Inhibitory Effect of PGE 2 on EGF-Induced MAP Kinase Activity and Rabbit Corneal Epithelial Proliferation

Transcription

1 Inhibitory Effect of PGE 2 on EGF-Induced MAP Kinase Activity and Rabbit Corneal Epithelial Proliferation Sylvia S. Kang, 1 Tie Li, 2 Dazhong Xu, 2 Peter S. Reinach, 1 and Luo Lu 1,2 PURPOSE. To determine in rabbit corneal epithelial cells in culture whether epidermal growth factor (EGF)-induced increases in prostaglandin (PG) E 2 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. METHODS. Serum starvation for 24 to 36 hours was used to synchronize cultures of SV40-transformed rabbit corneal epithelial (RCE) cells. The effects of exogenous PGE 2, inhibition of PGE 2 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. PGE 2 synthesis was measured by using enzyme-linked immunosorbent assay. [ 3 H]-Thymidine incorporation was used to measure RCE cell proliferation rates. RESULTS. EGF (5 ng/ml) significantly increased PGE 2 production in a time-dependent manner up to 94% 8% after 3 hours. EGF-induced PGE 2 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 PGE 2 (50 g/ml) or 8-CPT camp (100 M), the EGF-induced Erk-2 activation was lessened. PKA was activated by applications of EGF or PGE 2 and suppressed by AACOCF3. On the other hand, either inhibition of PGE 2 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 PGE 2 or 8-CPT camp present the mitogenic response to EGF was totally suppressed. CONCLUSIONS. EGF-induced increases in PGE 2 production dampened the mitogenic response to this growth factor. This suppression appears to be a consequence of PGE 2 -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. (Invest Ophthalmol Vis Sci. 2000;41: ) From the 1 Department of Biological Sciences, SUNY College of Optometry, New York, New York; and the 2 Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio. Supported by NIH Grants EY11653 (LL) and EY04795 (PSR). Submitted for publication September 1, 1999; revised December 15, 1999; accepted January 18, Commercial relationships policy: N. Corresponding author: Luo Lu, Department of Physiology and Biophysics, School of Medicine, Wright State University, Dayton, OH luo.lu@wright.edu Growth factors stimulate cell proliferation by initiating G1 progression to S phase of the cell cycle. 1 Growth factors transmit their mitogenic signals via the activation of a series of kinase cascades that eventually activate mitogen-activated protein kinases (MAPKs). The MAPKs that primarily respond to growth factor stimulation are extracellular-signal-response kinases 1 and 2 (Erk-1 and Erk-2). 2 4 There is emerging evidence that a host of cytokines may be involved in epithelial renewal or epithelial cell proliferation as a consequence of their regulatory effects on rates of proliferation and differentiation. In particular, epidermal growth factor (EGF) is one of the many factors involved in this renewal process. 5 This cytokine has been identified in a variety of studies to be a very potent and efficacious mitogen. 6 Therefore, it would appear that EGF makes a substantive contribution to mediating the control of corneal epithelial renewal through its role in stimulating proliferation. The receptor for EGF, EGFR, is activated by this cytokine and undergoes dimerization. This leads to activation of the tyrosine kinase domains and autophosphorylation of the receptor. 7 Src homology domains of adapter proteins recognize the phosphorylated tyrosine residues, which leads to activation of Ras, a GTP-binding protein, followed by activation of the MAPK pathway. In this sequential chain, Raf is the entry point of the MAPK cascades. Raf is also referred to as MAPKKK or MEKK, and it ultimately phosphorylates MAPK. 8 There are two isoforms of MAPK, the p44 MAPK (Erk-1) and the p42 MAPK (Erk-2), which are expressed in most cell types. The substrates of MAPK include nuclear transcription factors and nonnuclear Investigative Ophthalmology & Visual Science, July 2000, Vol. 41, No Copyright Association for Research in Vision and Ophthalmology

2 IOVS, July 2000, Vol. 41, No. 8 Effect of PGE 2 on EGF-Induced RCE Cell Proliferation 2165 substrates such as the protein, serine/threonine kinase p90sk, cytoskeletal proteins, and phospholipase A2 (cpla2). 9 cpla2 catalyzes the release of arachidonic acid from phospholipids in membranes and is one of the rate-limiting steps in the synthesis of prostaglandins (PGs) and other eicosanoids. In cultured rabbit corneal endothelial (RCE) cells, it was shown that PGE 2 inhibits mitosis. 10 This effect of PGE 2 suggests that in this tissue EGF could have a role in removing cells from the cell cycle and thereby promoting differentiation. However, the cellular signaling pathways linked to EGF receptor stimulation and to the effect of PGE 2 in the corneal epithelium have not been characterized. We report here on the cell signaling pathways in RCE cells linking EGF receptor stimulation to the control of growth and differentiation. Our results indicate that EGF induces concomitant increases in MAPK activity and PGE 2 synthesis. The increases in PGE 2 levels lead to the elevation of camp-dependent protein kinase activity, which in turn has a negative feedback effect on Raf-1 activity and results in the inhibition of EGFinduced increases in MAPK activity and corneal epithelial cell proliferation. METHODS Culture of SV40-Immortalized RCE Cells SV40 large T antigen transformed RCE cells were a generous gift from Kaoru Araki Sasaki, MD. RCE cells were cultured in supplemented hormone epithelial medium (SHEM) containing DMEM/F-12 (Life Technologies, Grand Island, NY), 10% fetal bovine serum (Life Technologies), 5 g/ml insulin, 10,000 U/ml penicillin, and 10,000 mg/ml streptomycin. The cultures were placed in 75-cm 2 culture flasks and maintained in an incubator supplied with 95% air and 5% CO 2 at 37 C. The medium was replaced every 2 days. The cultures were passed using 0.05% trypsin EDTA (Life Technologies) for experimental use, serial passage, or storage. RCE cells were stored in 80 C in SHEM with 10% dimethylsulfoxide for periods ranging from 1 to 6 months. Other tissue culture supplies were supplied by Fisher (Pittsburgh, PA), and chemicals were supplied by Sigma (St. Louis, MO) unless otherwise noted. Quantitation of PGE 2 Production RCE cells were cultured in 12-well plates with cells/ well. The cultures were serum-starved for 24 hours and treated with experimental agents. After incubation of the RCE cultures in experimental conditions, the medium of each sample was collected and assayed for PGE 2 synthesis according to the manufacturer s protocol using a commercial enzyme-linked immunosorbent assay (R&D Systems, MN) and calibrated spectrophotometrically with a standard curve. The experiments were performed in triplicate. [ 3 H]-Thymidine Incorporation RCE cells were cultured in six-well plates at a density of cells/well with SHEM to approximately 60% confluence. Then the cultures were serum-starved for 24 hours and treated with experimental agents for another 24 hours. [ 3 H]-Thymidine was added to cultures at 1 Ci/ml for 2.5 hours. After incubation, the samples were washed with PBS and placed on ice for 30 minutes with 1 ml of 10% trichloroacetic acid. The samples were washed with absolute ethanol and allowed to air-dry. The cells were then lysed with 0.1 M NaOH in 1% sodium dodecyl sulfate (SDS) and transferred to scintillation vials containing 10 ml of scintillation fluid. The samples were read in a beta counter, and results were statistically analyzed using the Origins program (version 5.0). The experiments were performed in triplicate. Measurements of Protein Kinase A Activity Activity of camp-dependent protein kinase A (PKA) in RCE cells was measured with a kit (SignaTECT PKA assay system, Promega, Madison, WI). Briefly, RCE cells were treated with 5 ng/ml of EGF or 10 g/ml of PGE 2 and 100 g/ml of 8-CPT camp (adenosine-3,5 -cyclic monophosphate-sodium salt; Calbiochem, La Jolla, CA) as a positive control. RCE cells (10 7 ) were collected and washed with phosphate-buffered saline (PBS), resuspended in 0.5 ml of extraction buffer (25 mm Tris HCl, ph 7.4; 0.5 mm EDTA; 0.5 mm EGTA; 10 mm -mercaptoethanol; 1 g/ml leupeptin; and 1 g/ml aprotinin), and homogenized using a Dounce homogenizer. Lysate (5 l) was added to a reaction mixture containing 10 Ci -[ 32 P]ATP, 5 M camp, 100 M PKA biotinylated peptide substrate, 40 mm Tris HCl (ph 7.4), 25 mm MgCl 2, and 100 g/ml bovine serum albumin. Each reaction was immediately incubated at 30 C for 5 minutes and then terminated by adding 12.5 l of 7.5 M guanidine hydrochloride. Terminated reactions were spotted onto a SAM 2 membrane. After the membrane was washed with 2 M NaCl, 2 M NaCl in 1% H 3 PO 4, and deionized water, respectively, the amount of 32 P-labeled substrate in the membrane was measured by a scintillation counter (model LS 6000 TA, Beckman, Fullerton, CA). Immunoprecipitation, Kinase Assay, and Western Immunoblot Analysis RCE cells were cultured in 60-mm petri dishes at a density of cells/dish with SHEM until approximately 60% confluent. The cultures were then serum-starved for 24 hours, washed with PBS, and treated with EGF (5 ng/ml) for 5, 15, 30, 90, or 180 minutes. The cells were lysed with lysis buffer containing 50 mm HEPES (ph 7.5), 150 mm NaCl, 1.5 mm MgCl 2, 1 mm EGTA, 10 mm sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mm NaF, 1 mm Na-orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 250 M p-nitrophenylphosphate, 10 g/ml aprotinin, and 10 g/ml leupeptin. The lysates were centrifuged at 12,000 rpm for 30 minutes, and the supernatant was transferred to a new tube containing either 2 g/ml rabbit Erk-2 antibody or rabbit Raf-1 antibody (New England Biolabs, Beverly, MA) and incubated overnight at 4 C. Protein A Sepharose beads were added and incubated at 4 C overnight. The beads were washed twice with lysis buffer and once with kinase buffer containing 20 mm HEPES (ph 7.6), 20 mm MgCl 2, 25 mm -glycerophosphate, 100 M sodium orthovanadate, and 2 mm dithiothreitol. The samples were then divided into aliquots and used for either a kinase assay or Western immunoblot assay. For the kinase assay, 2 g/ml of myelin basic protein (MBP; Upstate Biotechnology, Lake Placid, NY) was used as the substrate for the Erk-2 assay, and 1 g/ml of MEK-1 (Upstate Biotechnology) was added for the Raf-1 assay. The fusion protein reaction was started by the addition of 10 Ci [ 32 P] ATP to each sample. The phosphorylation of the MBP substrate by Erk-2 ran for 10 minutes and was terminated by the addition of an equal volume of 2 SDS sample buffer.

3 2166 Kang et al. IOVS, July 2000, Vol. 41, No. 8 SDS polyacrylamide gel electrophoresis (SDS PAGE) was performed in a 12% acrylamide gel and visualized on X-ray film. The other aliquot was used for Western immunoblot analysis by performing SDS PAGE in a 12% acrylamide gel and transferred to a polyvinylidene fluoride membrane. The membrane was then incubated overnight in 4 C with either Erk-2 or Raf-1 antibody in 5% nonfat milk in TBST (Tris Buffer Saline- Tween). The membrane was washed twice with TBST to remove the residual primary antibody and incubated with a alkaline phosphatase labeled anti-rabbit secondary antibody (New England Biolabs) for 1 hour at room temperature. The proteins were visualized using the CDP-Star Chemilumiscence Substrate system (New England Biolabs). RESULTS EGF-Induced Increases in PGE 2 Production To determine whether EGF can induce increases in PGE 2 levels, a concentration of 5 ng/ml EGF was used in our experiments because this dosage has been previously found to maximally stimulate proliferation in bovine corneal epithelial cells. 11 RCE cells were synchronized in the cell cycle by culturing them in serum-deprived medium for at least 24 hours. Figure 1 shows that PGE 2 levels in the medium significantly increased from baseline level as soon as 30 minutes after initiating exposure to EGF (5 ng/ml). The levels continuously increased to reach a value of 94% 8% (n 6) above the control value after 180 minutes. AACOCF3 (5 M), a selective inhibitor of cpla2, significantly suppressed EGF-induced increases throughout this period. This result indicates that EGF stimulates PGE 2 production in RCE cells through the activation of cpla2. FIGURE 1. Effects of EGF on PGE 2 production in RCE cells. RCE cells were treated with 5 ng/ml EGF (F), EGF plus AACOCF3 (5 M; AAC; Œ), and controls (f). Intracellular PGE 2 levels were measured by enzyme-linked immunosorbent assay and plotted as a function of time. Data were collected from 6 groups of cells and plotted as mean SE. *Represents a significant difference (P 0.05) compared with respective control values. FIGURE 2. Inhibitory effects of PGE 2 on EGF-induced RCE cell proliferation. [ 3 H]-Thymidine incorporation was measured at 24 hours in control cells (C) and in cells treated with 5 ng/ml EGF stimulated (E), EGF plus 10 or 50 g/ml PGE 2 (E P), EGF plus 100 M 8-CPT camp (E 8cpt), and EGF plus 5 M AACOCF3 (E AAC). RCE cells were synchronized in the cell cycle by serum starvation for 24 hours before experimentation. Data are shown as mean SE (n 4). Symbols * and # represent significant differences (P 0.05) from control and EGF induced cells, respectively. Effect of PGE 2 on EGF-Induced RCE Cell Proliferation After a 24-hour period of serum starvation, over the next 24 hours EGF significantly increased proliferation by 97% 8% (Fig. 2). In contrast, the mitogenic response to EGF was totally suppressed if the cells were treated with either 10 or 50 g/ml of exogenous PGE 2. Because PGE 2 interacts with prostaglandin receptor subtypes, which are linked to the stimulation of adenylate cyclase and modulation of PGE 2 levels altered proliferation, 12 we determined whether the effect of PGE 2 on the suppression of the EGF-induced RCE cell proliferation could be mediated through an increase in camp levels. After the application of 100 M 8-CPT camp, a permeable camp analogue, to the medium, the mitogenic response to EGF was markedly inhibited (Fig. 2). In contrast, this response was significantly enhanced, by 64% 1% (n 4), above the control level through the inhibition of cpla2 activity with AACOCF3 (5 M). These results are consistent with the notion that EGFinduced increases in PGE 2 level exert a negative feedback effect on the mitogenic response to EGF through the stimulation of adenylate cyclase. Effect of PGE 2 on EGF-Induced MAPK Activation EGF-induced stimulation of Erk-2 activation was characterized 5, 15, 30, 90, and 180 minutes after EGF addition to the medium. The results shown in Figure 3A indicate that Erk-2 was activated at 5 minutes and reached a maximum level after 15 minutes. This level was sustained for 90 subsequent minutes followed by a decline after another 90 minutes. To assess whether PGE 2 could suppress any of these increases, we measured the effect of exogenous PGE 2 (10 or 50 g/ml) on EGF-induced Erk-2 activation at 15 and 180 minutes. As shown in Figure 3B, the addition of PGE 2 suppressed EGF-induced increases in Erk-2 activity nearly to the baseline level at 15 and 180 minutes. Because the results shown in Figure 1 indicated

4 IOVS, July 2000, Vol. 41, No. 8 Effect of PGE 2 on EGF-Induced RCE Cell Proliferation 2167 FIGURE 3. Effects of PGE 2 on EGF-induced Erk-2 activation in RCE cells. (A) Time course of Erk-2 kinase activation stimulated by EGF. RCE cells were treated with EGF, and kinase activities were measured at 5, 15, 30, 90, and 180 minutes. Erk-2 activity was determined by the phosphorylation level of MBP by using kinase assay in vitro. (B) Inhibitory effects of PGE 2 on EGF-induced Erk-2 activation. Exogenous PGE 2 was added in EGF-stimulated RCE cells, and kinase assay was performed at 15 and 180 minutes after stimulation. (C) Enhanced Erk-2 activity in response to EGF stimulation by AACOCF3. EGF-induced Erk-2 activity was measured at 15 and 180 minutes after the application of AACOCF3. (D) Enhancement of EGF-induced Erk-2 activity by suppression of cpla2 and PKA. RCE cells were treated with EGF (5 ng/ml) with or without the addition of PGE 2 (10 g/ml; E PGE2), the cpla2 inhibitor AACOCF3 (5 M; EGF AAC), or the PKA inhibitor H-89 (1 M; EGF H89). that AACOCF3 lowered EGF-induced increases in PGE 2 levels, we determined whether suppression of cpla2 by AACOCF3 (5 M) could accentuate EGF-induced Erk-2 activation. The results shown in Figure 3C are supportive of this possibility because at 15 and 180 minutes the EGF-induced Erk-2 activation was enhanced more in the presence than in the absence of AACOCF3. Further assessment of a role for PKA activity in mediating a negative feedback effect on EGF-induced Erk-2 activation was obtained with H-89 (1 M), an inhibitor of PKA. As can be seen in Figure 3D (last lane on the right), the EGF-induced Erk-2 activation was enhanced by H-89, suggesting that changes in PKA activity can alter Erk-2 activity. EGF-Induced Raf Activation Enhanced by Suppression of PKA To study the effect of PKA activity on EGF-induced Raf activation, PKA activity in RCE cells was measured in the presence and absence of AACOCF3. RCE cells were synchronized in G 1 phase of the cell cycle by serum starvation and then stimulated with 10% fetal bovine serum (FBS), 5 ng/ml EGF, or 10 M PGE 2. PKA activity in stimulated RCE cells was markedly increased for 2.0 to 2.8 times compared with serum-deprived cells (Fig. 4A). The increase of PKA activity in response to EGF was completely suppressed by the inhibition of cpla2 with AACOCF3. This result indicates that inhibition of EGF-induced endogenous PGE 2 production resulted in a negative feedback regulation of PKA activity. It has been known that one of the upstream components from Erk-1/2 in the MAP kinase cascades is Raf (MAPKK kinase), which sequentially activates Mek-1/2 (MAPK kinase). 13 Because changes in PGE 2 levels can alter EGF-induced Erk-2 activation, we next determined whether the PGE 2 effect is through its interaction with upstream events in the MAPK cascade at the level of Raf. Raf-1 kinase activity was determined subsequent to immunoprecipitation using the anti-raf antibody and using the hypophosphorylated Mek-1 fusion protein as the substrate for the kinase assay. Application of EGF induced an observable increase in Raf-1 activity at 5 minutes, which then progressively declined at 15, 30, and 60 minutes toward the untreated control level (Fig. 4B). To determine whether negative feedback control of the EGF-induced MAPK activation involves camp-mediated modulation of Raf-1 activity, AACOCF3 (5 M) and H-89 (1 M) were added before the addition of EGF to inhibit cpla2 and PKA activities, respectively. Raf-1 activity induced by EGF was inhibited by the application of PGE 2 (10 g/ml) and enhanced in the presence of AACOCF3 and H-89 after 15 minutes in a manner similar to that obtained in the Erk-2 assay (Fig. 4C). Therefore, the inhibitory effect of PGE 2 on EGF-induced Erk-2 activation seems to reflect the inhibition of Raf-1 activity by PKA.

5 2168 Kang et al. IOVS, July 2000, Vol. 41, No. 8 FIGURE 4. Effects of suppressing PKA with H-89 on EGF-induced Erk-2 and Raf-1 activation. (A) Measurement of PKA activity. RCE cells were treated with FBS (10%), EGF (5 ng/ml), PGE 2 (10 g/ml), or EGF and AACOCF3 (5 M; AAC). Normalized PKA activity was calculated as fractions of the baseline PKA activity in serum-deprived cells. (B) Time course of EGF-induced Raf-1 activation in RCE cells. RCE cells were treated with EGF, and kinase activities were measured at 5, 15, 30, and 60 minutes. Raf-1 activity was determined by the phosphorylation level of MEK-1 fusion protein by using kinase assay in vitro. (C) Enhancement of EGF-induced Raf-1 activity by suppression of cpla2 and PKA. EGF-induced Raf-1 activity was measured at 15 minutes in the presence and absence of PGE 2 (10 g/ml), the cpla2 inhibitor AACOCF3 (5 M), or the PKA inhibitor H-89 (1 M). IgG, immunoglobulin G. DISCUSSION The maintenance of normal corneal epithelial thickness and function is dependent on a balance between the rates of basal cell layer proliferation and the loss of terminally differentiated cells in the suprabasal layers. This interplay assures that the corneal epithelium can function as a barrier against noxious agents and contribute to the maintenance of deturgescence. 14 Our results indicate that the EGF-induced mitogenic response in RCE cells appears to be regulated by multiple factors through different signaling pathways, because this cytokine activates both the MAPK cascade, COX-2, and cpla2. Increases in cpla2 and COX-2 activity result in turn in rises in PGE 2 levels, which elicit a negative feedback effect on EGF-induced MAPK activation. This feedback appears to result from PGE 2 - mediated increases in camp, which through the stimulation of PKA activity suppress EGF-induced Raf-1 activation. This type of feedback has been described in a number of tissues (see the Introduction section). We previously obtained some suggestive evidence for such a type of feedback in bovine corneal epithelial cells. In this case, the stimulatory effects of EGF on wound closure were concentration dependent. At lower concentrations, proliferation increased and reached a maximum at 5 ng/ml. However, at higher concentrations the mitogenic response was reduced. Using the RCE cell line rather than a primary culture was advantageous, because the cells were synchronized in the G 1 phase of the cell cycle before the growth factor stimulation. Our results reveal that in vivo the response of proliferating basal layer corneal epithelial cells to EGF is dependent on its activation of MAPK, which is in turn modulated through a negative feedback effect that is associated with the level of concomitant EGF-induced PKA activation. Activation of EGF receptor by EGF can induce a mitogenic response through activation of MAPK cascades. Our measurements of Erk-2 activity show that the EGF-induced Erk-2 activation was time dependent. Furthermore, there was an association between the magnitudes of the EGF-induced activation of the MAPK cascade and cell proliferation. The mitogenic responses to EGF were inversely related to both the cellular levels of PGE 2 and to the presence of exogenous PGE 2. Within the first 15 minutes, activation of Erk-2 reached a maximal level followed by a decline after 180 minutes toward its control level. Indicative of the inverse relationship between the mitogenic response to EGF and PGE 2 levels, EGF alone increased DNA synthesis by 97%, whereas subsequent to the inhibition of cpla2 with AACOCF3 the mitogenic response to EGF was enhanced by 64% relative to the effect of EGF alone (Fig. 2). This enhanced mitogenic response correlates with increases in Erk-2 kinase activity that occurred at all times shown in Figure 3C. Conversely, the decreases in Erk-2 activity that occurred with 10 and 50 g/ml PGE 2 were consistent with the inability of EGF to elicit a mitogenic response in their presence (Figs. 2 and 3B). In addition, EGF-induced mitogenic response may also be the result of the induction of increases in PKC activity through activating phospholipase C. Activation of PKC can activate Erks by activating MAPK kinase kinase, Raf-1, in many cell types A schematic diagram of EGF-induced signaling pathways in RCE cells is proposed in Figure 5. There is evidence in vascular endothelial cells, PC12 cells, fibroblasts, and renal mesangial cells that increases in PGE 2 levels can inhibit activation of the EGF-linked MAPK cascade at the level of Raf This inhibition occurs as a result of PGE 2 binding to a prostaglandin receptor followed by activation of adenyl cyclase and subsequent increases in camp levels. These increases in camp lead to stimulation of PKA and suppression of Raf activation. However, the cell signaling pathways linked to EGF receptor stimulation in the corneal epithelium have not been characterized. Our results indicate that the suppression of the mitogenic response to EGF could actually be a consequence of PGE 2 -mediated increases in intracellular camp that lead to the stimulation of PKA. As shown in Figure 2, exoge-

6 IOVS, July 2000, Vol. 41, No. 8 Effect of PGE 2 on EGF-Induced RCE Cell Proliferation 2169 FIGURE 5. Schematic diagram of EGF-induced signaling pathways in RCE cells. EGFR, epidermal growth factor receptor; Erk, MAP kinase; PGE 2 R, prostaglandin E 2 receptor; PLA, phospholipase A; PLC, phospholipase C; and Raf, MAPK kinase kinase. nous camp had inhibitory effects on Erk-2 activation and cell proliferation, which were equivalent to those caused by PGE 2, and EGF-induced RCE cell proliferation was markedly suppressed either in the presence of exogenous camp or PGE 2. Therefore the mitogenic response to EGF appears to be related to PGE 2 -mediated increases in camp, which in turn have a negative feedback effect on Erk-2 activation. We next investigated the effects of changes in camp levels on EGF-induced Raf-1 activation to determine whether PGE 2 - mediated increases in camp levels could have a negative feedback on EGF-induced upstream events above the Erk-2 kinase. Similar to the inhibitory effect of PGE 2 on EGF-induced Erk-2 activation, exogenous PGE 2 had a comparable effect on Raf-1. Other evidence for suggesting that the site of camp-mediated inhibition of the mitogenic response to EGF lies at the level of Raf-1 is that inhibition of PKA activity with H-89 accentuated at 15 minutes the EGF-induced increases in Raf-1 activity (Fig. 4B). This effect of H-89 is comparable to that of AACOCF3 on Raf-1 and Erk-2, further revealing that the site of the negative feedback effect lies at the level of Raf-1 rather than Erk-2. References 1. Beck TW, Magnuson NS, Rapp UR. Growth factor regulation of cell cycle progression and cell fate determination (review with 40 Refs.). Curr Topics Microbiol Immunol. 1995;194: Waskiewicz AJ, Cooper JA. Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast (review with 79 Refs.). Curr Opin Cell Biol. 1995;7: Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol. 1996;8: Davis RJ. The mitogen-activated protein kinase signal transduction pathway (review with 55 Refs.). J Biol Chem. 1993;268: Wilson SE, Schultz GS, Chegini N, Weng J, He YG. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59: Tripathi RC, Raja SC, Tripathi BJ. Prospects for epidermal growth factor in the management of corneal disorders. Surv Ophthalmol. 1990;34: Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990;61: Seger R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995; 9: Davis S, Gale NW, Aldrich TH, et al. Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. Science. 1994;266: Jumblatt MM. Autocrine regulation of corneal endothelium by prostaglandin E2. Invest Ophthalmol Vis Sci. 1994;35: Tao W, Liou GI, Wu X, Abney TO, Reinach PS. ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells [published erratum appears in Invest Ophthalmol Vis Sci. 1996;37:1937]. Invest Ophthalmol Vis Sci. 1995; 36: Ortmann R, Perkins JP. Stimulation of adenosine 3 :5 -monophosphate formation by prostaglandins in human astrocytoma cells. Inhibition by nonsteroidal anti-inflammatory agents. J Biol Chem. 1977;252: Neary J. MAPK cascades in cell growth and death. News Physiol Sci. 1997;12: Beuerman R, Crosson CE, Kaufman HE. Healing processes in the cornea. In: Advances in Applied Biotechnology Series. Houston: Gulf Publishing; Marais ER, Light Y, Mason C, et al. Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science. 1998;280: Marquardt B, Frith D, Stabel S. Signalling from TPA to MAP kinase requires protein kinase C, raf and MEK: reconstitution of the signalling pathway in vitro. Oncogene. 1994;9: Ueda Y, Hirai S, Osada S, et al. Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem. 1996;271: Ueffing M, Lovric J, Philip A, Mischak H, Kolch W. Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity. Oncogene. 1997;15: D Angelo G, Lee H, Weiner RI. camp-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation. J Cell Biochem. 1997;67: Hsi LC, Eling TE. Inhibition of EGF-dependent mitogenesis by prostaglandin E2 in Syrian hamster embryo fibroblasts. Prostaglandins Leukot Essent Fatty Acids. 1998;58: Li X, Zarinetchi F, Schrier RW, Nemenoff RA. Inhibition of MAP kinase by prostaglandin E2 and forskolin in rat renal mesangial cells. Am J Physiol. 1995;269:C986 C Mark MD, Storm DR. Coupling of epidermal growth factor (EGF) with the antiproliferative activity of camp induces neuronal differentiation. J Biol Chem. 1997;272: