nonproliferating culture, suggesting TGF-beta prevents the cells from proceeding through the G1 /S phase transition. This suppression was also seen with BrdU labeling. Together, these results indicate that TGF-beta could be one of the pathways that leads to G1 phase arrest in corneal endothelial cells.