Human Corneal Endothelial Cells Employ Phosphorylation of p27Kip1 at Both Ser10 and Thr187 Sites for FGF-2-Mediated Cell Proliferation via PI 3-Kinase

Corneal endothelium (CE) is the single layer of cells forming a boundary between the corneal stroma and anterior chamber. The major function of the corneal endothelial cells (CECs) is not only to maintain corneal transparency by regulating corneal hydration through their barrier and ionic pump functions, but also to facilitate the passage of nutrients from the aqueous humor to the cornea stroma.1–3 Human CECs (hCECs) are considered nonproliferative in vivo and are arrested at the G1 phase of the cell cycle throughout their lifespan.4,5 Therefore, corneal endothelial wound healing is predominantly maintained by cell migration and increase in cell size. This repair process differs from that of most cell types, in which both cell proliferation and migration are involved in the wound healing process. In contrast, in the nonregenerative (pathologic) wound healing process, CECs are transformed into mesenchymal cells that subsequently produce a fibrillar extracellular matrix (ECM) in the basement membrane environment. Thus, corneal fibrosis induces a significant pathophysiological problem; that is, it causes blindness by physically blocking light transmittance. One clinical example of corneal fibrosis observed in CE is the development of a retrocorneal fibrous membrane (RCFM) in Descemet's membrane. In RCFM, the contact-inhibited monolayer of CECs is lost, cell proliferation is markedly increased, and fibrillar ECM is deposited in the basement membrane.6,7 Our previous studies using a rabbit system demonstrated that fibroblast growth factor-2 (FGF-2) is the direct mediator for such endothelial mesenchymal transformation (EMT); FGF-2 signaling upregulates the steady state level of α1(I) collagen RNA by stabilizing the message and subsequently facilitates synthesis and secretion of type I collagen into the extracellular space; FGF-2 signaling induces a change in cell shape from a polygonal to a fibroblastic morphology and causes loss of the contact-inhibited phenotypes; and lastly, FGF-2 signaling directly regulates cell cycle progression through phosphorylation of p27Kip1 (p27) by the action of PI 3-kinase.8–12 The negative cell cycle regulators, such as p16INK4a, p21Cip, and p27, are all expressed in CECs of several species and are important for maintenance of the G1-arrested phenotype through inhibition of cell cycle progression.4,13,14 When cells are induced to express these negative regulators of G1/S transition, the cell cycle is sustained at the G1 phase and senescence phenotypes are increased in various cell types. In contrast, downregulation of their expression turns on cell cycle progression and induces cell proliferation.15–18 Especially in hCECs, reduction of negative cell cycle regulators by small interference RNA (siRNA) induced cell proliferation, resulting in an increase in the number of cells entering the cell cycle and in an increase in total cell numbers.14,19 For these reasons, studying the regulatory mechanism of these negative cell cycle regulators is important to understanding of cell proliferation pathways in hCECs. Our previous data showed that FGF-2 regulates the cell cycling pathway of rabbit CECs (rCECs) through degradation of p27 by its phosphorylation mechanism.9,12,20 To be degraded, p27 must be phosphorylated at the threonine 187 (Thr187) and serine 10 (Ser10) sites. The cycle-dependent kinase 2 (Cdk2)-Cyclin E complex is responsible for phosphorylation of p27 at Thr187,12,21,22 whereas Ser10 site phosphorylation is mediated by kinase-interacting stathmin (KIS; a nuclear serine-threonine kinase) or protein kinase B (Akt).20,23,24 Our kinetic studies using rCECs12,20,25 showed that the phosphorylated p27 at Ser10 (pp27Ser10) mediated by KIS was detected and degraded by the Kip1 ubiquitination-promoting complex 1/2 (KPC1/2) ubiquitin-proteosomal machinery in the cytoplasm at the early G1 phase of the cell cycle. In contrast, phosphorylated p27 at Thr187 (pp27Thr187) mediated by Cdk2 activated through cell division cycle 25A (Cdc25A) was degraded by nuclear ubiquitin ligase complex in the nucleus at the late G1 phase. Our recent study identified that extracellular signal-regulated kinase 1/2 (ERK1/2) is involved in G1/S transition parallel to and independent of the PI 3-kinase/Rac1 pathway and that both the ERK1/2 and PI 3-kinase/Rac1 pathways participate in activation of KIS and activation of Cdk2 through Cdc25A in rCECs.20 However, the regulatory mechanism for degradation of p27, leading to the induction of cell proliferation, in hCECs has not yet been defined. In the present study, we investigated the correlation between human and rabbit CECs in cell proliferation pathways. In hCECs, FGF-2 stimulated cell proliferation through the ERK1/2 pathway, as a downstream regulator of PI 3-kinase, and phosphorylation of p27 is mediated by KIS for the Ser10 site and Cdc25A for the Thr187 site. The differences between human and rabbit CECs in the regulatory pathway of FGF-2-mediated cell proliferation lie in the cross talk between PI 3-kinase and ERK1/2 for the phosphorylation event of p27: hCECs employ a linear signal transduction involving PI 3-kinase-dependent ERK1/2 activation, while rCECs use parallel and independent ERK1/2 and PI 3-kianse pathways.