Because degradation of p27 through its phosphorylation by FGF-2-stimulation is a major signaling observed in the cell proliferation pathway in rCECs,
10,12,25 we determined whether this is also true for FGF-2 to induce cell proliferation in hCECs. In ex vivo staining of corneal endothelium with anti-p27 antibody, 50% of cells maintained with DMEM were positive for p27 staining, whereas this positive staining was dramatically decreased in FGF-2-treated corneal endothelium (
Fig. 2A). The differential phosphorylation was further confirmed using immunocytochemistry in vitro. When cells were stained with anti-pp27Thr187 antibody, positive staining of pp27Thr187 was only observed in the cells maintained in DMEM with FGF-2, but not in the cells maintained with D-0 (
Fig. 2B). We then investigated whether PI 3-kinase and ERK1/2 participate in phosphorylation of p27 at both Ser10 and Thr187 sites in hCECs. Because our previous data with rCECs showed that phosphorylation of p27 at the Ser10 and Thr187 sites was maximized at 4 hours and 16 hours after FGF-2 stimulation, respectively,
12 we chose two FGF-2 treatment time points: 4 hours and 16 hours. As expected, pp27Ser10 was detected at 4 hours, but not at 16 hours, whereas pp27Thr187 was detected at 16 hours but not at 4 hours in response to FGF-2 stimulation (
Fig. 2C). The phosphorylation kinetics of p27 in hCECs are identical to those observed in rCECs. When pathway specific inhibitors were used, PI 3-kinase and ERK1/2 inhibition completely blocked phosphorylation of p27 at either the Ser10 or the Thr187 site induced by FGF-2 stimulation, suggesting that FGF-2 signaling also induced phosphorylation of p27 at both the Ser10 and Thr187 sites through PI 3-kinase and ERK1/2 in hCECs, as observed in rCECs. We then investigated whether the PI 3-kinase and ERK1/2 pathways are parallel and independent, similar to those defined in rCECs.
20 When hCECs were stimulated with FGF-2 for 8 hours, phosphorylation of Akt and ERK1/2 was observed similar to that in rCECs
20 (
Fig. 3). In contrast, pretreatment of hCECs with PI 3-kinase inhibitor for 2 hours before FGF-2 stimulation blocked phosphorylation of Akt at Ser473 and Thr308 (the downstream product of activated PI 3-kinase) and of ERK1/2, whereas MEK1/2 inhibitor blocked the ERK1/2 activation but did not block Akt activation after FGF-2 stimulation. These findings indicate that ERK1/2 is the downstream effecter to the PI 3-kinase/Akt pathways triggered by FGF-2 in hCECs (
Fig. 3). These results demonstrate that hCECs employ the signaling molecules similar to those of rCECs, but that there is a hierarchy in signal transduction in hCECs unlike in rCECs.