epidermal-growth-factor and arachidonyltrifluoromethane

Physiological level of reactive oxygen species (ROS) has been shown to play an important role in mitogen-stimulated cell signaling in many cell types. Both EGF and bFGF can induce ROS generation in human lens epithelial cells. But the role of ROS and Redox signaling on EGF and bFGF-stimulated cell proliferation is not clear. This study was to investigate the control of EGF and bFGF-induced cell proliferation by Redox signaling in human lens epithelial cells (SRA 01/04), using specific inhibitors to Redox signaling.. EGF and bFGF-induced cell proliferation was measured by [methyl-3H] thymidine incorporation assay. In some experiments, cell proliferation was also measured by trypan blue negative cell counting parallel with 3H-thymidine incorporation assay. The inhibitors used in this study include: catalase (specific enzyme to detoxify hydrogen peroxide), N-acetyl-L-cysteine (free radical scavenger), DPI (inhibitor for NADPH oxidase) and AACOCF3 (specific inhibitor for cytosolic phospholipase A2, which had been shown to play important role in ROS generation in our previous study). Serum starved SRA 01/04 cells were pretreated with these inhibitors for 30 minutes before exposure to EGF or bFGF (20 microg/L). In short term study, all these inhibitors were removed before adding growth factor, while in long term study, inhibitors were maintained in the medium along with growth factor. Cells were kept growing in the medium with 20 microg/L EGF or 20 microg/L bFGF for 48 hours. Then cell proliferation was quantified by [methyl-3H] thymidine incorporation assay or by cell counting.. We found that catalase, NAC, DPI and AACOCF3 were able to suppress EGF and bFGF-induced cell proliferation in both short term and long term study. In EGF study, 20 microg/L EGF produced about 26% (t = 7.093, P <0.01) increase in DNA synthesis after 48 hours. Pretreatment of the cells for 30 minutes with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 inhibited EGF-stimulated DNA synthesis by 18.0% (t=6.132, P<0.01), 24.6% (t=6.188, P<0.01), 28.5% (t=6.386, P<0.01) and 16.4% (t =3.705, P =0.001) respectively. The inhibition was dose-dependent and was proved by trypan-blue negative cell counting. If the cells were treated with inhibitors for 48.5 hours (long term study), the lowest concentrations to inhibit cell proliferation were much lower than those used in short term study. Treatment of the cells with 0.5 x 10(5) U/L catalase, 0.2 mmol/L NAC, 0.01 micromol/L DPI and 0.1 micromol/L AACOCF3 led to suppression on DNA synthesis significantly. Similar results were detected in bFGF study. 48 hours treatment with 20 microg/L bFGF induced about 28.8% (t =9.523, P <0.01) increase in cell proliferation. If the cells were pretreated with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 for 30 minutes, bFGF-stimulated cell proliferation was suppressed by 24.5% (t = 6.697, P < 0.01), 22.2% (t = 6.693, P<0.01), 23.9% (t =6.661, P<0.01) and 30.5% (t =8.959, P <0.01) respectively. If cells were treated with inhibitors for 48.5 hours, the lowest concentration of catalase, NAC, DPI and AACOCF3 to inhibit cell proliferation significantly was 0.5 x 10(5) U/L( t =21.641, P <0.01), 0.2 mmol/L (t =11.218, P < 0.01), 0.01 micromol/L (t = 4.570, P <0.01) and 0.1 micromol/L (t = 5.426, P < 0.01) respectively, lower than those used in short term study.. We conclude that mitogenic stimulus function of EGF and bFGF in human lens epithelial cells appears to be mediated via ROS to activate cell proliferation. Inhibition of Redox signaling, either by removal of ROS (the role of catalase and NAC) or blocking ROS generation (the role of DPI and AACOCF3), eradicate EGF and bFGF-stimulated cell proliferation. It is proposed that Redox signaling may play an important role in cell proliferation in human lens epithelial cells.

Topics: Acetylcysteine; Arachidonic Acids; Catalase; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Fibroblast Growth Factor 2; Humans; Lens, Crystalline; Male; NADP; Oxidation-Reduction; Reactive Oxygen Species; Signal Transduction

To determine in rabbit corneal epithelial cells in culture whether epidermal growth factor (EGF)-induced increases in prostaglandin (PG) E2 production inhibit both the extracellular signal-regulated kinase 2 (Erk-2), a mitogen-activated protein kinase (MAPK), cascade activation, and the mitogenic response to this growth factor.. Serum starvation for 24 to 36 hours was used to synchronize cultures of SV40-transformed rabbit corneal epithelial (RCE) cells. The effects of exogenous PGE2, inhibition of PGE2 synthesis, and modulation of protein kinase A (PKA) activity on EGF-induced Erk-2 activation were assessed by immunoprecipitation, kinase assays, and Western blot analysis. PGE2 synthesis was measured by using enzyme-linked immunosorbent assay. [3H]-Thymidine incorporation was used to measure RCE cell proliferation rates.. EGF (5 ng/ml) significantly increased PGE2 production in a time-dependent manner up to 94%+/-8% after 3 hours. EGF-induced PGE2 production was suppressed by AACOCF3, a phospholipase A2 (cPLA2) inhibitor. EGF-induced Erk-2 activation reached a maximal level at 15 minutes, followed by a decline toward the control level after 3 hours. In the presence of either PGE2 (50 microg/ml) or 8-CPT-cAMP (100 microM), the EGF-induced Erk-2 activation was lessened. PKA was activated by applications of EGF or PGE2 and suppressed by AACOCF3. On the other hand, either inhibition of PGE2 production with AACOCF3 or H-89, a PKA inhibitor, enhanced EGF-induced Erk-2 activity. Raf-1 activity was stimulated by EGF to maximal activity at 5 minutes and returned toward its control level after 60 minutes. As with the dependence of Erk-2 activity on PKA activity, in the presence of H-89, the EGF-induced Raf-1 activation was significantly enhanced. DNA synthesis was increased 59%+/-5% (n = 4) after EGF stimulation, indicating a mitogenic effect of EGF in RCE cells. Inhibition of cPLA2 activity with AACOCF3 increased DNA synthesis in RCE cells by another 64% relative to the effect of EGF alone. In contrast, with either PGE2 or 8-CPT-cAMP present the mitogenic response to EGF was totally suppressed.. EGF-induced increases in PGE2 production dampened the mitogenic response to this growth factor. This suppression appears to be a consequence of PGE2-elicited increases in PKA activity, which leads to inhibition of EGF-induced activation of MAPK cascades at the level of Raf-1 and further affects downstream events including Erk-2. These results indicate that the mitogenic response to EGF in vivo in the proliferating basal cell layer may be dependent on the level of its PKA activity.

Topics: Animals; Arachidonic Acids; Blotting, Western; Cell Division; Cell Line, Transformed; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; DNA; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelium, Corneal; Isoquinolines; Mitogen-Activated Protein Kinase 1; Proto-Oncogene Proteins c-raf; Rabbits; Sulfonamides; Time Factors