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INTRODUCTION |
UV radiation from the sun is the major environmental factor
responsible for a high incidence of nonmelanoma skin cancer (1-4). The
electromagnetic spectrum of UV can be divided into three parts: UVA
(320-400 nm), UVB (290-320 nm), and UVC (100-290 nm) (2). In animal
experiments, both UVB and UVC can act as complete carcinogens, whereas
UVA can only act as a tumor promoter (2-6). Because UVC light does not
penetrate the atmosphere (2), UVB radiation is believed to be
responsible for most of the carcinogenic effects of sun exposure (2,
7-9). Irradiation by UVB or UVC is known to damage DNA and could cause
gene mutations such as p53 or ras mutations (10-12).
Because of the importance of these genes in growth and differentiation,
a DNA-damaging effect has been proposed as the mechanism of UV-induced
initiation (2-9). The mechanism behind the tumor-promoting ability of
UV, however, is not well understood.
A number of reports have established that UVC and UVB induce certain
gene expression (13-16). These "UV responses" activate several
signal transduction pathways and transcription factors (14, 17). There
are two transcription factor complexes implicated in mediating the UV
response, AP-1 and NF
B (18, 19). In light of the important role of
AP-1 and NF
B activation in apoptosis and tumor promotion, the
UV-induced signal transduction pathways may be involved in the
UV-induced apoptosis and tumor promotion (18, 19). Previously, several
laboratories have reported that UVC-activated signal transduction
pathways extend from the cell membrane to the nucleus (19-22). It was
assumed that the epidermal growth factor receptor, but not protein
kinase C (PKC),1 plays a
major role in the UV-induced response (23). Very recently, we
demonstrated that atypical PKC is involved in UV-induced AP-1 activation, whereas the epidermal growth factor receptor is not required for UV-induced signal transduction (15, 24). In the present
study, we investigated whether other types of PKC isozymes of classical
PKC or novel PKC are activated and/or involved in UVB-induced signal
transduction and apoptosis.
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EXPERIMENTAL PROCEDURES |
Materials--
Eagle's minimum essential medium (EMEM) and
fetal bovine serum (FBS) were from Whittaker Biosciences;
L-glutamine was from Life Technologies, Inc.; gentamicin
was from Quality Biological, Inc.; luciferase assay substrate was from
Promega. Aprotinin and leupeptin were from Sigma; rottlerin, GF109203X,
and safingol were from Calbiochem. The phosphospecific antibodies
against phosphorylated sites of Erks, JNKs, and p38 kinase were from
New England Biolabs; the antibodies against protein kinase C subtypes
were from Santa Cruz.
UV Irradiation of Cells--
UVB irradiation was performed on
serum-starved monolayer cultures utilizing a transluminator emitting
UVB. Because the normal UVB lamp also generates a small amount of UVC
light, the UVB irradiation was carried out in a UVB exposure chamber
fitted with a Kodak Kodacel K6808 filter that eliminates all
wavelengths below 290 nm (14).
Cell Culture--
Mouse epidermal JB6 promotion
sensitive Cl 41 and its dominant negative mutant cell lines for
PKC
, PKC
, and PKC
were grown at 37 °C in EMEM supplemented
with 5% heat-inactivated FBS, 2 mM
L-glutamine, and 25 mg/ml gentamicin (25).
Stable Transfection and Cell Culture--
Cl 41 PKC
-DNM and
PKC
-DNM, the stable transfectants with dominant negative PKC
and
PKC
, were established and reported previously (26). PKC
mutants
and vector plasmid P
MTH (26-28) were received from Dr. Peter
Blumberg. JB6 promotion sensitive cells, Cl 41, were
cultured in a 6-well plate until they reached 85-90% confluence. We
used 0.3 µg of cytomegalovirus-neo vector and 2 µg of
AP-1 luciferase plasmid with 6 µg of a dominant negative mutant of
PKC
plasmids or vector P
MTH plasmid DNA and 15 µl of
LipofectAMINE reagent to transfect each well in the absence of serum.
After 10-12 h, the medium was replaced with 5% FBS/MEM. Approximately
30-60 h after the beginning of the transfection, the cells were
treated with 0.033% trypsin, and the cell suspensions were transferred
to 75-ml culture flasks and cultured for 24-28 days with geneticin
selection (300 µg/ml). Stable transfectants were screened by Western
blotting with rabbit polyclonal IgG against PKC
, PKC
, and PKC
.
The stable transfectants and PKC
-DNM were cultured in geneticin-free
EMEM for at least two passages before each experiment (26).
PKC Translocation Assay--
2 × 105 cells
were seeded in a 10-cm dish. After they reached 85-90% confluence,
the cells were starved for 24-48 h in 0.1% FBS. After irradiation,
the cells were washed one time with ice-cold phosphate-buffered saline
(without Ca2+). Two hundred µl of homogenization buffer A
(20 mM Tris-HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 10 µg/ml
leupeptin) was added to each dish, and the cells were scraped into a
1.5-ml tube with a rubber policeman. The suspension was sonicated for
10 s at output 4 with a sonicator (Ultrasonics Inc., NY) and
centrifuged at 100,000 × g for 1 h at 4 °C.
The supernatant was collected as the cytosol fraction. The pellet was
resuspended in 200 µl of homogenization buffer B (1% Triton X-100 in
buffer A) and sonicated for 10 s. The suspension was centrifuged
at 15,000 × g for 15 min at 4 °C. The supernatant
was collected as a membrane fraction. Protein concentration of each
sample was determined, and 100 µl of 3× Laemmli sample buffer (187.5 mM Tris-HCl, pH 6.8, 6% SDS, 30% glycerol, 150 mM dithiothreitol, 0.3% bromphenol blue) was added (29).
Western Blotting--
Samples containing equal amount of protein
were loaded in each lane for 8% SDS-polyacrylamide gel
electrophoresis. The gel was transferred and analyzed as described
previously (30). Immunoblots for phosphorylated proteins of p38
kinases, Erks, and JNKs were carried out using phosphospecific
mitogen-activated protein kinase antibodies against phosphorylated
sites of p38, Erks and JNKs as described previously (30). Antibodies
for phosphorylated MAP kinases were from New England Biolabs, and
antibodies for PKC subtypes were from Santa Cruz. Antibody-bound
proteins were detected by chemiluminescence (ECL of New
England Biolabs or ECF of Amersham Pharmacia Biotech) and analyzed
using the Storm 840 (Molecular Dynamics).
DNA Fragmentation Assay--
Cells were grown in a 15 cm-dish
and treated with various PKC inhibitors and UVB irradiation when cell
density reached 50-70% confluence. Both detached and attached cells
were harvested by scraping and centrifuging. Then the cells were lysed
with lysis buffer (5 mM Tris-HCl, pH 8.0, 20 mM
EDTA, 0.5% Triton X-100) on ice for 45 min. Fragmented DNA in the
supernatant after centrifugation at 14,000 rpm (45 min at 4 °C) was
extracted twice with phenol/chloroform/isopropanol (25:24:1, v/v) and
once with chloroform and then precipitated with ethanol and 5 M NaCl. The DNA pellet was washed once with 70% ethanol
and resuspended in Tris-EDTA buffer (pH 8.0) with 100 µg/ml RNase at
37 °C for 2 h. The DNA fragments were separated by 1.8%
agarose gel electrophoresis (31, 32).
MAP Kinases Activity Assay--
JNKs, Erks, and p38 kinase
activities were assayed as described previously (25).
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RESULTS |
UVB-induced Translocation of PKC
and PKC
, but Not PKC
, to
Membranes--
Translocation of PKC to a particulate fraction is the
key step for the activation of this enzyme (33). Determination of PKC
content in membranes or the ratio of membranes to cytosol can reflect
PKC activity. The following three PKCs can be dominantly detected in
JB6 cells: PKC
, PKC
, and PKC
(15). To investigate whether
these PKCs are involved in UVB-induced signal transduction, we analyzed
the membrane/cytosol distribution of the three main subtypes of PKC in
JB6 cells (15). 12-O-tetradecanoylphorbol-13-acetate was
used as a positive control for stimulating PKC translocation. Our
results showed that UVB markedly induced the translocation of PKC
and PKC
, but not PKC
, from cytosol to membrane. The UVB-induced
PKC
and PKC
translocation was dose-dependent (Fig. 1) and reached a high level 5-15 min
after UVB irradiation (Fig. 2).

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Fig. 1.
The dose response of
PKC , PKC , and
PKC translocation by UVB. JB6 Cl 41 cells
(2 × 105) were cultured in monolayer in 10 cm-diameter dishes until 90% confluent. Then the cells were starved
for 48 h in 0.1% FBS/EMEM. The cells were treated with various
doses of UVB irradiation and harvested 10 min later. The samples were
fractionated as described under "Experimental Procedures." The
sample was analyzed by 8% SDS-polyacrylamide gel electrophoresis and
Western blotting. The same membrane was stripped and reprobed with the
different isotype-specific antibodies. The immunoblots were visualized
using the ECF detection reagents and scanned by Storm imager. The
experiments were repeated three times and similar results were
obtained. A, Western blotting. B, blots were
scanned and quantified by Storm 840. Each value is the relative ratio
of membrane to cytosol PKC. TPA,
12-O-tetradecanoylphorbol-13-acetate.
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Fig. 2.
The time course of
PKC , PKC , and
PKC translocation by UVB. JB6 Cl 41 cells
(2 × 105) were cultured in monolayer in 10-cm dishes
until 90% confluent. Then the cells were starved for 48 h in
0.1% FBS/EMEM. The cells were harvested at 5, 15, and 30 min after UVB
irradiation at 8 kJ/m2. The membrane and cytosol fractions
were obtained as described under "Experimental Procedures" and were
analyzed by 8% SDS-polyacrylamide gel electrophoresis and Western
blotting. The same membrane was stripped and reprobed with the
different isotype-specific antibodies. The immunoblots were visualized
using the ECF detection reagents and scanned by Storm imager. The
experiments were repeated three times and similar results were
obtained. A, Western blotting. B, blots were
scanned and quantified by Storm 840. Each value is the relative ratio
of membrane to cytosol PKC.
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Dominant Negative Mutant of PKC
and PKC
Blocked PKC
Translocation and UVB-induced MAP Kinases--
The above results
suggest that PKC
and PKC
, but not PKC
, were involved in
mediating UVB-induced signal transduction. To address this question, we
used JB6 stable transfectants with the DNM of PKC
, PKC
, or PKC
(26). These DNMs were constructed by site-directed mutagenesis of
lysine residues in the ATP binding site (located in the catalytic
domain) (26). The protein level of PKC
in these transfectants was
determined using rabbit polyclonal IgG against PKC
. The results
showed a high level of the introduced mutated protein of PKC
in this
transfectant (Fig. 3). Fig.
4 showed that the DNM PKC
or PKC
blocked UVB-induced translocation of PKC
and PKC
, respectively.
Further, we have analyzed the effect of these dominant negative PKC
mutants on the UVB-induced activation of Erks, JNKs, and p38 kinases.
We found that the total protein content of Erks, JNKs, and p38 kinases
in three DNM PKC transfectants was lower than in Cl 41 cells, although
the equal volume in each sample was loaded (Fig.
5). Further, we demonstrated that the
total protein in each sample was equal by dying the blotting membrane
with Coomassie Blue (data not shown). However, UVB-induced phosphorylation of Erks and JNKs was strongly inhibited by DNM PKC
and PKC
, whereas the DNM of PKC
was less effective on the UVB-induced phosphorylation of Erks and JNKs. The results suggest that
the DNM of PKC
or PKC
, but not the DNM of PKC
, inhibited UVB-induced activation of Erks and JNKs, but not p38 kinases.

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Fig. 3.
Overexpression of dominant negative
PKC in stable transfectants. JB6 Cl 41 stable transfectants as indicated were cultured in each well of 6-well
plates with 5% FBS/EMEM medium. After cells reached 95% confluent,
the cells were washed once with ice-cold phosphate-buffered saline and
extracted with SDS-sample buffer. Then the cell extracts were separated
on an 8% polyacrylamide-SDS gel, transferred, and probed with the
rabbit polyclonal IgG against PKC . CMV-neo,
cytomegalovirus-neo.
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Fig. 4.
Effect of DNM PKC
and DNM PKC on UVB-induced PKC
translocation. JB6 Cl 41 cells and Cl 41 stable transfectants with
DNM PKC and DNM PKC (2 × 105) were cultured in
monolayer in 10-cm dishes until 90% confluent. Then the cells were
starved for 48 h in 0.1% FBS/EMEM. The cells were harvested at 5, 15, and 30 min after UVB irradiation at 8 kJ/m2. The
samples were fractionated as described under "Experimental
Procedures." The samples were analyzed by 8% SDS-polyacrylamide gel
electrophoresis and Western blotting. The same membrane was stripped
and reprobed with the different isotype-specific antibodies. The
immunoblots were visualized using the ECF detection reagents and
scanned by Storm imager. Each value is the relative ratio of membrane
to cytosol PKC. This is one of three similar experiments. A,
relative ratio of membrane to cytosol PKC . B, relative
ratio of membrane to cytosol PKC .
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Fig. 5.
Effect of DNM PKC ,
PKC , and PKC on
UVB-induced phosphorylation of Erks, JNKs, and p38 kinases. JB6 Cl
41 cells and Cl 41 stable transfectants with DNM PKC , PKC , and
PKC (5 × 104/well) were cultured in monolayer in
6-well plates until 90% confluent. The cells were harvested at
different times after UVB irradiation at 8 kJ/m2 as
indicated. The samples were analyzed by Western blotting with
antibodies for nonphosphorylated and phosphorylated Erk, JNK, and p38
proteins using a PhosphoPlus MAP kinase kit from New England
Biolabs.
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Further, we also compared protein phosphorylation and enzyme activity
of MAP kinases after treatment with UVB. The results in Fig.
6 showed that the phosphorylation of MAP
kinases induced by UVB is well correlated with the activity of these
kinases.

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Fig. 6.
Effect of UVB on phosphorylation and activity
of MAP kinases. For testing the phosphorylation of MAP kinases,
JB6 Cl 41 were starved for 48 h in 0.1% FBS/EMEM. The cells were
irradiated by UVB at 8 kJ/m2 and harvested with SDS sample
buffer. The samples were analyzed by Western blotting with antibodies
for phosphorylated Erk, JNK, and p38 proteins using a PhosphoPlus MAP
kinase kit from New England Biolabs. For testing the activity of MAP
kinases, the cells treated by UVB were lysed and centrifuged. The three
MAP kinase proteins were immunoprecipitated using specific MAP kinase
antibodies and were detected by Western blot.
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Inhibitors of PKC
, but Not of PKC
, Blocked UVB-induced
Activation of Erks and JNKs--
It has been reported that some PKC
inhibitors can selectively inhibit certain PKC isozymes without
inhibiting other subtypes of PKC or other protein kinases (34). The
above results suggest that the role of PKC in mediating UVB stimulation
is isozyme-specific. We therefore used several PKC inhibitors to
investigate the role of these PKC subtypes on UVB-induced MAP kinase
activity. GF109203X is an inhibitor mainly of classical PKC and novel
PKC (35, 36); rottlerin is a selective inhibitor of PKC
(34), and
safingol is only active for inhibition of PKC
(37).
The results in Fig. 7 showed that
rottlerin could markedly inhibit the UVB-induced phosphorylation of
Erks and JNKs, but not p38 kinases. We also found that the protein
levels of Erks and JNKs in rottlerin treatment groups were lower than
in the control group, although the total p38 protein level was not
affected. Safingol, the selective inhibitor for PKC
, did not inhibit
UVB-induced activation of Erks, JNKs, or p38 kinases. At a low
concentration of GF109203X (<10 µM) there was no effect
on UVB-induced activation of Erks or JNKs (data not shown). However,
higher concentrations of GF109203X (>20 µM) inhibited
activation of all three MAP kinases (Fig. 7). Because the affinity of
GF109203X on classical PKC is stronger than on novel PKC and it
inhibits PKC
and PKC
at higher concentrations, the results
obtained using this inhibitor provide additional evidence that PKC
and PKC
, but not PKC
, are involved in UVB-induced signal
transduction.

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Fig. 7.
Effect of PKC inhibitors on UVB-induced
phosphorylation of Erks, JNKs, and p38 kinases. JB6 Cl 41 cells
(5 × 104/well) were cultured in monolayer in 6-well
plates until 90% confluent. Then the cells were starved for 48 h
in 0.1% FBS/EMEM. The cells were pretreated with inhibitors at various
concentrations for 30 min before irradiation with UVB at 8 kJ/m2 for different times as indicated and harvested. The
samples were analyzed by Western blotting with antibodies for
nonphosphorylated and phosphorylated Erk, JNK, and p38 proteins using a
PhosphoPlus MAP kinase kit from New England Biolabs.
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Based on these results, together with the effect of DNM of PKC, we
conclude that the activation of PKC
and PKC
, but not PKC
,
plays an important role in mediating UVB-induced phosphorylation of
Erks, JNKs, and p38 kinases.
PKC Inhibitors Inhibit UVB-induced Apoptosis of JB6 Cells--
UV
radiation is a strong inducer of cell apoptosis. We have found that
UVB-induced apoptosis in JB6 cells is dose-dependent (data
not shown). To further assess the biological significance of PKC in
mediating the UVB-induced signal transduction, we investigated the role
of PKC in UVB-induced apoptosis in JB6 cells. The results in Fig.
8 show that UVB-induced apoptosis of JB6
cells was markedly inhibited by rottlerin and GF109203X, whereas
safingol had little inhibitory effect. These results suggest that the
regulation by PKC of UVB-induced signal transduction may mediate
UVB-induced apoptosis.

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Fig. 8.
Effect of PKC inhibitors on UVB-induced
apoptosis. JB6 cells were grown in 15-cm dishes and treated with
various PKC inhibitors and UVB irradiation. Both detached and attached
cells were harvested by scraping and centrifuging. The cells were
harvested, and fragmented DNA was extracted and precipitated. The DNA
fragments were separated by 1.8% agarose gel electrophoresis.
A, effect of safingol and rotterlin on UVB-induced
apoptosis. B, effect of GF109203X on UVB-induced
apoptosis.
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Erks and JNKs, but Not p38 kinases, May Be Involved in UVB-induced
Apoptosis--
Because rottlerin inhibits UVB-induced Erks and JNKs,
but not p38 kinases, and inhibits UVB-induced apoptosis, we hypothesize that Erks and JNKs, but not p38 kinases, play a role in mediating UVB-induced apoptosis. We therefore used PD98059, a selective inhibitor
of mitogen-activated protein kinase/extracellular signal-regulated kinase that is a specific upstream activator of Erks as well as dominant negative mutant Erk2, to investigate the role of Erks in
UVB-induced apoptosis. We also used dominant negative mutant JNK1 and a
selective inhibitor of p38 kinase, SB202190, to study the role of JNK1
and p38 kinases in UVB-induced apoptosis, respectively (Fig.
9). The results showed that inhibiting
Erk activation by PD98059 and DNM-Erks inhibited UVB-induced apoptosis.
Further, cells expressing the dominant negative mutant of JNK1 also
blocked UVB-induced apoptosis, whereas SB202190 was not inhibitory. The concentration of SB202190 applied in the apoptosis assay has been shown
to inhibit the UV-induced phosphorylation of p38 kinases in JB6
cells.2 These results suggest
that Erk2 and JNK1, but not p38 kinases, mediate UVB-induced
apoptosis.

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Fig. 9.
Role of MAP kinases in mediating UVB-induced
apoptosis. JB6 cells, DNM-Erks, and DNM-JNK1 cells were grown
in 15-cm dishes, and JB6 cells were treated with various concentrations
of PD98059 and SB202190. After treatment with UVB irradiation, both
detached and attached cells were harvested by scraping and
centrifuging. The cells were harvested, and fragmented DNA was
extracted and precipitated. The DNA fragments were separated by 1.8%
agarose gel electrophoresis.
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DISCUSSION |
In the present work, we found that UVB irradiation induced PKC
and PKC
, but not PKC
, translocation from cytosol to membrane and
that this translocation was partially blocked in the cells that express
DNM PKC
or DNM PKC
. In cells transfected with DNM PKC
and
PKC
, UVB-induced phosphorylation of JNKs and Erks was markedly
attenuated. A selective inhibitor of PKC
completely blocked
UVB-induced phosphorylation of JNKs and Erks, but not p38 kinases.
However, the PKC
-specific inhibitor had no effect on the UVB-induced
phosphorylation of MAP kinases (Erks, JNKs, and p38 kinases). These
findings indicate that the UVB-induced phosphorylation of Erks and JNKs
requires PKC
and PKC
activation.
PKC belongs to a large kinase family consisting of at least 11 members,
which are divided into three groups on the basis of their biochemical
properties and sequence homologies (38). The different PKC isotypes may
have specific roles in signal transduction (39). It has been reported
that PKC may be involved in UVA-induced signal transduction (40), but
not UVC-induced signal transduction (23). Here, we used mouse epidermal
JB6 cells to study the role of PKC in UVB-induced signal transduction.
JB6 cells express PKC
, PKC
, and PKC
(15), and PKC
and
PKC
both belong to novel PKC (33, 38). From our present results, it
appears that membrane-bound PKC
and PKC
were higher than PKC
before cells were stimulated. Upon UVB irradiation, PKC
and PKC
,
but not PKC
, were translocated to the particulate fraction. When the
cells were treated with rottlerin, a specific antagonist for PKC
(34), UVB-induced phosphorylation of Erks and JNKs, but not p38
kinases, was markedly blocked. GF109203X, a potent inhibitor for PKC
and PKC
, produced an inhibitory effect on UVB-induced Erks or JNKs
at a high concentration (20 µM). Safingol, a specific
inhibitor of PKC
(37), did not display any inhibitory effect on
these three MAP kinases. Cells transfected with the dominant negative
mutants of PKC
or PKC
inhibited the stimulation of Erks, JNKs,
and p38 kinases induced by UVB. These results indicate that activated
PKC
and PKC
are necessary in mediating UVB-induced signal transduction.
Activation of PKC is associated with the translocation of enzymes from
the cytosol to the cell particulate fraction (41). UVB stimulation of
membrane-associated PKC could result from several possible mechanisms.
The translocation of PKC is triggered by diacylglycerol or
12-O-tetradecanoylphorbol-13-acetate interacting with the C1
domain of the PKC protein (33). It was reported that UVB induces
phospholipase A2 activation and arachidonic acid release
(42), a reaction that also produces lysophospholipid. It has been found
that lysophosphatidylcholine and arachidonic acid are also activators
for PKC (42). Punnonen and Yuspa (43) reported that UVB irradiation of
cultured cells increases levels of diacylglycerol. On the other hand,
UV energy generates oxygen radicals such as
H2O2, which may activate PKC (44-46). Indeed, it has been reported that reactive oxygen species directly activate purified PKC in vitro (47).
It is interesting that DNM PKCs decrease the total protein level of MAP
kinases. We also found that rottlerin inhibits the total protein levels
of Erks and JNKs, but not p38 kinases. The mechanisms of the inhibitory
effect on MAP kinase protein level are currently under investigation in
our laboratory.
Considerable attention has recently been focused on the role played by
different kinase cascades in the control of apoptosis. In the present
report, we found that both rottlerin and GF109203X inhibited
UVB-induced apoptosis. However, rottlerin blocked UV-induced Erks and
JNKs, but not p38, whereas GF109203X can block all three MAP kinases.
MAP kinase signaling cascades are involved in the many cellular
responses, including apoptosis, to extracellular stimuli. For example,
Jimenez et al. (48) reported that the mitogen-activated
protein kinase/extracellular signal-regulated kinase 1 inhibitor
PD98059 blocked asbestos-induced apoptosis in rat pleural mesothelial
cells. Activation of JNKs plays a causal role in the induction of
apoptosis in numerous cells when stimulated by some stresses, whereas
the inhibition of the Erks pathway has been observed in a number of
cell systems undergoing programmed cell death (49). In the present
study, we found that the inhibition of Erks or JNKs, but not p38
kinases, blocks UVB-induced apoptosis. This suggests that activation of
PKC
and PKC
and phosphorylation of Erks and JNKs, but not p38
kinases, are important in mediating UVB-induced apoptosis.
In summary, UVB induces activation of PKC
, PKC
, and MAP kinases
in JB6 cells. Inhibition of PKC
and PKC
blocks UVB-induced MAP
kinases and apoptosis. We conclude that UVB-induced apoptosis appears
to be mediated by PKC
, PKC
, Erks, and JNKs.