Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo

Purpose. Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. Methods. Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27^kip, p16^INK4a, and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. Results. All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27^kip and p16^INK4a. Conclusions. This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.