Common and Distinct Pathways for Cellular Activities in FGF-2 Signaling Induced by IL-1␤ in Corneal Endothelial Cells

PURPOSE. To determine the mechanism by which IL-1␤ induces FGF-2 and to elucidate the signaling pathways of IL-1␤-induced FGF-2 in corneal endothelial cells (CECs). METHODS. Expression and/or activation of FGF-2, p38, ERK1/2, and Akt was analyzed by immunoblot analysis. Cell proliferation was measured by MTT assay. Pharmacologic inhibitors were used to block PI 3-kinase, p38, or ERK1/2. RESULTS. Brief stimulation of CECs with IL-1␤ activated PI 3-kinase and p38 in a biphasic fashion. The first wave of activation, triggered by IL-1␤, involves the inductive activity of IL-1␤ on FGF-2 production; the second wave of activation, triggered by the induced FGF-2, involves the promotion of cellular activities. In both pathways, p38 acts downstream to PI 3-kinase. The inductive activity of IL-1␤ on FGF-2 is further evidenced by the conditioned medium, which contains a large amount of FGF-2. Stimulation of CECs with IL-1␤ also activated ERK1/2 in a delayed fashion. The IL-1␤-induced FGF-2 exerted cellular activities using distinct pathways: the second wave of activation of PI 3-kinase and p38 was involved in cell migration, whereas cell proliferation was simultaneously stimulated by ERK1/2 and the second wave of PI 3-kinase. Likewise, the conditioned medium demonstrated cellular activities and pathways identical with those observed in cells treated with IL-1␤. CONCLUSIONS. These data suggest that CECs produce FGF-2 by IL-1␤ stimulation through PI 3-kinase and p38. The IL-1␤-induced FGF-2 facilitates cell migration via PI 3-kinase and p38, whereas it stimulates cell proliferation using PI 3-kinase and ERK1/2 in parallel pathways. (Invest Ophthalmol Vis Sci

Regulatory Role of FGF-2 on Type I Collagen Expression during Endothelial Mesenchymal Transformation

PURPOSE. To investigate the regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation (EMT). METHODS. Corneal endothelial cells (CECs) treated with FGF-2 from the primary culture to the third passage were transformed and designated as fibroblastic CECs (fCECs). Steady state levels of both type I collagen RNAs were measured using reverse transcription-real-time PCR, and their half lives were determined in the presence of inhibitor of RNA synthesis. Limited proteolysis with pepsin was used to determine secretion of type I collagen. Protein-protein interaction was determined by coimmunoprecipitation, and subcellular localization was studied by immunofluorescence. RESULTS. fCECs were characterized by greatly stimulated proliferative potential, loss of contact inhibition, and multilayer fibroblastic cells. The steady state level of ␣1(I) collagen RNA was greatly upregulated through stabilization of the message in fCECs, whereas steady state level and half-life of the ␣2(I) collagen RNA were slightly increased compared with the corresponding levels in normal CECs. Of interest, fCECs predominantly secreted homotrimeric type I collagen, [␣1(I)] 3 , with heterotrimeric type I collagen as a minor species. Type I collagen in fCECs was preferentially associated and colocalized with Hsp47 at Golgi apparatus as opposed to its association with protein disulfide isomerase in CECs. LY294002 (a specific PI 3-kinase inhibitor) greatly reduced the steady state levels and stability of ␣1(I) and ␣2(I) collagen RNAs and the secretion of type I collagen. CONCLUSIONS. FGF-2 directly mediates corneal EMT through the action of PI 3-kinase, which acts on posttranscriptional regulation by affecting the stability of type I collagen RNA. (Invest Ophthalmol Vis Sci. 2005;46:4495-4503

Regulatory role of cAMP on expression of Cdk4 and p27(Kip1) by inhibiting phosphatidylinositol 3-kinase in corneal endothelial cells

PURPOSE. Fibroblast growth factor (FGF)-2 is a potent mitogen of corneal endothelial cells (CECs). Results in an earlier study showed that FGF-2 activates phosphatidylinositol (PI) 3-kinase to stimulate the cell cycle machinery. The current study was designed to determine whether adenosine 3Ј,5Ј-monophosphate (cAMP) antagonizes FGF-2 by inhibiting PI 3-kinase/Akt pathways, thus leading to regulation of cyclin-dependent kinase 4 (Cdk4) and p27 Kip1 (p27) expression. METHODS. Cell proliferation was assayed by counting cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of Cdk4, p27, PI 3-kinase, Akt, and ␤-actin was analyzed by immunoblot analysis. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. RESULTS. 8-Bromoadenosine cAMP (8-Br-cAMP), a diffusible cAMP analogue, inhibited the PI 3-kinase/Akt signaling pathways. The 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels. They also equally inhibited cell proliferation, nuclear translocation of Cdk4, and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP was mediated by a direct inhibition of PI 3-kinase activity, which subsequently blocked phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP promoted a rapid turnover of Akt protein, and 8-Br-cAMP markedly reduced the half-life of Cdk4 protein. This inhibitory activity of cAMP was not mediated by PKA, but 8-Br-cAMP inhibited membrane localization of the p85 regulatory subunit of PI 3-kinase. CONCLUSIONS. These data support the hypothesis that cAMP inhibits the proliferation of CECs, preventing them from entering the S phase by negatively regulating PI 3-kinase. (Invest Ophthalmol Vis Sci. 2003;44:3816 -3825

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

FGF-2 required both PI 3-kinase/Rac1 and ERK activation for cell proliferation, after which the two signals used the common pathways for phosphorylating p27 but used differential kinetics and pathways: Ser-10 by KIS and Thr-187 by Cdc25A/Cdk2

Induction of FGF-2 Synthesis by IL-1β in Aqueous Humor through P13-Kinase and p38 in Rabbit Corneal Endothelium

IL-1β released by polymorphonuclear leukocytes into aqueous humor stimulates FGF-2 synthesis in corneal endothelium via PI3-kinase and p38

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

FGF-2 stimulates proliferation of human CECs through PI 3-kinase and its downstream target ERK1/2 pathways. This signal transduction downregulates p27 through its phosphorylation at both Ser10 and Thr187 sites mediated by KIS and Cdc25A, respectively

Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+)/K(+)-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions