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INTRODUCTION |
Extracellular signals regulate cellular proliferation,
differentiation, and death through activation of kinase cascades of the
mitogen-activated protein kinases
(MAPKs)1 including ERK, JNK,
and p38 (1-4). The ERK MAPKs are most frequently activated by
mitogenes, whereas the JNK and p38 MAPKs are strongly responsive to
stress and inflammatory signals. The then individual MAPK activities
often either collaborate or oppose each other in the regulation of
biological responses to different stimuli. ERK activity, for example,
has been shown to antagonize the p38 and JNK activities in regulation
of apoptosis in PC-12 cells (5). At the level of a target gene
expression, ERK kinase stimulates cyclin D1 transcription, which is
suppressed by p38 MAPK (6). A similar opposing effect between ERKs and
p38 MAPK is also observed in regulation of chondrogenesis of
mesenchymes (7). Moreover, an antagonizing effect can occur between the
JNK and p38 kinases, despite the fact that both pathways frequently
respond to the same classes of stimuli (8, 9). Hypertrophic agonists
including endothelin-1 and phenylephrine, for example, stimulate p38
and JNK kinases in myocytes, in which p38 promotes but JNK suppresses the development of myocyte hypertrophy (10). Our previous work of
analyzing Ras signal transduction pathways demonstrated that oncogenic
Ras stimulates ERK, JNK, and p38 in NIH 3T3 cells, but the p38
activation in this process blocks the Ras signal through its inhibition
of Ras-induced JNK downstream effects (11). In addition to their
opposing activities, under certain circumstances ERK and p38 kinases
cooperate in regulation of c-fos expression in
response to UV light (12, 13). It appears, therefore, that signaling
cross-talks and integrations among the ERK, JNK, and p38 MAPK pathways
are important steps to regulate the final signaling output from MAPK
pathway activations. Since each of MAPKs has several subfamily members,
it is possible that this signaling cross-talk and integration might
also occur inside each type of MAPK among its isoforms to meet cellular
requirements for various intricate and delicate biological processing
of signals from stimuli.
AP-1 (activating proteins 1) are sequence-specific transcription
factors composed of homodimers or heterodimers of the Jun family
(c-Jun, JunD, and JunB) or heterodimers of the Jun family member with
any of the Fos family members (c-Fos, FosB, Fra1, and Fra2) or other
transcription factors such as activating transcription factor-2 (ATF2,
cAMP-response element-binding protein, and NFAT (14-16). AP-1
transcription factors are key regulatory molecules that play a central
role in control of cell proliferation and transformation (14, 16, 17)
by converting MAPK signals into expression of specific target genes (3,
18). Members of the AP-1 family are regulated at both the
transcriptional and posttranscriptional levels by MAPKs (14, 16).
c-fos expression, for example, is regulated by all of the
MAPK pathways including the ERK (19), JNK (20), and p38 pathways (12).
c-Jun, on the other hand, is phosphorylated at Ser-63 and Ser-73
by JNK (21, 22). The activated c-Jun can then autoregulate its own
expression through a c-Jun/AP-1 enhancer element in its promoter (23).
p38 can also stimulate c-Jun expression by activation of the
transcription factor ATF2 (24, 25), which forms a heterodimer with
c-Jun that acts on the AP-1 enhancer element in the c-jun
gene (23). Moreover, p38 may activate transcription factors
myocyte-enhancing factor 2C (MEF2C) and MEF2A, which transactivate
c-Jun through a MEF-responsive element also present in the
c-jun gene regulatory region (26-29). Thus, AP-1 can be
activated by all three MAPK pathways in a selective manner and serve as
a common integrator of MAPK signaling to specific target gene
expressions (14, 16, 30).
p38 activation is known to trigger pleiotropic biological effects,
including cell death, differentiation, and proliferation, by mechanisms
mostly unknown (1-3). The signal relay along the p38 pathway involves
a kinase cascade, which generally consists of the upstream stimulators
MAPK kinase 6 (MKK6) and 3 (MKK3), the p38 kinases (
,
,
, and
), and the downstream effectors (3). p38 kinases downstream
phosphorylate protein kinases including MAPK-activated protein kinase
2 and p38-related/activated protein kinase as well as the
nonkinase small heat shock protein 27 (Hsp27) and transcription factors
such as AP-1 family proteins ATF2, c-fos, and MEF2 (3).
p38
, also called p38, was first cloned as a 38-kDa protein (31-33).
Three other p38 isoforms (p38
, -
, and -
) were isolated later
and have more than 60% identity in sequence in comparison with p38
.
p38
and p38
are ubiquitously expressed, whereas the p38
and
p38
products are only detected in certain cell types (3). The most
important functions of the p38 pathway so far deduced were largely
discovered by analyzing p38
, despite the fact that each p38 family
member may have distinct functions (3). p38
, for example, is known
to be essential for mammalian embryonic development as demonstrated by
knock-out studies (34), is widely involved in cytokine production in
response to inflammatory stimuli (32, 33, 35), and regulates
cardiomyocyte differentiation/apoptosis (36, 37). Whereas some of these
functions have also recently been ascribed to p38
in some cell types
(15, 27, 38), the contributions of other p38 family members remain
mostly obscure. Since p38 family members are expressed differently in
different cell types and tissues, and each of the family members may
have distinct functions, we sought to test the hypothesis that the cell
type-specific isoform expression profiles of p38 family members determine the biological effect of p38 pathway activation in that cell
type. Our results demonstrated that p38
has an opposing effect to
p38
and p38
in mediating MKK6 and sodium arsenite (ARS) signaling
to activate AP-1 trans-activation of target genes through
mechanisms involving c-Jun regulation. These results suggest that an
upstream signal of the p38 pathway is interpreted by the p38 isoform
expression pattern through differentially regulating AP-1 transcription
factor activity, leading to a specific cellular outcome that is
dependent on the spectrum of isoform expression but independent of the
upstream pathway stimulator.
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MATERIALS AND METHODS |
cDNA Constructs and Expression
Plasmids--
pcDNA3-HA-MKK6 is a constitutively active
mutant of MKK6 with Ser-207 and Thr-211 replaced by Glu (MKK6/2E) (39,
40). This plasmid as well as the FLAG-tagged wild type and dominant negative forms (AF or KM) of human p38
, p38
, p38
, and p38
have been previously described (32, 39-41). The AFs are p38 mutants that cannot be phosphorylated, since the TGY dual phosphorylation site
has been changed to AGF, whereas the kinase-dead KM mutants were
generated by a mutation of the ATP-binding site (Lys to Met (K to M)).
Mutations were generated by PCR-based techniques using the QuikChange
site-directed mutagenesis kit from Stratagene as described (40, 41). A
p38
double mutant (p38
/AF) was created by substituting
Thr180 with Ala and Tyr182 with Phe
(underlined) using a PCR-based procedure (the primer sequence:
GCAGACGCCGAGATGGCTGGCTTCGTGGTGACCCGCTGG). pGEX
for GST-ATF2-(1-109) was kindly provided by Dr. Roger Davis (25).
pSV-GFP was purchased from Invitrogen. A full length of mouse c-Jun was
cloned into a mammalian expressing vector pHM6-HA (Roche Molecular
Biochemicals). An AP-1 luciferase construct (AP-1-Luc) was kindly
provided by Craig Hauser, which was generated by cloning three AP-1
repeats into a luciferase reporter gene containing a minimal Fos
promoter (42). The mouse VDR promoter (VDR-Luc, 0.5 kb) was cloned and inserted into a pGL2 basic vector (Promega) in front of the luciferase gene as previously described (43, 44). A c-fos luciferase reporter (c-fos Luc, containing
356 to +109 of the murine
c-fos promoter) (45) and c-jun luciferase
promoter (c-jun Luc, containing base pairs
225 to
+150 of the promoter) (26) have been previously described.
Other Reagents--
Minimum essential medium,
L-glutamine, and antibiotics were supplied by
Invitrogen. Fetal bovine serum was obtained from BioWhittaker. DNA was prepared using an Endofree Kit from Qiagen. A DNA transfection kit (calcium phosphate) and a dual luciferase kit were purchased from
Promega. Fugene 6 reagent for transfection was purchased from Roche
Molecular Biochemicals. Glutathione-agarose beads were from Sigma.
Proteinase inhibitors and other chemicals including ARS and anisomycin
(ANI) are from Sigma. Protein G-Sepharose 4B and protein A-Sepharose 4B
beads were purchased from Zymed. Rabbit antisera against p38
,
p38
, p38
, and p38
have been previously described (38).
Anti-phospho-c-Jun (Ser-63) and anti-HA antibody were purchased from
Cell signaling and Roche Molecular Biochemicals, respectively.
Anti-FLAG M2 affinity gel and anti-FLAG mouse monoclonal antibody were
from Sigma. [
-32P]ATP was from Amersham Biosciences.
The GST-ATF2-(1-109) was prepared by affinity chromatography
over GSH-agarose beads (Sigma).
Cell Culture, Transfection, and Cell Proliferation and Luciferase
Assay--
Human breast cancer cell line MCF-7 and MDA-MB-468 were
obtained from ATCC and maintained in minimal essential medium
containing 10% fetal bovine serum and antibiotics at 37 °C with 5%
CO2. Mouse fibroblasts NIH 3T3, the Ras-transformed
counterpart, and mammary carcinoma cells EMT-6 have been previously
described (11, 46). The wild type and c-Jun knockout MEF were kindly
provided by Ron Wisdom (47). For promoter analyses, the protocol
of calcium phosphate-mediated transfection from Promega was followed.
To increase transfection efficiency, Fugene 6 was used for cell
proliferation, kinase assay, and MKK6-p38s binding. The luciferase
construct (AP-1-Luc or VDR-Luc) or FLAG-tagged p38 (wild type or the AF or KM mutant p38
, p38
, p38
, and p38
) was expressed at a 1:1 ratio with vector or the active MKK6 (MKK6/E). For cell proliferation, MCF-7 cells were transfected with p38
or p38
together with a marker plasmid pSV-GFP and, after 48 h, were pulse-labeled with 5-bromo-2'-dexoyuridine (BrdUrd) (Roche Molecular Biochemicals) and
fixed in 4% paraformaldehyde. The GFP-positive and
BrdUrd-positive cells were counted under fluorescence microscope
(Leica). For luciferase assay, cells were collected 48 h later in
the lysis buffer, and the luciferase activity of the promoter was
assayed with a dual luciferase kit from Promega by using pRL-TK
(encoding Renilla luciferase) as a normalization control in
a TD-20/20 Luminometer (Turner Designs). To assess ARS-induced AP-1
activity, cells were treated with 2 mM ARS for 30 min
24 h before the luciferase assay. The results from at least three
separate experiments were analyzed with Student's t test
for the statistically significant difference.
Western Blotting Analyses--
For Western blot analyses, cells
in good growth condition were lysed in modified radioimmune
precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 10 mM NaF, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml aprotinin, leupeptin, and pepstatin).
Protein concentration was determined by a DC Protein Assay kit
(Bio-Rad). Typically, 50 µg of protein was separated on a SDS-PAGE,
which was transferred to a nitrocellulose membrane for detection of the
molecule of interest, using ECL (Amersham Biosciences) as previously
described (48). For detection of endogenous or FLAG-tagged p38s, the
experimental conditions of previous publications (38, 49) were followed by using either p38 isoform-specific or anti-FLAG antibodies. The
different dilutions of the polyclonal rabbit anti-p38 antibodies have
been previously titrated and shown to yield similar binding affinities
to their respective proteins (38). The antibody dilutions used in this
work were as follows: anti-p38
, 1:5,000; p38
, 1:5,000; p38
,
1:2,000; p38
, 1:1,000. Under the these experimental conditions, the
dilutions of the antibodies showed similar affinities toward their
respective bacterially expressed recombinant p38 isoform proteins with
little cross-reactions (data not shown).
Immunoprecipitation and Protein Kinase Assay--
Following
transfection, cells were collected by trypsinization, and pellets were
quick-frozen in liquid nitrogen after washing and spin. For ARS-induced
p38 activation, cells transiently expressing different FLAG-tagged p38s
were treated with 2 mM ARS for 30 min, followed by cell
collection in the lysis buffer. The cell pellet was either stored at
80 °C or used immediately for protein preparation (resuspended in
100 µl of the lysis buffer and incubated on ice for 30 min with
vortexing for 30 s every 10 min). Protein (200 µg) was incubated
with 20 µl of anti-FLAG M2 affinity gel (Sigma) for FLAG-tagged p38
at 4 °C in a rotating plate overnight. The precipitates were washed
two times with respective lysis buffer and two times with kinase
binding buffer (20 mM HEPES, pH 7.6, 50 mM
NaCl, 0.05% Triton X-100, 0.1 mM EDTA, 2.5 mM
MgCl2). The kinase reaction was carried out at 30 °C for
30 min in 25 µl of kinase reaction buffer (20 mM HEPES,
pH 7.6, 20 mM MgCl2, 15 µM ATP,
20 mM
-glycerolphosphate, 20 mM
p-nitrophenyl phosphate, 0.5 mM
Na3VO4, 2 mM dithiothreitol) as
described previously (11, 48). [
-32P]ATP (5 µCi) and
1 µg of GST-ATF2-(1-109) were used for each sample. Following a
30-min reaction at 30 °C, an equal volume of 2× Laemmli buffer was
added to stop the reaction. The phosphorylated protein was separated in
a SDS-PAGE. The gels were dried, scanned, and quantitated in a
PhosphorImager (Amersham Biosciences). Each kinase assay,
starting at transfection and ending with the determination of the
protein kinase activity, was repeated two or three times with similar results.
 |
RESULTS |
Expression of Endogenous p38s in Human Breast Cancer Cells and
Their Contribution to MKK6-induced AP-1 Activation--
p38 kinase is
known to stimulate AP-1 via activation of one or more its components
(12, 25, 26). To assess the contribution of each p38 family member to
transduction of AP-1 stimulatory signaling, Western blot analyses was
carried out to determine the expression of endogenous p38 family
members in the two human breast cancer cell lines, MCF-7 and MDA-MB-468
(468). Cell lysates were prepared and examined for p38 isoform
expressions using the isoform-specific antibodies as previously
described (38). A similar ECL staining intensity was shown for these
antibodies toward the same amount of the purified recombinant p38
isoform proteins under the antibody dilutions utilized here (see
"Materials and Methods"). The results of Fig.
1 (upper panel)
reveal that all four p38 isoforms are present in MCF-7 and 468 cells,
which are migrated differently on SDS-PAGE due to their different
molecular weights (3, 38). p38
displayed a major band at 38 kDa in these two human breast cancer cell lines, followed by p38
(39 kDa),
p38
(43 kDa), and p38
(40 kDa). The higher level of p38
than
other isoforms in both cell lines is consistent with the previous
literature, indicating that p38
is the major predominant form of the
p38 family (3). However, a considerable amount of the p38
and p38
proteins is also present in these cells, which isoforms are known to be
selectively expressed only in certain tissues or cell types (3). This
is perhaps the first report showing that proteins of all p38 family
members are expressed in the same cell lines.

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Fig. 1.
Expression of p38 family members in human
breast cancer cells and their contribution to MKK6 stimulation of AP-1
activity. Top panel, expression of p38 isoforms
in human breast cancer cells. MCF-7 and 468 cells were lysed, 50 µg
of protein was separated on an SDS-PAGE, levels of p38 isoform proteins
were detected by Western blot analyses using the isoform-specific
antiserum, and the same membrane was reprobed with actin antibody for
loading control. Antibody was titrated to give equivalent stain
intensity for equal concentrations of recombinant p38 isoform proteins
(see "Materials and Methods"). Bottom, stimulation of
AP-1 trans-activation by MKK6 and its inhibition by dominant
negative forms of p38s. AP-1-Luc was co-expressed with vector or active
MKK6 (MKK6/E) in the presence of vector or the dominant negative genes
of the p38 isoforms. AP-1 luciferase activity was determined 48 h
later by using the dual luciferase kit from Promega. Results shown are
mean of three separate experiments (bars, S.E.; *,
significantly different compared with MKK6 alone).
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Our previous work demonstrated that expression of the p38 stimulator
MKK6 in these breast cancer cells increases AP-1
trans-activity (50). Expression of all four p38 family
members promoted us to determine which of these family members
contribute to transduction of the MKK6 signaling to AP-1. The dominant
negative mutant form of each p38 member was co-expressed with or
without MKK6, together with AP-1-Luc (a minimal c-fos
promoter containing an additional three AP-1 enhancer elements
regulating the luciferase gene transcription (42). Since the AF mutants
do not have the dual phosphorylation residue TGY and the KM mutant is
inactivated in the ATP-binding site, both should function as a
kinase-dead form of p38 to block the upstream signals, as previously
demonstrated (51). Expression of these kinase-dead forms of p38 alone
has different effects on AP-1 reporter activity, with a slight
stimulation by p38
/AF and p38
/AF but not by p38
/AF and
p38
/KM (data not shown). All four mutant forms, however, are able to
block MKK6-induced AP-1 activity (Fig. 1, bottom
panel). The strongest inhibitions were observed for
p38
/AF (p < 0.01 versus MKK6 alone) and
the p38
/KM (p < 0.01), followed by a moderate
suppression of MKK6-induced AP-1 activity by p38
/AF and the
p38
/AF (p < 0.05 in both cases). Application of p38
/AF also
achieved a significant inhibition of the AP-1 activation by MKK6, as in
the case of p38
/KM (p < 0.02, data not shown). Of
interest, the inhibitory activity of these dominant negatives on
MKK6-induced AP-1 activity does not correlate with the expression
levels of their endogenous proteins. For example, p38
and p38
are
expressed at a similar level, yet transfection of the same amounts of
p38
/AF and p38
/AF shows that the p38
/AF is much more efficient
in inhibition of MKK6 stimulation of AP-1. These results suggest that
each of these family isoforms may have different roles in mediating
MKK6 signaling to AP-1.
p38
Potentiates but p38
and p38
Inhibit or Have No Effects
on MKK6- and ARS-induced AP-1 Activation--
MKK6 is a specific
upstream kinase that activates all four family members of p38 (1, 3,
25, 39). To further evaluate roles of each p38 family member in
transduction of signaling to activate AP-1-dependent
transcription, each of the wild-type p38 family members were
individually expressed with or without the active MKK6, and their
effects were determined on the stimulated AP-1 reporter activity (Fig.
2). Transfection of either wild-type p38
or p38
but not p38
and p38
alone increases the basal
AP-1-Luc activity about 5-fold (p < 0.05). To our
surprise, whereas co-expression of wild-type p38
had no effect on
the MKK6-induced AP-1 activity, compared with either MKK6 or with
p38
alone, although its endogenous content is most predominant.
Unexpectedly, transfection of the same amount of wild-type p38
synergistically increased the AP-1 activity by more than 30-fold
(p < 0.05 versus MKK6). In contrast, both
p38
and p38
significantly inhibited the stimulation by MKK6
(p < 0.01, both versus MKK6 alone in 468 cells), indicating an opposing effect of p38
with p38
and p38
.
The enhancing effect of p38
is kinase-dependent, since
this effect is not seen with the phosphorylation-dead form p38
/AF
(Fig. 1), but the inhibition by p38
and p38
occurs regardless
whether the mutants (Fig. 1) or the wild-type forms of the p38s (Fig.
2) are applied.

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Fig. 2.
Opposing effects of p38
with p38 and p38
in MKK6 and/or ARS stimulation of AP-1 activity in human breast
cancer 468 (top panel) and MCF-7
(bottom panel) cells. Each of the
wild-type p38 family members was expressed alone or together the active
MKK6, in the presence of the AP-1-Luc construct in 468 and MCF-7 cells.
The luciferase activity was measured 48 h after transfection. For
regulation of ARS-mediated AP-1 activity, cells were transfected and
treated with 2 mM ARS for 30 min, and the luciferase
activity was assayed 24 h later. Results are the mean of three
independent experiments (bars, S.E.). *, significantly
different compared with vector; **, compared with MKK6 alone; ***,
compared with ARS alone.
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To confirm this result independently of MKK6 overexpression, ARS, a
well known chemical p38 activator (11, 52-54), was utilized. Our
previous work demonstrated that ARS stimulates AP-1
trans-activity and AP-1 binding in these breast cancer cells
(50). Different p38 isoforms were expressed in 468 cells, which were
then treated with 2 mM ARS for 30 min 24 h before the
assessing of AP-1 luciferase activity. As for MKK6 activation, ARS
treatment alone increased AP-1 activity of about 2-fold, but this
stimulation was increased to 10-fold when p38
was also transfected
into the cells (p < 0.01 versus ARS alone).
Expression of other p38
and p38
, however, failed to have a
significant impact on the ARS-induced AP-1 activation, implying that
these isoforms play a different role in transmitting MKK6 and ARS
signaling to AP-1 (Fig. 2, upper panel). The
results suggest that p38
is the only p38 family member to transmit
both MKK6 and ARS signal to AP-1. To determine whether the opposing effect of p38
with p38
and p38
is cell type-specific, another human breast cancer cell line, MCF-7, which has a similar expression profile of p38 family members (Fig. 1), was examined for effects of p38
isoform expression on MKK6 activation of AP-1 activity. As shown in the
bottom panel of Fig. 2, expression of p38
again increased MKK6 stimulation of AP-1 from 3.2- to 16.5-fold
(p < 0.01 versus MKK6 alone). Expression of
p38
or p38
, on the other hand, failed to inhibit the AP-1
stimulation by MKK6 (p > 0.05, both versus
MKK6 alone). Together, these results demonstrate that at least in these
two human breast cell lines in which all of the isoforms are expressed,
p38
increases MKK6 and/or ARS signaling to stimulate
AP-1-dependent transcription, whereas p38
and p38
are
either inhibitory or have no effects on the AP-1 activation by MKK6
and/or ARS.
Opposing Effects of p38
with p38
and p38
on MKK6-mediated
VDR trans-Activation--
AP-1 is a key transcription factor that
converts MAPK signaling into target gene expression (14, 16). Our
previous work has established that MKK6 trans-activates VDR
in these human breast cancer cells in a manner dependent on
c-Jun/AP-1 activity (50). It is therefore critical to determine whether
the opposing effects of p38
with p38
and p38
also occur in
regulation of this p38/AP-1 target gene transcription. A reporter
plasmid containing 0.5 kb of the mouse VDR promoter (43, 44) in front
of the luciferase gene was co-transfected with each isoform of the p38
family members in the presence or absence of the active MKK6, and the
VDR-luciferase activity was assessed. As illustrated in Fig.
3 (upper panel) in
468 cells, the VDR promoter activity conferred by MKK6 expression was
significantly increased from 2.2- to 11-fold by p38
(p < 0.05, versus either MKK6 or p38
alone). Expression of each of the other family members (p38
, p38
,
and p38
with MKK6), on the other hand, reduced the MKK6 stimulation
of the VDR gene promoter (p < 0.05, the p38 isoform
and MKK6 combination versus MKK6 alone). These results
further support an opposing role of p38
with p38
and p38
(probably also p38
) in regulation of an AP-1-dependent VDR gene transcription by MKK6. When the same experiment was carried out in MCF-7 cells (Fig. 3, bottom panel), an
inhibitory effect was similarly observed for p38
, p38
, and p38
(p < 0.05 versus MKK6 alone) but not for
p38
on the MKK6 activation of the VDR promoter. The stimulation of
AP-1 and VDR-Luc activities by expression of p38
or p38
alone
observed in the estrogen receptor-negative 468 cells but not in
estrogen receptor-positive MCF-7 cells (Figs. 2 and 3) implies either a
cell line-specific phenomenon or estrogen receptor-related effect. The
enhancing effect of p38
on MKK6 activation of AP-1 (Fig. 2) but not
VDR promoter (Fig. 3) activity in MCF-7 cells may suggest that p38
can mediate MKK6 signaling to VDR via AP-1-independent mechanisms in
this cell line. Nevertheless, a clearly different effect is observed
for p38
-mediated transduction on MKK6 stimulation of VDR
transcription than from the rest of other three p38 isoforms in both
human breast cancer cell lines.

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Fig. 3.
Different regulation of MKK6 stimulation of
VDR promoter activity by p38 family members in 468 (top
panel) and MCF-7 cells (bottom
panel). Cells were transfected and assessed for
luciferase activity by procedure described in the legend to Fig. 2,
except that the AP-1-Luc was replaced with VDR-Luc containing 0.5 kb of
mouse VDR gene promoter. The results shown are the mean of three
separate experiments (bars, S.E.). **, significantly
different compared with MKK6 alone.
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|
Expression Profiles of Endogenous p38 Family Members Determine a
Specific Response of a p38-activating Signal--
Our results of the
antagonizing activities of p38
with p38
/
in MKK6 activation of
AP-1/VDR suggest that effects of p38 activation on
AP-1-dependent transcription are determined by the expression pattern of endogenous p38 isoforms. p38 activation would be
stimulatory to the AP-1-dependent transcription if p38
is the major form, whereas it would be inhibitory in cells
predominantly expressing p38
and/or p38
. To directly test this
possibility, a series of cell lines were screened for expression of p38
isoforms by Western blot analyses as described above. As shown in Fig. 4A, the p38
protein level
is about 2 times higher in mouse NIH 3T3 cells compared with mouse
mammary carcinoma EMT-6 cells, and p38
, on the other hand, is only
detected in EMT-6 but not in the other cell lines screened. Exactly as
predicted, MKK6 expression in 3T3 cells significantly activates AP-1
(Fig. 4B) and VDR promoter activity (Fig. 4C,
p < 0.05 in both cases), but the same MKK6 transfection in EMT-6 cells has no effects on the basal AP-1 activity (Fig. 4B) and inhibits the VDR promoter activity by more
than 50% (p < 0.05, Fig. 4C). These
results provide direct evidence that the endogenously expressed p38
isoforms may determine the final outcome of the p38 activation
signaling on AP-1-dependent gene transcription.

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Fig. 4.
Determinant roles of endogenous expression
profiles of p38 isoforms in MKK6-induced AP-1 transcription.
A, expression of p38 isoforms in different murine cell
lines. Note that p38 is higher in NIH 3T3 than EMT-6, whereas p38
is only detected in EMT-6 but not 3T3 cells. B, MKK6
stimulates AP-1 reporter activity in 3T3 cells but not in EMT-6 cells
(bar, S.E., n = 3). C, MKK6
stimulates the basal VDR promoter activity in 3T3 and inhibits the
basal VDR promoter activity in EMT-6 cells, respectively
(bar, S.E., n = 3). *, significantly
different compared with vector.
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Increase by p38
but Suppression by p38
of Cell Proliferation
by MKK6--
In addition to regulation of target gene expression, AP-1
activity can be either growth-inhibitory or growth-stimulatory by itself and/or as a result of integration of the effects on multiple cellular AP-1-dependent target genes (14). To examine
whether the AP-1 activity in our analyses corresponds to a
proliferative or growth-inhibitory signal, cell proliferation assays
were performed in which p38
or p38
was co-expressed with MKK6 in
MCF-7 cells, together with a marker plasmid pSV-GFP. Following
transfection, cells were pulse-labeled with BrdUrd and examined for
BrdUrd-positive cells in the transfected population (GFP-positive,
about 10-20%) under a fluorescence microscope. Expression of MKK6 or
p38
or p38
alone had no significant effect on BrdUrd labeling
over vector control (Fig. 5).
Co-expression of MKK6 with p38
, however, increased the labeling, and
that with p38
decreased BrdUrd incorporation (p < 0.05 for MKK6 plus p38
or MKK6 plus p38
versus MKK6
alone). The slight inhibition of BrdUrd incorporation by MKK6 alone
(26%, p > 0.05 versus control) may reflect
the net effects of transfected MKK6 on the four p38 endogenous isoforms
shown to be expressed in these cells. The lack of significant
growth-regulatory effects of p38
or p38
by itself suggests that
these activities require activation by upstream signals such as MKK6.
The significant increase of growth promoted by co-transfection of MKK6
plus p38
, and the growth inhibition by co-transfection of MKK6 plus
p38
indicates that the positive or negative growth regulatory
effects of the isoforms can be observed when these proteins are
overproduced on a background in which all four isoforms are detected.
Furthermore, in order to see an effect, the p38 pathway must be
activated by an upstream activator such as MKK6. These
growth-regulatory activities correlate with the changes of AP-1
transcriptional activity observed in these human breast cancer cells
(Fig. 2). Although alterations of AP-1 activity may correspond to
either cell proliferation, differentiation, or apoptosis, depending
on cellular contents, our results suggest that the activity
corresponds to cell proliferation under our experimental
conditions.

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Fig. 5.
p38 increases but
p38 decreases cell proliferation in
combination with MKK6. Cells were transfected with various
constructs as indicated, together with a marker plasmid pSV-GFP.
Transfected cells were labeled with BrdUrd, immunostained, and
quantitated under a fluorescence microscope. Results shown are means of
three independent experiments (bar, S.E.), with the
exception of p38 or p38 alone, in which only one experiment was
performed. *, significantly different compared with MKK6 alone.
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Roles of the p38 Kinase Activity and MKK6-p38 Binding in the
Opposing Effects of p38
and p38
/
--
The Thr-Gly-Tyr (TGY)
dual phosphorylation motif presents in all four members of the p38
family (3, 41). So far, all regulatory and biological activities of
p38s have been ascribed to the tyrosine and threonine phosphorylation
on this motif (3). The p38 activator MKK6 has been reported to
phosphorylate and activate all p38 family members (1, 25, 39). We
wished to determine whether the opposing effects between p38
and
either p38
or p38
are due to difference in isoform kinase
activations by MKK6/ARS in these two human breast cancer cell lines.
FLAG-tagged p38s were expressed with or without the active MKK6 in
these breast cancer cells. Confirmation of their expressions was
determined by immunoprecipitation and Western analysis using anti-FLAG
antibody. Results in Fig. 6A
showed that all transfected p38 isoforms are expressed in both 468 and
MCF-7 cells. The similar levels of these four exogenous p38s in 468 cells and of transfected p38
, -
, and -
in MCF-7 cells
demonstrated that the opposing effects of p38
with p38
/
are
not due to the differences in expression of these transfected isoforms.
The higher level of p38 expression in MCF-7 cells, on the other hand,
most likely results from higher transfection efficiency in these
cells.

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Fig. 6.
Activation of p38 kinases by MKK6 and
ARS. A, expression of FLAG-tagged p38s in MCF-7 and 468 cells. FLAG-tagged p38s were expressed and immunoprecipitated with
anti-FLAG antibody. The precipitates were examined for expression of
p38s by Western analysis using anti-FLAG antibody. B,
activation of p38s by MKK6 or ARS. Wild-type p38s were expressed alone,
co-expressed with MKK6, or treated with ARS 24 h after the
transfection, as described in the legend to Fig. 2. Transfected p38s
were isolated by anti-FLAG immunoprecipitation and assessed for their
in vitro kinase activity against ATF2. The results at the
top represent the basal activity of transfected p38
(control) in comparison with p38 stimulated by MKK6
overexpression (middle) and by ARS (bottom). A
similar result was obtained from a separate experiment.
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Activation of the transfected p38 isoforms by MKK6 and ARS was examined
next. Cells were cotransfected with the active MKK6 and p38s, and
48 h later, the FLAG-p38s were immunoprecipitated with anti-FLAG
antibody, and their in vitro kinase activity was determined
using recombinant GST-ATF2 as the substrate. The kinase activity of
p38
and p38
was most strongly activated by MKK6 in MCF-7 cells
compared with their respective controls (Fig. 6B, top panel) in a manner similar to that observed
in COS-7 cells (55), whereas MKK6 stimulated p38
and p38
activations to a lesser extent at similar expressed levels. Of
interest, in 468 cells, p38
was most highly activated by MKK6,
followed by p38
, p38
, and p38
. Whereas previous in
vitro data indicated that p38
can not phosphorylate ATF2 (49),
the results in COS-7 (55) as well as in these 468 human breast cancer
cells suggest that ATF2 can be phosphorylated by p38
. Co-expression
of the mutant forms of p38s (p38
/AF, p38
/AF, p38
/AF, and
p38
/KM) with MKK6 did not reveal any kinase activity compared with
vector controls (data not shown). These results demonstrated that MKK6
activates ATF2 phosphorylation by all four forms of p38s in both breast cancer cell lines, with p38
and p38
more strongly in MCF-7 cells but p38
more efficiently in 468 cells. In none of these cases, however, does the kinase activity of p38s stimulated by MKK6 offer an
explanation for their effects on the AP-1-dependent
transcription. Whereas p38
is mostly strongly activated by MKK6 in
MCF-7 cells, for example, it has no obvious effect on the induction of
AP-1 or VDR promoter activity. This result further consolidates our conclusion that the effect of p38s on transduction of MKK6 signaling to
AP-1 is isoform-specific and does not correlate with the observed differences between their MKK6 activations as measured against the p38
substrate ATF2.
To further assess the role of the kinase activity of p38s in
ARS-induced AP-1 activation, groups of cells transfected with FLAG-tagged p38 isoforms were also treated with ARS, the FLAG-tagged p38s were isolated by immunoprecipitation, and their in
vitro kinase activity was determined as above. Results shown in
Fig. 6B (bottom panel) demonstrated
that ARS activates all p38s similarly in 468 cells, whereas the effect
on p38
and p38
was greater than p38
and p38
in MCF-7 cells,
similar to the activation by MKK6 (middle panel).
Once again, none of these kinase activities explain the opposing
effects of p38
with p38
and p38
in AP-1-dependent transcription. Since the kinase-dead form of p38
does not increase MKK6-induced AP-1 transcription, whereas the dominant negatives of
either p38
or p38
may inhibit the AP-1 stimulation by MKK6 (Fig.
1), these results suggest that p38
mediates MKK6 and/or ARS
signaling to AP-1 dependent on its kinase activity, whereas p38
and
p38
inhibit the AP-1-dependent transcription in a manner independent of kinase activity.
Enzyme binding to a substrate is required for MKK6 activation of the
p38s (56, 57). To examine whether the opposing effects may be due to
different associations of the p38 isoforms with MKK6, an in
vivo binding experiment was performed by co-expression of HA-MKK6
with wild-type and dominant negative FLAG-p38s in MCF-7 cells.
Transfected p38s were isolated by immunoprecipitation with anti-FLAG
antibody and examined for the presence of co-precipitated HA-MKK6. As
in Fig. 7A, HA-MKK6 was
detected in every group whether the wild-type or dominant negative p38
isoforms were transfected. If the FLAG-p38 isoform was normalized by
co-precipitated HA-MKK6 in each of the transfections (p38/MKK6 ratio
constant), there appeared to be more p38
and p38
binding to MKK6
with both wild-type and the dominant negative p38 mutant forms (Fig.
7A). These results may explain why the in vitro
kinase activity of both p38
and p38
is relatively higher in these
cells after co-expression with MKK6 (Fig. 6B), but once more
this result does not correlate with their effects on
AP-1-dependent transcription.

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Fig. 7.
Contributions of c-Jun activation to the
opposing effects on AP-1 activity. A, direct binding of
MKK6 to all four isoforms of p38 family members of both wild type and
dominant negatives. HA-MKK6 was cotransfected with each FLAG-tagged p38
in MCF-7 cells, and the transfected p38s were purified by anti-FLAG
immunoprecipitation. The precipitates were examined for the presence of
HA-MKK6 by Western analysis with anti-HA antibody (bottom)
and FLAG-p38s (top). B, selective activation of
c-jun transcription by MKK6 and its regulation by p38
isoforms. c-jun Luc containing residues 225 to +150 of the
c-jun promoter (left) and c-fos Luc
containing residues 356 to +109 of the c-fos promoter
(right) were cotransfected with MKK6 in the presence or
absence of p38 isoforms, and the luciferase activity was assessed as
above. The open and filled columns
indicate transfections without and with MKK6, respectively. Results of
one representative experiment from two similar experiments are shown.
C, increase of MKK6-induced c-Jun phosphorylation by p38
and its inhibition by p38 or p38 . The active MKK6 was
cotransfected with HA-c-Jun in the absence or presence of wild type or
dominant negative p38 isoforms. Transfected c-Jun was isolated by
anti-HA immunoprecipitation, and its phosphorylation status was
determined by Western blot using anti-phospho-c-Jun (Ser-63) antibody.
The same membrane was stripped off and reprobed with anti-HA antibody
for c-Jun expression (bottom).
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Contributions of c-Jun Transcription and c-Jun Phosphorylation to
the Opposing Activities--
AP-1 activity most frequently consists of
a heterodimer of c-Jun and c-Fos, both of which can be activated by p38
kinases (3, 14, 16). In order to dissect whether c-Fos or c-Jun is
involved in p38 isoform-specific regulations of AP-1 activity by MKK6,
the luciferase activity driven by either the c-fos (45) or
c-jun (26) promoter was analyzed. Results in Fig.
7B showed that MKK6 alone selectively
trans-activates c-Jun (left) but not c-Fos
(right). These results are consistent with our published data that c-Jun but not c-Fos or ATF2 is the major component of the
AP-1 activity induced by MKK6 in these breast cancer cells (50). Of
interest, co-transfection of p38
or p38
inhibited the
c-jun promoter activity induced by MKK6, whereas p38
appears to increase the stimulation. These results indicate that the
opposing effects of p38
with p38
and p38
correlate with their
different effects on MKK6-induced c-jun transcription.
Alterations in c-jun transcription by p38 isoforms in
combination with MKK6 could contribute to the opposing effects on AP-1 activity. The p38 isoform-specific effect on c-jun
transcription may be a result of their effects on regulation of other
AP-1 components or regulators such as MEF2s (26, 29). Alternatively, it
could occur as a consequence of AP-1 regulation via the c-Jun/AP-1
enhancer element in c-jun promoter by its positive
autoregulatory loop (23). Besides transcriptional regulation, c-Jun
phosphorylation is the second major mechanism by which c-Jun/AP-1 is
activated by MAPKs, especially by JNK (14, 16). Whereas p38s were
reported by several investigators not to phosphorylate c-Jun in
vitro (32, 41, 58), recent studies suggest that immunoprecipitated
and transfected human p38 (59) or murine p38
in mammalian cells (60)
phosphorylates c-Jun in an in vitro kinase assay.
We have previously demonstrated that adenovirus-mediated MKK6 gene
delivery in these breast cancer cells phosphorylates endogenous c-Jun
(50). To determine whether p38 isoforms may differently regulate c-Jun
phosphorylation by MKK6, HA-c-Jun was transfected with MKK6 in the
absence or presence of wild-type or dominant negative p38s. Following
immunoprecipitation with anti-HA antibody, c-Jun phosphorylation status
was examined by Western blot using specific anti-phospho-c-Jun (Ser-63)
antibody, and the same membrane was stripped off and reprobed with anti
c-Jun antibody. As shown in Fig. 7C, MKK6 induced higher
levels of both c-Jun and phosphorylated c-Jun, the later consistent
with the observation with the adenovirus infection (50). Of great
interest, wild-type p38
increased phosphorylated c-Jun, but c-Jun
levels were equal (p38
plus MKK6 versus MKK6 alone). Both
p38
and p38
, on the other hand, inhibited the MKK6 induction of
c-Jun as well as phosphorylated c-Jun levels. The decrease of c-Jun
phosphorylation by either p38
or p38
in this case may represent a
consequence of inhibition of the total c-Jun protein. These results
indicate that p38
may increase the AP-1 activity predominantly by
stimulation of the c-Jun phosphorylation, whereas p38
or p38
may
inhibit primarily by suppression of the c-Jun
trans-activation by MKK6. The moderate inhibitory effects on
c-Jun phosphorylation and to a lesser extent c-Jun levels by each of
the dominant negative p38 isoforms, on the other hand, may explain
their moderate inhibitory activities on MKK6-induced AP-1 activation
(Fig. 1). The different c-Jun phosphorylation is not due to the
potential presence of active JNK or p38 in the anti-HA complex, as
detected with phosphor-JNK or phosphor-p38 antibody (data not shown).
Since c-Jun but not c-Fos or ATF2 is the major component of
MKK6-induced AP-1 activity in these cells as shown by Western blotting
and gel retardation (50), these results strongly suggest that the p38
isoform-specific regulation of c-Jun represents one mechanism for the
opposing effects of p38
with p38
and p38
on
AP-1-dependent transcription.
Correlations of Expression of Endogenous p38 Isoforms with
Endogenous c-Jun Expression and Phosphorylations--
The c-Jun
regulation by MKK6 and p38s may provide an important mechanism for the
opposing effects of p38
with p38
/
in
AP-1-dependent transcription. Results obtained above,
however, are based on experiments with co-transfection and
overexpression and consequently need to be further confirmed in
physiologically relevant conditions. Mouse NIH 3T3 and EMT-6 cells were
applied here again to assess the total and phospho-c-Jun levels by p38
activation by virtue of their distinct expression pattern of endogenous
p38 isoforms. NIH 3T3 cells contain higher concentrations of p38
,
and p38
is only detected in EMT-6 cells (Fig. 4). If these two p38
isoforms indeed contribute to regulations of the endogenous c-Jun
expression and phosphorylations, the total and phosphorylated c-Jun
should be higher in NIH 3T3 than in EMT-6 cells. Total and
phospho-c-Jun levels were determined in NIH 3T3 and EMT-6 by Western
analysis. Exactly as expected, the level of total c-Jun protein is
about 3 times higher in NIH 3T3 cells over that in EMT-6 cells
(p < 0.01, Fig. 8,
A and B). These results thus consolidate our
conclusion obtained above with the c-jun promoter analysis
(Fig. 7B) that p38
is also suppressive to endogenous
c-Jun expression. More importantly, after treatment with anisomycin (10 µg/ml for 30 min), a well known p38 stimulus that activates all four
p38 isoforms (41, 55), phosphorylated c-Jun is only detected in NIH 3T3 cells in which p38
is higher and p38
is undetectable (Fig.
8C). ANI also increases the phospho-p38 but does not
increase total c-Jun protein expression in both cell lines (data not
shown). These results thus provide strong evidence supporting the
notion that the expression profiles of p38 isoforms regulate the level of endogenous c-Jun expression and phosphorylated c-Jun.

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Fig. 8.
Higher levels of total and phosphorylated
c-Jun in NIH 3T3 cells. A and B, higher
levels of endogenous c-Jun protein in NIH 3T3 cells. Total cell lysates
were prepared, and the total c-Jun protein expression was determined by
Western analysis with a representative result shown in A,
and means of three separate experiments were plotted in B
(bar, S.E.). C, ANI induces c-Jun phosphorylation
in NIH 3T3 but not EMT-6 cells. The phosphorylated c-Jun was detected
by using anti-phospho-c-Jun (Ser-63) antibody by Western blot, and the
same membrane was reprobed with actin antibody as a loading
control.
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DISCUSSION |
The p38 MAPKs are universal signaling cascades that transmit
extracellular signals into target gene expressions through their regulation of transcription factor activities. Whereas various mechanisms for signaling specificity such as complex formation and
selective recognition of specific docking sites and the activation the
T-loop of the p38s (56, 61) have been proposed, it remains unclear why
the same p38 activation in different cellular contents triggers
different biological response (1-4). Here our results demonstrate that
upstream stimulatory signals of the p38 pathway may induce different
cellular outcomes, depending on the expression profile of p38 isoforms
and in a manner independent of the upstream pathway stimulator. This
conclusion is supported by our finding that p38
increases, but
p38
and/or p38
inhibit, AP-1-dependent transcription
and cell proliferation induced by MKK6 in human breast cancer cells
(Figs. 2, 3, and 5). A similar effect of the p38 isoforms on regulation
of AP-1 activity was also observed with activation of the pathway by
ARS. The stimulatory activity of p38
and the inhibition effect of
p38
were further confirmed in NIH 3T3 and EMT-6 cells in which
endogenous p38
protein level is higher in 3T3 than that in EMT-6
cells, respectively, whereas p38
is expressed in EMT-6 but not NIH
3T3 cells. Moreover, evidence is presented indicating that the
enhancing effect of p38
requires the kinase activity, whereas the
inhibition by p38
and p38
is independent of p38 phosphorylation.
These effects were further demonstrated by both transfection and in
endogenous systems, at least in part, to be due to the isoform-specific
regulations of c-jun transcription/expression and
phosphorylations. The results thus show that specific outcomes of p38
MAPK activation in regulation of AP-1-dependent
transcription and proliferation in a given cell type depend on the
expression profile of p38 family members in those cells.
Different activities of p38 isoforms have been previously suggested,
but the opposing effects within the same family members have not been
described (3). p38
is the first isolated family member and has been
mostly studied and characterized (3). It is not clear why its
expression has the least effect on AP-1 stimulation by the upstream
stimulators MKK6 and/or ARS. Published studies suggest that p38
may
be mitogenic and/or antiapoptotic, whereas p38
may be involved in
stress response. In HeLa cells, for example, adenovirus-mediated p38
delivery was demonstrated to protect SB202190-induced apoptosis
(62). Furthermore, p38
but not p38
was shown to protect
mesangilal cells from tumor necrosis factor-
-induced apoptosis (63).
Our results, however, showed that expression of either p38
or p38
alone has no substantial effects on cell proliferation, and their
opposing effects only became obvious when their upstream activator,
MKK6, is also co-expressed (Fig. 5). The role of p38
in stress
response was, on the other hand, suggested by the observation in which
inhibition of p38
by p38
/AF expression suppressed
-radiation-induced G2 arrest in human osteosarcoma U2OS
cells, whereas inhibition of other family members by the dominant
negatives had no effect (38). There are also, however, exceptions to
this distinction; both p38
and p38
are involved in
hypoxia-induced down-regulation of cyclin D1 in PC12 cells (64). Green
tea polyphenol, on the other hand, selectively stimulates p38
phosphorylation, but this effect is not associated with inhibition, rather activation of AP-1 activity in human keratinocytes as a result
of simultaneous stimulation of the Ras/MEKK pathways (65). These
results are consistent with the concept that each p38 family member has
a distinct function. However, whether these effects are mitogenic or
proapoptotic may depend on the individual cellular content.
p38s are serine/threonine protein kinases, and the biological effects
of p38 MAPKs have been so far exclusively ascribed to the kinase
activities (1-3). Our result that the increase in c-Jun
phosphorylation and AP-1-dependent transcription is
observed with wild-type but not kinase-dead mutant p38
is consistent
with this notion. Paradoxically, the kinase activities may not be
required for the inhibitory effects of p38
and p38
on
MKK6-induced c-Jun phosphorylation and AP-1 activation, although the
wild-type p38
and p38
appear to be more effective (Figs. 1, 2,
and 7C). However, since all dominant negatives of p38s
inhibit c-Jun phosphorylation (Fig. 7C), which more or less
correlates with their inhibition on AP-1-dependent
transcription (Fig. 1, bottom), the inhibitory effects by
these mutants do not appear to be isoform-specific. These results
suggest that p38
increases the AP-1 by stimulation of c-Jun
phosphorylation, whereas p38
and p38
inhibit this by mechanisms
involving suppression of the c-jun transcription. Mechanisms operative in this process are unclear at present but may involve p38
isoform-specific protein-protein interactions in which the wild-type
and
isoforms act as dominant negatives. Efforts in this
research direction will facilitate identification of novel functions of
p38 MAPKs.
The opposing activity of p38
to p38
and p38
in AP-1
trans-activation of transcription has important implications
for understanding p38 signaling specificity. p38 activation would
correspond to an increase in AP-1 activity if p38
is the dominant
form. However, if p38
and/or p38
are the major isoforms, p38
activation would correspond to an inhibition of
AP-1-dependent gene expression. AP-1 activity induced by
p38 activation in a cell type in which all four p38 family members are
expressed, as in the case of 468 and MCF-7 cells, would represent the
net output of the signal integration among its family members (Fig.
9). AP-1 plays a key role in regulation
of many target genes whose expression levels control cell
proliferation, cell differentiation, and apoptosis, including cyclin
D1, c-Jun, p53, p16ARF (14), and VDR (50). Since many cell
lines only express some of p38 family members (Fig. 4) (3), the p38
isoform-specific effect on AP-1-dependent gene expression
would provide an explanation as to why the same p38 activator could
lead to various biological outcomes in different cell types.

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Fig. 9.
An experimental model describing a
determinant role of expression profiles of p38 family members in p38
signal specificity. p38-activating signal (MKK6 or ARS) stimulates
AP-1-dependent transcription through c-Jun regulation via
p38 , but p38 or p38 inhibits this process. p38-activating
signal would be stimulatory to AP-1-dependent activities in
cells expressing high levels of p38 , but it is inhibitory in cells
predominantly expressing p38 and/or p38 . The net response in
cells expressing all p38 family members is determined by integrations
of the positive (p38 ) and the negative (p38 and - ) AP-1
regulatory signaling.
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