(Investigative Ophthalmology and Visual Science. 2000;41:2164-2169.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Inhibitory Effect of PGE2 on EGF-Induced MAP Kinase Activity and Rabbit Corneal Epithelial Proliferation
Sylvia S. Kang1,
Tie Li2,
Dazhong Xu2,
Peter S. Reinach1 and
Luo Lu1,2
1 From the 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.
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Abstract
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PURPOSE. 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.
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 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.
RESULTS. 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 µg/ml)
or 8-CPT–cAMP (100 µM), 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.
CONCLUSIONS. 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.
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Introduction
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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
3
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 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 PGE2 inhibits mitosis.10
This effect of PGE2 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
PGE2 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 PGE2 synthesis. The increases in
PGE2 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
EGF-induced increases in MAPK activity and corneal epithelial cell
proliferation.
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Methods
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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 µg/ml insulin, 10,000 U/ml penicillin, and 10,000
mg/ml streptomycin. The cultures were placed in
75-cm2 culture flasks and maintained in an
incubator supplied with 95% air and 5% CO2 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 PGE2 Production
RCE cells were cultured in 12-well plates with 1 x
104 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 PGE2 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.
[3H]-Thymidine Incorporation
RCE cells were cultured in six-well plates at a density of 5 x 104 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.
[3H]-Thymidine was added to cultures at 1
µCi/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
µg/ml of PGE2 and 100 µg/ml of 8-CPT–cAMP
(adenosine-3',5'-cyclic monophosphate-sodium salt; Calbiochem,
La Jolla, CA) as a positive control. RCE cells
(107) 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
ß-mercaptoethanol; 1 µg/ml leupeptin; and 1 µg/ml aprotinin), and
homogenized using a Dounce homogenizer. Lysate (5 µl) was added to a
reaction mixture containing 10 µCi
-[32P]ATP, 5 µM cAMP, 100 µM PKA
biotinylated peptide substrate, 40 mM Tris–HCl (pH 7.4), 25 mM
MgCl2, and 100 µg/ml bovine serum albumin. Each
reaction was immediately incubated at 30°C for 5 minutes and then
terminated by adding 12.5 µl of 7.5 M guanidine hydrochloride.
Terminated reactions were spotted onto a SAM2
membrane. After the membrane was washed with 2 M NaCl, 2 M NaCl in 1%
H3PO4, and deionized water,
respectively, the amount of 32P-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
1 x 105 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
MgCl2, 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 µM
p-nitrophenylphosphate, 10 µg/ml aprotinin, and 10 µg/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
µg/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 MgCl2, 25
mM ß-glycerophosphate, 100 µM 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 µg/ml of myelin basic protein (MBP; Upstate Biotechnology,
Lake Placid, NY) was used as the substrate for the Erk-2 assay, and 1
µg/ml of MEK-1 (Upstate Biotechnology) was added for the
Raf-1 assay. The fusion protein reaction was started by the addition of
10 µCi –[32P]–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 2x SDS sample
buffer. 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).
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Results
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EGF-Induced Increases in PGE2 Production
To determine whether EGF can induce increases in
PGE2 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 PGE2 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 µM), a selective
inhibitor of cPLA2, significantly suppressed EGF-induced increases
throughout this period. This result indicates that EGF stimulates
PGE2 production in RCE cells through the
activation of cPLA2.
Effect of PGE2 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 µg/ml of exogenous
PGE2. Because PGE2
interacts with prostaglandin receptor subtypes, which are linked to the
stimulation of adenylate cyclase and modulation of
PGE2 levels altered proliferation,12
we determined whether the effect of PGE2 on the
suppression of the EGF-induced RCE cell proliferation could be mediated
through an increase in cAMP levels. After the application of 100 µM
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 µM). These results are consistent with the notion
that EGF-induced increases in PGE2 level exert a
negative feedback effect on the mitogenic response to EGF through the
stimulation of adenylate cyclase.
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Figure 2. Inhibitory effects of PGE2 on EGF-induced RCE cell
proliferation. [3H]-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 µg/ml PGE2 (E + P), EGF
plus 100 µM 8-CPT–cAMP (E + 8cpt), and EGF plus 5 µM 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.
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Effect of PGE2 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 PGE2 could suppress any of these
increases, we measured the effect of exogenous
PGE2 (10 or 50 µg/mL) on EGF-induced Erk-2
activation at 15 and 180 minutes. As shown in Figure 3B
, the addition
of PGE2 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 that AACOCF3 lowered
EGF-induced increases in PGE2 levels, we
determined whether suppression of cPLA2 by AACOCF3 (5 µM) 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 µM), 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.
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Figure 3. Effects of PGE2 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 PGE2
on EGF-induced Erk-2 activation. Exogenous PGE2
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 PGE2 (10 µg/ml;
E + PGE2), the cPLA2 inhibitor AACOCF3 (5 µM; EGF + AAC), or
the PKA inhibitor H-89 (1 µM; EGF + H89).
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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
G1 phase of the cell cycle by serum starvation
and then stimulated with 10% fetal bovine serum (FBS), 5 ng/ml EGF, or
10 µM PGE2. 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
PGE2 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 PGE2
levels can alter EGF-induced Erk-2 activation, we next determined
whether the PGE2 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 µM)
and H-89 (1 µM) 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 PGE2 (10
µg/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 PGE2 on
EGF-induced Erk-2 activation seems to reflect the inhibition of Raf-1
activity by PKA.
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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), PGE2 (10
µg/ml), or EGF and AACOCF3 (5 µM; 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 PGE2 (10 µg/ml), the cPLA2 inhibitor AACOCF3
(5 µM), or the PKA inhibitor H-89 (1 µM). IgG, immunoglobulin G.
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Discussion
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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 PGE2 levels, which elicit a
negative feedback effect on EGF-induced MAPK activation. This feedback
appears to result from PGE2-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
G1 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 PGE2 and to the presence of exogenous
PGE2. 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 PGE2
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 µg/ml
PGE2 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.15
16
17
18
A schematic
diagram of EGF-induced signaling pathways in RCE cells is proposed in
Figure 5 .
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Figure 5. Schematic diagram of EGF-induced signaling pathways in RCE cells. EGFR,
epidermal growth factor receptor; Erk, MAP kinase; PGE2R,
prostaglandin E2 receptor; PLA, phospholipase A; PLC,
phospholipase C; and Raf, MAPK kinase kinase.
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There is evidence in vascular endothelial cells, PC12 cells,
fibroblasts, and renal mesangial cells that increases in
PGE2 levels can inhibit activation of the
EGF-linked MAPK cascade at the level of Raf-1.19
20
21
22
This
inhibition occurs as a result of PGE2 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 PGE2-mediated increases in
intracellular cAMP that lead to the stimulation of PKA. As shown in
Figure 2
, exogenous cAMP had inhibitory effects on Erk-2 activation and
cell proliferation, which were equivalent to those caused by
PGE2, and EGF-induced RCE cell proliferation was
markedly suppressed either in the presence of exogenous cAMP or
PGE2. Therefore the mitogenic response to EGF
appears to be related to PGE2-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
PGE2-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 PGE2
on EGF-induced Erk-2 activation, exogenous PGE2
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.
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Footnotes
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Supported by NIH Grants EY11653 (LL) and EY04795 (PSR).
Submitted for publication September 1, 1999; revised December 15, 1999; accepted January 18, 2000.
Commercial relationships policy: N.
Corresponding author: Luo Lu, Department of Physiology and Biophysics, School of Medicine, Wright State University, Dayton, OH 45435. luo.lu{at}wright.edu
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Top
Abstract
Introduction
), and controls (
). Intracellular PGE2 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.