Jeong Goo Lee

Endothelial mesenchymal transformation mediated by IL-1β-induced FGF-2 in corneal endothelial cells.

This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemets membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1β (IL-1β) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1β pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes.

NF-κB Is the Transcription Factor for FGF-2 That Causes Endothelial Mesenchymal Transformation in Cornea

PURPOSE To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs). METHODS Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. RESULTS Brief stimulation of CECs with IL-1β upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. CONCLUSIONS These data suggest that IL-1β signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.

Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser-10 and Thr-187 via Phosphatidylinositol 3-Kinase in Response to Fibroblast Growth Factor-2 Stimulation

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G1/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G1/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G1 phase of the cell cycle.

Involvement of Two Distinct Ubiquitin E3 Ligase Systems for p27 Degradation in Corneal Endothelial Cells

PURPOSE p27(Kip1) (p27) is an important regulator of G(1) progression. For cells to proliferate, p27 must undergo proteolysis. FGF-2 enables phosphorylation of p27 at both the Thr-187 and Ser-10 sites, an event that is prerequisite for polyubiquitination. This study was undertaken to determine whether degradation of the two phosphorylated p27s is mediated by a distinct ubiquitin E3 ligase complex at different subcellular locations. METHODS Expression of p27, KPC1, KPC2, Skp1, Skp2, and Cul1 was analyzed by immunoblot analysis. Association of p27 with ubiquitin E3 ligase was determined with coimmunoprecipitation followed by immunoblot analysis. Inhibitors were used to inhibit proteasomal degradation and nuclear export of the phosphorylated p27. DNA synthesis was measured by BrdU incorporation into DNA. RESULTS Among ubiquitin ligase complex proteins, Cul1, KPC1, and KPC2 were constitutively expressed, whereas expression of Skp1 and Skp2 was temporally induced by FGF-2. Skp1, Skp2, and Cul1 were involved in polyubiquitination of phosphorylated p27 at Thr-187 (pp27Thr187) in nuclei. Maximum association of pp27Thr187 with the ubiquitin E3 ligase occurred 24 hours after FGF-2 stimulation. pp27Ser10 used the cytoplasmic ubiquitin E3 ligases KPC1 and KPC2, with maximum protein interaction observed at 8 hours. MG132 effectively blocked degradation of both pp27Thr187 and pp27Ser10, whereas leptomycin B blocked the nuclear export of pp27Ser10. Both inhibitors blocked BrdU incorporation into DNA. CONCLUSIONS The findings demonstrate distinct polyubiquitination pathways for pp27Thr187 and pp27Ser10; the former is ubiquitinated through the nuclear ubiquitin E3 ligase system during late G(1) phase; the latter by cytosolic ubiquitin E3 ligase during early G(1) phase.

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

PURPOSE To determine the mechanism of p27 phosphorylation through common and differential pathways triggered by FGF-2 in corneal endothelial cells (CECs). METHODS A GTP pull-down assay was performed to identify Rac1-GTP. Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by an MTT assay. Transfection of CECs with kinase-interacting stathmin (KIS) siRNA was performed. RESULTS FGF-2 activated Rac1 through Akt, and Rac1 inhibitor greatly inhibited the FGF-2-stimulated cell proliferation. Rac1 inhibitor reduced p27 phosphorylation at both serine 10 (Ser10) and threonine 187 (Thr187). ERK1/2 was also involved in FGF-2-stimulated CEC proliferation and phosphorylation of p27 at Ser10 and Thr187 in parallel to phosphatidylinositol (PI) 3-kinase. In both PI 3-kinase/Rac1 and ERK1/2 pathways, Ser10 of p27 is phosphorylated by KIS, confirmed by siRNA to KIS, which subsequently hampered the FGF-2-stimulated cell proliferation, while Thr187 of p27 was phosphorylated through Cdk2 activated by Cdc25A. Cdc25A inhibitor blocked activation of Cdk2, phosphorylation of p27 at Thr187, and cell proliferation. FGF-2 induced both KIS and Cdc25A during the G1 phase; the maximum KIS expression was observed 4 hours after FGF-2 stimulation, while the maximum Cdc25A expression was observed at 12 hours. Blockade of ERK1/2 and Rac1 greatly reduced KIS and Cdc25A expression. CONCLUSIONS Results suggest that FGF-2 uses both PI 3-kinase/Rac1 and ERK pathways for cell proliferation; two signals employ common pathways for phosphorylating p27 according to the sites (KIS for Ser10 and Cdc25A/Cdk2 for Thr187) with their characteristic kinetics (early G1 for Ser10 and late G1 for Thr187).

Common and distinct pathways for cellular activities in FGF-2 signaling induced by IL-1β in corneal endothelial cells.

PURPOSE To determine the mechanism by which IL-1beta induces FGF-2 and to elucidate the signaling pathways of IL-1beta-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-1beta activated PI 3-kinase and p38 in a biphasic fashion. The first wave of activation, triggered by IL-1beta, involves the inductive activity of IL-1beta 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-1beta on FGF-2 is further evidenced by the conditioned medium, which contains a large amount of FGF-2. Stimulation of CECs with IL-1beta also activated ERK1/2 in a delayed fashion. The IL-1beta-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-1beta. CONCLUSIONS These data suggest that CECs produce FGF-2 by IL-1beta stimulation through PI 3-kinase and p38. The IL-1beta-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.

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

PURPOSE FGF-2 stimulates cell proliferation of rabbit corneal endothelial cells (rCECs) by degrading the cyclin-dependent kinase inhibitor p27(Kip1) (p27) through its phosphorylation mechanism. The authors investigated whether the cell proliferation of human CECs (hCECs) is also induced by FGF-2 stimulation through the p27 phosphorylation pathway. METHODS Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Transfection of hCECs with small interference RNA (siRNA) was performed using a transfection reagent. RESULTS FGF-2 stimulated cell proliferation in hCECs; the FGF-2 action was completely blocked by pathway-specific inhibitors for PI 3-kinase (LY294002) and MEK1/2 (U0126), respectively. Using immunoblotting, the authors showed that FGF-2 induced phosphorylation of p27 at both serine 10 (Ser10) and threonine 187 (Thr187) sites. These effects were also completely blocked by LY294002 or U0126. The authors then determined cross-talk between PI 3-kinase and extracellular signal-regulated kinase (ERK)1/2; blocking of ERK1/2 activation by LY294002 indicated that in hCECs ERK1/2 works as a downstream effector to PI 3-kinase for cell proliferation induced by FGF-2, whereas the ERK1/2 pathway in rCECs is parallel to the PI 3-kinase pathway. However, the downstream mechanism involved in cell cycle progression in hCECs is identical to that of rCECs: phosphorylation of p27 at Ser10 was mediated by kinase-interacting stathmin (KIS), confirmed with siRNA to KIS, and phosphorylation of p27 at Thr187 was mediated by cell division cycle 25A (Cdc25A), confirmed using Cdc25A inhibitor. CONCLUSIONS; FGF-2 stimulates proliferation of hCECs through PI 3-kinase and its downstream target ERK1/2 pathways. This linear signal transduction significantly downregulates p27 through its phosphorylation at both Ser10 and Thr187 sites mediated by KIS and Cdc25A, respectively.

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

PURPOSE To determine whether the elevated level of interleukin (IL)-1beta in aqueous humor after transcorneal freezing upregulates FGF-2 synthesis in rabbit corneal endothelium through PI3-kinase and p38 pathways. METHODS Transcorneal freezing was performed in New Zealand White rabbits to induce an injury-mediated inflammation. The concentration of IL-1beta was measured, and the expression of FGF-2, p38, and Akt underwent Western blot analysis. Intracellular location of FGF-2 and actin cytoskeleton was determined by immunofluorescence staining. RESULTS Massive infiltration of polymorphonuclear leukocytes (PMNs) to the corneal endothelium was observed after freezing, and IL-1beta concentration in the aqueous humor was elevated in a time-dependent manner after freezing. Similarly, FGF-2 expression was increased in a time-dependent manner. When corneal endothelium was stained with anti-FGF-2 antibody, the nuclear location of FGF-2 was observed primarily in the cornea after cryotreatment, whereas FGF-2 in normal corneal endothelium was localized at the plasma membrane. Treatment of the ex vivo corneal tissue with IL-1beta upregulated FGF-2 and facilitated its nuclear location in corneal endothelium. Transcorneal freezing disrupted the actin cytoskeleton at the cortex, and cell shapes were altered from cobblestone morphology to irregular shape. Topical treatment with LY294002 and SB203580 on the cornea after cryotreatment blocked the phosphorylation of Akt and p38, respectively, in the corneal endothelium. These inhibitors also reduced FGF-2 levels and partially blocked morphologic changes after freezing. CONCLUSIONS These data suggest that after transcorneal freezing, IL-1beta released by PMNs into the aqueous humor stimulates FGF-2 synthesis in corneal endothelium via PI3-kinase and p38.