Common and Distinct Pathways for Cellular Activities in FGF-2 Signaling Induced by IL-1 in Corneal Endothelial Cells (original) (raw)
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Journal of Biological Chemistry, 2004
Our previous work demonstrated that both polymorphonuclear leukocytes (PMNs) and protein fractions released from PMNs induced de novo synthesis of fibroblast growth factor 2 (FGF-2), which in turn becomes the direct mediator of endothelial mesenchymal transformation observed in corneal endothelial cells (CECs). To identify the protein factor, we used ProteinChip Array technology. Protein fractions obtained from the conditioned medium released by PMNs were resolved by twodimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 17 kDa, were sequentially subjected to in-gel trypsin digestion and mass spectrometry. The 17-kDa peptide band was identified as interleukin-1 (IL-1). Biological activities of IL-1 were further determined; IL-1 altered the shape of CECs from polygonal to fibroblastic morphologies in a time-and dose-dependent manner, whereas neutralizing IL-1 antibody, neutralizing antibody to FGF-2, and LY294002 blocked the action of IL-1. IL-1 greatly increased the levels of FGF-2 mRNA in a time-and dose-dependent manner; IL-1 stimulated expression of all isoforms of FGF-2. IL-1 initially induced nuclear accumulation of FGF-2 and facilitated translocation of FGF-2 to plasma membrane and extracellular matrix. IL-1 activated phosphatidylinositol (PI) 3-kinase, the enzyme activity of which was greatly stimulated after a 5-min exposure to IL-1. This early and rapid activation of PI 3-kinase greatly enhanced FGF-2 production in CECs; pretreatment with LY294002 hampered the induction activity of IL-1. These observations suggest that IL-1 takes part in endothelial to mesenchymal transformation of CECs through its inductive potential on FGF-2 via the action of PI 3-kinase. Corneal fibrosis represents a significant pathophysiological problem 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) 1 in Descemet's membrane
Investigative Ophthalmology & Visual Science, 2010
PURPOSE. To determine whether the elevated level of interleukin (IL)-1 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-1 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-1 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-1 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-1 released by PMNs into the aqueous humor stimulates FGF-2 synthesis in corneal endothelium via PI3-kinase and p38. (Invest Ophthalmol Vis Sci.
Experimental Eye Research, 2001
After wounding, the corneal endothelium heals primarily by migration of adjacent cells into the denuded wound area. In this study, it has been attempted to identify elements of the intracellular signaling pathway activated through basic Fibroblast Growth Factor (FGF-2)-and Protein Kinase C (PKC)-modulated migration, using speci®c inhibitors and stimulators of second messengers in a cell culture model. Bovine corneal endothelial cells (BCEC) were grown to con¯uency and experiments performed with ®rst passage cells under serum-free conditions. A central circular`wound' was made with a specially designed trephine. In different experiments, cells were incubated with either FGF-2 (10 ng ml À1 ), pertussis toxin (PTX; 1±50 ng ml À1 ), phorbol 12-myristate 13-acetate (PMA; 50 ng ml À1 ), 2,4 H -di-bromoacetophenone (DAP; 5 mM), 1-(5-iosquinolinesulphonyl)-2-methyl-piperazine dihydrochloride (H7; 10 mM), indomethacin (5 ng ml À1 ), nordihydroguaiaretic acid (NDGA; 10 ng ml À1 ), 2-(4-morpholinyl)-8-pheny-4H-1-benzopyran-4-one (LY294002; 10 mM) or different combinations of these agents. Unsupplemented cultures served as controls. Migration was quantitated by counting the cells inside the denuded area in one randomly chosen section from the wound edge 72 hr after wounding. Cell toxicity was determined with the trypan blue exclusion test. Results were statistically analysed by Student's t-test. FGF-2 and PMA (a protein kinase C activator) both stimulated migration of endothelial cells at 2 . 2-and 3 . 1-fold, respectively. The PLA 2 inhibitor DAP and the PKC inhibitor H7 both signi®cantly reduced PMA-stimulated migration to control levels but had no effect (DAP) or even stimulated (H7) FGF-2-modulated migration. PTX did not affect FGF-2-stimulated migration. The phosphoinositol (3)-kinase inhibitor LY294002 signi®cantly reduced FGF-2-mediated stimulation of endothelial migration similar to the rate of control cultures. LY294002 had no effect when applied together with PMA. The cyclooxygenase inhibitor indomethacin did not in¯uence migration rates of the cells added either alone or in combination with PMA and FGF-2, respectively. The lipoxygenase inhibitor NDGA signi®cantly reduced the number of migrating cells in cultures with no other supplements, or of those supplemented with either PMA or FGF-2. FGF-2-induced endothelial migration in vitro is not dependent on PKC/PLA 2 or pertussis-toxin sensitive G-protein pathways but rather requires activation of a phosphoinositol (3)-kinase-like enzyme and/or arachidonic acid release with subsequent liberation of lipoxygenase products. Independent of FGF-2, PKC is a major intracellular effector of corneal endothelial migration activity after wounding and stimulates migration via the PLA 2dependent generation of lipoxygenase metabolites.
Endothelial mesenchymal transformation mediated by IL1β-induced FGF2 in corneal endothelial cells
Experimental Eye Research
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/Descemet's 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-1b (IL-1b) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1b 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.
Investigative ophthalmology & visual science, 1994
Previously reported from this laboratory are two distinct factors responsible for corneal endothelium modulation: basic fibroblast growth factor (bFGF) and the corneal endothelium modulation factor (CEMF) that is released by inflammatory cells. The altered phenotypes mediated by these two distinct factors--marked increase in cell proliferation, cell shape changes, and synthesis of fibrillar collagens--are identical. The current study sought to determine if bFGF is the direct mediator for corneal endothelium modulation and if CEMF plays a role in inducing bFGF production. bFGF synthesis mediated by CEMF was analyzed by immunoblot assay; cycloheximide was used to block protein synthesis. bFGF-Specific antisense oligonucleotide primer was used to inhibit CEMF-mediated bFGF synthesis and to block further the autocrine activity of bFGF. Cell proliferation was measured by cell counting. The steady-state levels of RNA were determined by Northern blot analysis. CEMF was further purified to ...
Investigative Opthalmology & Visual Science, 2008
PURPOSE. Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-B and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-B activation is mediated by phosphorylation of the inhibitor IB by IB kinase (IKK)-2 and consequent IB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. METHODS. Rabbit corneal fibroblasts were cultured in threedimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IB␣ and the subcellular localization of NF-B were examined by immunoblot and immunofluorescence analyses, respectively. RESULTS. IL-1-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration-and timedependent manner. TPCA-1 inhibited the IL-1-induced expression of MMP-1,-3, and-9 in these cells at both the mRNA and protein levels and the IL-1-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IB␣ and the nuclear translocation of NF-B induced by IL-1. CONCLUSIONS. An IKK-2 inhibitor blocked IL-1-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-B signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.
Investigative Ophthalmology & Visual Science
Purpose. Fibroblast growth factor 2 (FGF-2) is not only a potent mitogen, it is a modulator for corneal endothelial cells. To define how the modulation activities of FGF-2 are mediated, we used pharmacologic inhibitors to examine the association of phospholipase C-yl (PLCy) with FGF receptor or with cytoskeleton. Methods. Cell proliferation was determined either by the incorporation of 3 H-thymidine into DNA or by counting cell numbers in the absence or presence of the inhibitors. The protein expression was analyzed by immunoprecipitation and immunoblot analysis. Cell shape change was determined by phase-contrast microscopy. Results. FGF-2 stimulated DNA synthesis, whereas genistein inhibited the FGF-2-mediated cell proliferation in a dose-dependent manner, regardless of the concentration of FGF-2. The PLC-yl specific antisense oligonucleotide primer was able to inhibit cell proliferation by 25% in the absence of FGF-2; however, the antisense primer was not able to override the action of FGF-2. Fibroblast growth factor receptor was phosphorylated on treatment of the cells with FGF-2; however, 24-hour treatment with the growth factor significandy reduced phosphorylation of the receptor. Phospholipase Cyl appears to be abundant in cytoplasm in the absence and presence of FGF-2, and a minor portion of the molecule is translocated to membrane after treatment with FGF-2; genistein inhibited the translocation. When the cytoskeleton fraction of the normal and the modulated corneal endothelial cells was immunoprecipitated with PLC-7I antibodies, PLC-7I, actin, and vinculin were coprecipitated in both cell cultures. Phospholipase C7I associated with cytoskeleton was phosphorylated on treatment of the cells with FGF-2. In the presence of FGF-2 of the modulated cells, cytochalasin B, which did not revert the modulated cell morphology, abolished the association of PLC-yl with actin and vinculin; colchicine, which did revert the modulated cell shape to the polygonal shape, did not block the association of these three molecules. Interestingly, colchicine slighdy enhanced the stimulatory effect of FGF-2 on corneal endothelial proliferation in contrast to die effect of cytochalasin B, which slighdy decreased die FGF-2 action on cell proliferation. Conclusions. The association of PLC-yl widi cytoskeleton plays a role in cell proliferation, whereas the association of PLC-yl with actin and vinculin has no effect on cell shape changes. These findings indicate that FGF-2 appears to use distinct signaling pathways for cell proliferation and cell shape changes in corneal endothelial cells. Invest Ophthalmol Vis Sci. 1996;37:2326-2334.