PI 3-Kinase/Rac1 and ERK1/2 Regulate FGF-2–Mediated Cell Proliferation through Phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdc25A/Cdk2

Human corneal endothelial cells (CECs) remain arrested at the G1 phase of the cell cycle throughout their lifespan.1,2 Such characteristic behavior of cell proliferation dictates most of the wound-healing processes occurring in the corneal endothelium: CECs do not use cell division to replace the lost cells but use migration and attenuation to cover the denuded area. On the other hand, in nonregenerative wound healing, CECs are transformed into mesenchymal cells that subsequently produce a fibrillar extracellular matrix (ECM) in the basement membrane environment. Thus, corneal fibrosis represents a significant pathophysiological problem, one that causes blindness by physically blocking light transmittance. One clinical example of corneal fibrosis observed in corneal endothelium is the development of a retrocorneal fibrous membrane (RCFM) in Descemet's membrane.3,4 We established an animal (rabbit) RCFM model, and we reported that CECs in RCFM are converted to fibroblast-like cells: The contact-inhibited monolayer of CECs is lost, resulting in the development of multilayers of fibroblast-like cells.5,6 These morphologically altered cells simultaneously resume their proliferation ability and deposit a fibrillar ECM in Descemet's membrane. Furthermore, our in vitro model using rabbit CECs (rCECs)7–10 elucidated the molecular mechanism of RCFM formation and demonstrated that fibroblast growth factor-2 (FGF-2) directly mediates the endothelial mesenchymal transformation (EMT) observed in rCECs. We reported that, among the phenotypes altered during EMT, FGF-2 signaling regulates cell cycle progression through phosphorylation of p27Kip1 (p27) by the action of phosphatidylinositol (PI) 3-kinase. Our kinetic studies11,12 demonstrated that phosphorylation of p27 at serine 10 (Ser10) occurred much earlier than phosphorylation of p27 at threonine 187 (Thr187) and that the subsequent polyubiquitination of the two phosphorylated p27s was carried out in the different subcellular localizations under the differential kinetics: phosphorylated p27 at Ser10 (pp27Ser10) is exported from nucleus to cytoplasm, followed by degradation through the KPC1/2 ubiquitin-proteasomal machinery in the cytoplasm, whereas phosphorylated p27 at Thr187 (pp27Thr187) is degraded through nuclear ubiquitin E3 ligase complex, Skp1-Cul1-F-box protein (SCFSkp2), in the nucleus.12 Thus, at least two respective populations of p27 undergo phosphorylation; each population functions at a different stage of the G1 phase of the cell cycle in response to mitogenic signals.11,12 The PI 3-kinase and the extracellular signal-regulated kinase (ERK) pathways are centrally involved in cell proliferation.13,14 The ERK signaling pathway regulates the subcellular localization of cyclin-dependent kinase 2 (Cdk2) to the nucleus and is necessary for Cdk activation through phosphorylation of Tyr160. The ERK signaling is also involved in upregulation of cyclin D1 and downregulation of p27.15–19 Likewise, the importance of p27 as a regulator of PI 3-kinase-mediated cell cycle progression is well established.11,13,20–24 Protein kinase B (commonly known as Akt) is an important downstream effector of the PI 3-kinase pathway. Akt has been shown to directly phosphorylate p27 on Ser-10, Thr-157, and Thr-198.25,26 Ser-10, which is the major phosphorylation site of p27, is also phosphorylated by kinase-interacting stathmin (KIS), a nuclear serine-threonine kinase.27,28 We have shown that phosphorylation of p27 at Ser-10 takes place in the nuclei within 2 hours after stimulation with FGF-2. The maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 hours after FGF-2 stimulation; nuclear export of pp27Ser10 was observed for up to 12 hours after FGF-2 stimulation. We further demonstrated that phosphorylation of p27 at Ser-10 is the major mechanism for FGF-2–mediated-G1/S transition leading to cell proliferation, while phosphorylation of p27 at Thr-187 acts as the second major mechanism of FGF-2–stimulated cell proliferation. We have shown that these actions of FGF-2 are mediated by PI 3-kinase.11 Because ERK1/2 is another mechanism for cell proliferation observed in many different cells, we decided to test whether this is the case in CECs stimulated with FGF-2. We also determined the downstream effector molecules for the distinctive phosphorylation events of p27. This study shows that PI 3-kinase uses Rac1 as the downstream to Akt and that FGF-2 employs the PI 3-kinase and ERK1/2 pathways in parallel for cell proliferation. We further showed that FGF-2 greatly induces both KIS and Cdc25A through PI 3-kinase/Rac1 and ERK1/2 pathways. Subsequent phosphorylation of p27 at Ser-10 is mediated by KIS, while phosphorylation of p27 at Thr-187 is mediated by Cdk2 activated by Cdc25A, a phosphatase that activates Cdk2.29 This study, thus, shows that phosphorylation of p27 is regulated by multiple mechanisms, but that it occurs within the context of the already determined pathway.