MEK7-dependent Activation of p38 MAP Kinase in
Keratinocytes*
Shervin R.
Dashti
,
Tatiana
Efimova
, and
Richard L.
Eckert
§¶
From the Departments of
Physiology and Biophysics and
§ Biochemistry, Reproductive Biology, Dermatology, and
Oncology, Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106-4970
Received for publication, December 12, 2000, and in revised form, January 4, 2001
 |
ABSTRACT |
Previous studies suggest that a PKC/Ras/MEKK1
cascade regulates involucrin (hINV) gene expression in
human epidermal keratinocytes. MEK7, which is expressed in epidermis,
has been identified as a member of this cascade (Efimova, T., LaCelle,
P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert,
R. L. (2000) J. Biol. Chem. 275, 1601-1607). However, the kinase that functions downstream of MEK7 has not been
identified. Our present studies show that MEK7 expression in
keratinocytes markedly activates p38
and modestly activates JNK.
Activation of p38 MAPK by MEK7 is a novel finding, as previous reports
have assigned MEK7 as a JNK regulator. We also demonstrate that
this regulation is physiologically important, as the p38
- and
JNK-dependent activities regulate hINV promoter
activity and expression of the endogenous hINV gene.
 |
INTRODUCTION |
Mitogen-activated protein kinase signal transduction pathways are
three kinase modules that include
MAPK1 kinase kinase (MEKK),
MAPK kinase (MEK), and MAPK (1). MAPK activation requires dual
phosphorylation at threonine and tyrosine residues separated by a
single amino acid (1, 2). Activated MAPKs, in turn, translocate to the
nucleus and phosphorylate nuclear transcription factors such as Elk-1
and AP1 (3, 4). These factors then modulate gene expression by binding
to DNA elements (5-7). Seven different mammalian MEKs have been
described (8). MEK1 and MEK2 activate ERK1 and ERK2, MEK3 and MEK6
activate p38 kinase, MEK4 activates p38 and the SAPK/JNK pathways, and
MEK5 and MEK6 regulate ERK5/BMK1 (8). MEK7 is a recently described MAPK
kinase (9). Studies in various cell types indicate that MEK7
activates JNK/SAPK but not ERK or p38 (9, 10).
Previous studies from our laboratory indicate that MAPK cascades have a
central role in the regulation of keratinocyte differentiation, as
measured by the effects of activation of this cascade on the expression
of a marker of keratinocyte differentiation, involucrin (6, 7). This
cascade, which includes the novel PKC isoforms, Ras, and MEKK1,
activates involucrin expression, by a mechanism that requires MEK1,
MEK3, and MEK7 activity (6). MEK3 appears to regulate hINV
gene expression via activation of p38 MAPK (6); however, the path of
signal transmission following activation of MEK1 and MEK7 is not known.
In the present experiments, we address the role of MEK7 as an activator
of hINV gene expression. These studies suggest that MEK7
increases hINV gene expression via activation of p38
.
 |
MATERIALS AND METHODS |
Tissue Culture, Cell Transfection, and Adenovirus
Infection--
Third passage normal human epidermal keratinocytes were
maintained in 35-mm dishes in keratinocyte serum-free medium (KSFM). For transfection studies, 50% confluent cells were incubated with 2 µg of plasmid mixed with 4 µl of FUGENE-6 reagent and 96 µl of
KSFM. For adenovirus infection studies, 75% confluent cells were
infected with 15 plaque-forming units of adenovirus per cell in 1 ml of
KSFM containing 2.5 µg/ml Polybrene. After 24 h, the medium was
replaced with 2 ml of fresh KSFM, and the cells were incubated for an
additional 24 h. The cells were then washed, and lysates were
prepared for immunoblot or detection of luciferase or kinase activity.
Luciferase Assay--
To assay involucrin
promoter-dependent luciferase activity, cells were
transfected with 2 µg of pINV-2473 reporter plasmid (5) mixed with 4 µl of FUGENE-6 reagent and 96 µl of KSFM, incubated for 24-48 h,
washed with phosphate-buffered saline, dissolved in 140 µl of cell
lysis reagent (Promega), and harvested by scraping. Luciferase activity
was assayed immediately using a Promega Luciferase assay kit and a
Berthold luminometer. All assays were performed in triplicate, and each
experiment was repeated at least three times. Luciferase activity was
normalized per µg of protein as previously described. We used a green
fluorescent protein-encoding expression plasmid to normalize
transfection efficiency (11, 12).
ERK1/2, p38, and JNK Kinase Activity Assays--
p38, ERK1/2,
and JNK activity were assayed using nonradioactive assay methods.
Lysates were prepared in lysis buffer (20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), and kinase activity was
monitored in kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol,
0.1 mM Na3VO4, and 10 mM MgCl2). To assay total p38 activity, a
monoclonal antibody (New England BioLabs 9219) that binds to the
phosphorylated form (Thr180/Tyr182) of all four
p38 isoforms was used to immunoprecipitate active p38 kinase from cell
lysates. The precipitated p38 was then assayed for activity based on
its ability to phosphorylate ATF-2. ATF-2 phosphorylation (at
Thr71) was detected by immunoblot using a rabbit
anti-[phospho-Thr71]ATF-2-specific antibody (New England
BioLabs 9221S). ERK1/2 activity was monitored by immunoprecipitation of
phosphorylated ERK1/2 using a monoclonal antibody (New England BioLabs
9109) that binds to
Thr202/Tyr204-phosphorylated ERK1/2. Activity
of the precipitated kinase was assayed based on its ability to
phosphorylate ELK-1. Phosphorylated ELK-1 (at Ser383) was
detected by immunoblot using a rabbit anti-phospho-ELK1 antibody (New
England BioLabs 9181S). To measure JNK/SAPK activity, endogenously
activated JNK was precipitated using c-Jun fusion protein beads
(New England BioLabs 9811, Ref. 13). Activity of the precipitated JNK
was monitored by using c-Jun as a substrate. Phosphorylated c-Jun was
detected by immunoblot using anti-phospho-c-Jun (New England BioLabs 9810).
Western Blot Analysis--
Cell monolayers were rinsed with
phosphate-buffered saline and lysed in Laemmli sample buffer, and an
equivalent amount of protein (10 µg) was electrophoresed on 8%
acrylamide gels. The blots were incubated with the primary antibody,
washed, and exposed to horseradish peroxidase-conjugated secondary
antibody. Specific antibody binding was visualized using
chemiluminescence detection reagents, and band intensity was estimated
by densitometry.
Adenoviruses and Plasmids--
Adenoviruses and/or plasmids
encoding wild-type (wt), constitutively active (ca), and
dominant-negative (dn) kinases were used in these studies. These
include wtMEK7; caMEK7 (14); and FLAG-tagged p38
, p38
, p38
,
and p38
; adenoviruses (14, 15); and plasmids encoding dnJNK and the
corresponding empty control plasmid (13, 16). An empty adenovirus was
generated by recombining pCA3 plasmid with the pJM17 adenovirus
backbone in 293 cells. This virus was purified and used as an empty
control adenovirus. The green fluorescent protein-encoding virus,
CMV-GFP, was used to define the optimal adenovirus infection
multiplicity. Recombinant adenoviruses were propagated in 293 cells and
purified by cesium chloride centrifugation. pINV-2473, a plasmid
encoding the human involucrin gene promoter fused to luciferase, was
constructed as previously described (5).
Detection of mRNA--
Total keratinocyte
poly(A+) RNA was isolated using the Oligotex Direct
mRNA protocol (Qiagen). Equivalent quantities of mRNA were
assayed by real time RT-PCR using an RNA amplification kit (SYBR Green
I, Roche Molecular Biochemicals). Melting curve analysis of the PCR
products distinguished the higher melting-specific PCR product from
nonspecific products. For quantification, following the elongation step
in each PCR cycle and just prior to the fluorescence reading, the
reaction temperature was stepped up to the temperature required to melt
nonspecific PCR products. Primers amplifying a 330-bp fragment of the
hINV gene, primers amplifying individual p38 isoforms, and
primers amplifying a 600-bp fragment of the human GAPDH gene
were used in parallel. hINV mRNA quantities were normalized
based on GAPDH mRNA levels. The GAPDH primers were 5'-CCACCCATGGCAAATTCCATGGCA and 5'-CCACCTGGACTGGACGGCAGATCT and the
hINV primers were 5'-CTCCACCAAAGCCTCTGC and 5'-CTGCTTAAGCTGCTGCTC. Primers for detection of p38
, p38
, p38
, and p38
were as
previously described (17). MEK7 mRNA was detected by real time
RT-PCR using MEK7-specific primers (5'-ATCCCCGAGCGCATTCTGG, and
5'-TTGATCTGGCCCCGCTCGTC). These primers amplify a 120-nucleotide
segment of human MEK7.
 |
RESULTS |
MEK7 Increases p38 MAPK Activity--
To determine which MAPK is
activated in the presence of MEK7, we delivered wild-type or
constitutively active MEK7 to keratinocytes using adenovirus. As shown
in Fig. 1A, p38 kinase
activity is not detected in mock-infected cells or cells infected with
empty adenoviral vector; however, the presence of wt or caMEK7 results in activation of endogenous p38 MAPK. To compare the relative activity
of wtMEK7 and caMEK7, we measured the level of each protein in infected
cells by immunoblot (Fig. 1B). When corrected for level of
expression, wt and caMEK7 appeared to be equally effective activators
of p38 activity. We further tested caMEK7 as a regulator of JNK and ERK
activity. In addition to the marked increase in p38 MAPK activity,
caMEK7 also produced a modest increase in JNK activity (3-fold), but
did not regulate ERK activity (Fig. 1C). In addition, as
shown in Fig. 1D, MEK7 did not alter p38, JNK, or ERK
protein level, suggesting that the changes in p38 and JNK activity are
caused by increased activity of pre-existing enzymes.

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Fig. 1.
MEK7 activates p38 MAPK activity. Normal
human keratinocyte were mock-infected, infected with empty adenovirus
(EV), or infected with adenovirus encoding wild-type or
constitutively active MEK7. After 48 h, the cells were lysed for
extract preparation. A, activated p38 was precipitated using
a mouse monoclonal anti-phospho-p38 antibody. Activity of the
precipitated kinase was monitored by immunoblot using rabbit
anti-phospho-ATF-2. B, to monitor MEK7 level, cells were
lysed, and equivalent amounts of protein were electrophoresed for
immunoblot using a goat anti-MEK7-specific antibody (Santa Cruz SC7104,
1:500). Binding of the primary antibody was detected using horseradish
peroxidase-conjugated donkey anti-goat IgG (Santa Cruz SC2020,
1:10,000). C, ERK1/2, p38, and JNK MAPKs were
immunoprecipitated respectively using mouse monoclonal
anti-phospho-ERK1/2, mouse monoclonal anti-phospho-p38 , - , - ,
- , and c-Jun fusion beads. Phosphorylation of substrates was
monitored by immunoblot using rabbit anti-phospho-ATF-2, rabbit
anti-phospho-c-Jun, and rabbit anti-phospho-ELK1, respectively.
D, p38, JNK1/2, and ERK1/2 levels were assayed in by
immunoblot using rabbit anti-p38 (Sigma M0800, 1:5000), rabbit
anti-JNK1/2 (Sigma J4500, 1:2000), and rabbit anti-ERK1/2 (Sigma M5670,
1:5000) followed by detection with horseradish peroxidase-conjugated
donkey anti-rabbit IgG (Amersham Pharmacia Biotech NA934, 1:10,000).
These experiments were repeated a minimum of three times with similar
results.
|
|
To identify the specific p38 isoforms that are regulated by MEK7, we
performed three types of experiments. First, we used real time RT-PCR
to measure the level of mRNA encoding each individual p38 isoform.
The results indicate that keratinocytes express all p38 isoforms except
p38
(Fig. 2A). p38
and
p38
are the most abundant, followed by p38
. To measure the
activity of individual p38 isoforms, we coinfected cells with
adenoviruses encoding FLAG-tagged forms of each p38 isoform in the
presence or absence of caMEK7. After 48 h, individual p38 isoforms
were precipitated using anti-FLAG antibody, and the activity of the
precipitated antibody was monitored based on ability to phosphorylate
ATF-2. As shown in Fig. 2B, in the absence of MEK7, the only
p38 isoform displaying substantial activity is p38
. A similar
profile is observed in the presence of caMEK7, except that p38
activity is slightly increased. As expected, no activity is observed in
cells not infected with FLAG-p38 expression virus. The results shown in
panels A and B suggest that p38
, p38
, and
p38
are not involved in mediating the MEK7-dependent activation. To further examine the role of p38
, we mock-infected cells, or infected cells with empty virus or caMEK7-encoding virus. After 48 h, we precipitated endogenous p38
using a specific
antibody, and monitored the level of activated enzyme based on its
ability to phosphorylate ATF-2. As shown in Fig. 2C, the
presence of caMEK7 increases endogenous p38
activity. In addition,
although not shown, a smaller increase in activity was also detected
for cells infected with wtMEK7-encoding adenovirus. To confirm the
specificity of the p38
antibody, we infected cells with empty vector
or a virus encoding p38
and then detected expression of p38
by
immunoblot using anti-p38
. As shown in Fig. 2D, the
antibody detects a single band in cells infected with empty vector.
This band corresponds to the migration of expressed FLAG-p38
, when
the migration is adjusted for the contribution of the FLAG tag
(lane
). The antibody also nonspecifically cross-reacts
with overexpressed FLAG-p38
but does not detect p38
.

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Fig. 2.
Abundance and regulation of p38 MAPK isoforms
in keratinocytes. A, the relative level of the p38
isoforms in keratinocytes were compared using real time RT-PCR and p38
isoform-specific primers (17). B, to measure the enzymatic
activity of individual p38 isoforms in response to MEK7, keratinocytes
were coinfected with empty vector (EV) or caMEK7, and
FLAG-p38 , - , - , or - . After 48 h, the p38 isoforms
were immunoprecipitated using mouse monoclonal anti-FLAG antibody
(Sigma F3165) followed by 30 µl of protein G/A-agarose (Oncogene
IP05). p38 activity was monitored based on ability of the precipitated
kinase to phosphorylate ATF-2. Phosphorylated ATF-2 was detected by
immunoblot as described in the legend to Fig. 1. C,
endogenous p38 activity was measured by mock-infecting
keratinocytes, infecting with empty (EV) adenovirus, or
infecting with adenovirus encoding wtMEK7 or caMEK7. After 48 h,
the cells were harvested, and endogenous p38 was immunoprecipitated
using rabbit anti-p38 (1:5000). p38 activity was monitored based
on ability to phosphorylate ATF-2 as described in Fig. 1. D,
keratinocytes were infected with adenoviruses encoding FLAG-p38 ,
- , - , or - . After 48 h, lysates were prepared for
immunoblot using rabbit anti-p38 (1:5000). Binding of the primary
antibody was detected using peroxidase-conjugated donkey anti-rabbit
IgG (1:10,000). Each of these experiments was repeated three
times.
|
|
MEK7 Induces hINV Gene Expression--
Our previous studies
suggest that a PKC-activated cascade can act via MEK1, MEK3, and MEK7
to regulate hINV gene expression (6, 7). To directly examine
the role of MEK7 as a regulator of hINV gene expression, we
transfected keratinocytes with the hINV gene promoter
reporter plasmid, pINV-2473 (5), followed by infection with empty or
MEK7-encoding adenoviruses. As shown in Fig.
3A, wtMEK7and caMEK7 increase
promoter activity by 2- and 8-fold, respectively. This regulation
appears to be physiological, because, as shown in Fig. 3B,
real time RT-PCR shows that hINV mRNA is increased by 4-fold in the
presence of caMEK7. hINV protein levels also increase. hINV protein is
not readily detected in mock- or empty vector-infected keratinocytes
(Fig. 3C); however, the level is increased in the presence
of caMEK7. Moreover, SB203580, an agent that inhibits p38
and p38
activity at concentrations less than 1-2 µM (18),
inhibits the caMEK7-dependent activation at a concentration
of 0.5 µM (19, 20).

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Fig. 3.
Effect of MEK7 expression on hINV
gene expression. A, to measure the effects of
MEK7 on hINV promoter activity, keratinocytes were
transfected with pINV-2473 (5). After 24 h, the cells were
mock-infected or infected with empty adenovirus or adenovirus encoding
wtMEK7 or caMEK7. After 48 h, the cells were harvested, and
extracts were prepared and assayed for luciferase activity as
previously described (5, 7). This experiment was repeated three times.
The error bars indicate the mean ± S.E. B,
to measure the effects of MEK7 on endogenous hINV gene
expression, cells were infected with empty adenovirus
(control) or caMEK7-encoding adenovirus. After 48 h,
mRNA was prepared and hINV mRNA levels were monitored by real
time RT-PCR. As a control, glyceraldehyde phosphate dehydrogenase
(GAPDH) mRNA levels were monitored in parallel. These
results are representative of two experiments. C, to provide
additional confirmation of the mRNA results, cells were
mock-infected or infected with empty adenovirus or adenovirus encoding
caMEK7. A parallel group was infected with caMEK7-expressing adenovirus
in the presence of treatment with 0, 0.5, and 1 µM
SB203580. Fresh SB203580 was added every 12 h. At 48 h after
infection, the cells were lysed, and the hINV protein level was assayed
by immunoblot (24). Identical results were observed in each of three
experiments.
|
|
MEK7 Regulates hINV Promoter Activity via p38- and
JNK-dependent Pathways--
MEK7 has been reported to
activate JNK (9). Consistent with this observation, MEK7 increases JNK
activity in keratinocytes (Fig. 1C). To test the importance
of this regulation, we monitored the effects of JNK inactivation on
hINV promoter activity. Keratinocytes were transfected with
pINV-2473 and in the presence or absence of plasmid-encoding dnJNK
followed 24 h later by MEK7-encoding adenovirus. As shown in Fig.
4A, inactivation of JNK by
dnJNK results in an increase in wtMEK7- and caMEK7-associated promoter activity. This result suggests that JNK is a negative regulator of
hINV promoter activity.

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Fig. 4.
MEK7-dependent activation of JNK
may inhibit hINV gene expression. A,
to identify the effect of the MEK7-dependent activation of
JNK, we cotransfected keratinocytes with pINV-2473 reporter plasmid in
the presence or absence of dominant-negative JNK (dnJNK).
Twenty-four hours after transfection, the cells were infected with
empty adenovirus (EV) or adenovirus encoding wtMEK7 or
caMEK7. After an additional 48 h, the cells were harvested and
assayed for luciferase activity. This experiment was repeated three
times. The error bars indicate the mean ± S.E.
B, a schematic describing the MEK7-associated pathway that
regulates hINV gene expression. A more complete presentation
of the overall regulatory scheme has been previously presented (6, 7).
The solid arrows indicate positive influences, whereas the
dotted arrows indicate proposed inhibition.
|
|
 |
DISCUSSION |
The MAPK kinase cascades provide an important mode of transferring
regulatory information from the cell surface to the nucleus (1). These
cascades include a 3-kinase module that includes a MAPK kinase kinase
(MEKK), a MAPK kinase (MEK), and a MAPK. Four major mitogen-activated
protein kinases (MAPK) have been identified including ERK1/2, ERK5, p38
MAPK, and the c-Jun NH2-terminal kinase (JNK, Refs. 8,
21-23). Dual phosphorylation of the regulatory loop of these kinases
by MEKs results in MAPK activation (8). The MEK family includes MEK1,
2, 3, 4, 5, 6, and 7. Each of these MEK enzymes has a preferred
downstream target. For example, MEK1/2 activates ERK1/2, MEK5 activates
ERK5/BMK1, MEK3 and MEK6 activate the p38 MAPKs, MEK4 activates p38 and
JNK kinases, and MEK7 has been reported to be a specific activator of
JNK (8-10, 1, 25).
We have investigated the role of these enzymes as regulators of
differentiation in epidermal keratinocytes, particularly their role in
regulating expression of the differentiation marker, involucrin. Involucrin is specifically expressed in the suprabasal layers in
stratifying squamous surface epithelia, and expression in cultured cells is increased by agents that enhance keratinocyte differentiation (26-28). A study of the regulatory inputs that regulate this gene suggest that the MAPK cascades play a central role in maintaining both
basal and regulated expression (6, 7, 11, 12, 29). Using
dominant-negative kinases and pharmacologic agents to identify important regulatory enzymes, the results suggest that p38 activity is
required for activation, and that ERK and JNK kinase activity are not
required. Expression of the dominant-negative form of p38
completely
inactivates normal regulation (6). An exploration of other candidate
enzymes in these cascades suggest that the novel protein kinase C
isoforms are involved, as are Ras and MEKK1 (7). Other enzymes,
including Raf1, do not appear to play a role. MEKK1, in turn, appears
to target several MEKs, including MEK1, MEK3, and MEK7, but not MEK4
(6). Expression of dominant-negative forms of MEK1, MEK3, or MEK7
eliminates MEKK1-dependent regulation. Downstream targets
of this pathway include C/EBP and AP1 transcription factors that, in
turn, bind to specific DNA elements that have been identified in the
hINV promoter proximal and distal regulatory regions to
regulate transcription (5-7, 12, 29, 30).
Taken together these results suggest that the MEKs transmit the
regulatory signal via the p38 MAPKs. MEK3 is a known activator of p38
MAPK isoforms (19, 20, 31), and so our results suggest that one major
pathway that mediates regulation includes MEKK1, MEK3, and p38.
However, the downstream target of MEK7 was not identified. Previous
studies suggest that MEK7 is a specific activator of JNK kinase (9,
32). However, because JNK does not appear to mediate the regulation in
keratinocytes (6, 7), it appeared unlikely that JNK could be the
downstream target. We therefore examined the role of MEK7 as an
activator of p38 MAPKs. The present studies show that
adenovirus-mediated expression of caMEK7 or wtMEK7 in keratinocytes
results in an increase in total p38 activity. Interestingly, both
wild-type and constitutively active MEK7 were able to active p38, a
result that is consistent with the ability of the wild-type form of
other signaling enzymes (e.g. MEKK1) to activate this
cascade (6). In addition, MEK7 produced a modest activation of JNK
(3-fold) but did not regulate ERK activity.
Among the p38 family members, p38
and p38
appear to be
ubiquitously expressed (23). In contrast, p38
is expressed in muscle
where it plays a role in differentiation regulation (33, 34), and
p38
is enriched in lung, kidney, testis, pancreas, and intestine
(19). To identify the p38 family members involved in the regulation, we
analyzed p38 mRNA levels in keratinocytes using quantitative real
time RT-PCR (17). Our studies show that p38
and p38
are the major
forms present in keratinocytes with p38
also present at significant
levels. p38
is absent, thus eliminating it as a possible MEK7
downstream target. Three types of functional studies support a role of
p38
MAPK as a mediator of MEK7-dependent signaling.
First, total p38 activity is minimally detected in cells infected with
empty adenovirus and markedly increased in cells infected with
MEK7-encoding adenovirus. Second, infection of cells with individual
FLAG-tagged p38 isoform reveal that p38
, p38
, and p38
are
inactive in the presence or absence of MEK7. Third, a specific role for
p38
was confirmed by immunoprecipitation of increased levels of
active p38
from cells expressing MEK7.
One puzzling feature of these data is the high level of p38
activity
in cells expressing FLAG-tagged p38
(Fig. 2B). We would have expected, based on the other results presented in the manuscript, that this activity would be low. However, similar results have been
reported in other systems (14, 35). For example, Alonso et
al., (35) showed that recombinant p38
exhibits significant activity in the absence of MEK kinases, and that this activity markedly
increased with a slight increase in MEK levels. In contrast, p38
,
p38
, and p38
were substantially less sensitive. Thus it is
conceivable that when present at high levels, p38
may become activated by the endogenous MEKs. Despite this, p38
activity did
increase in the presence of MEK7. Taken together with the involucrin
gene regulatory data, these results suggest that MEK7 is a physiologic
regulator of keratinocyte differentiation; it is expressed at high
levels in epidermis (9, 10), and it regulates hINV promoter
activity and expression of the endogenous involucrin gene. Real time
RT-PCR also confirms that MEK7 mRNA is present in cultured
keratinocytes (not shown).
MEK7 is 47 and 43% homologous to MEK6 and MEK3, respectively, proteins
that have been previously shown to regulate p38 MAP kinase (9). As
noted above, MEK7 has been reported to be a specific activator of JNK
in a variety of cell types. For example, previous reports show that
MEK7 activates JNK in 293T cells (10), in cultured neonatal rat
cardiomyocytes (14), and in CHO cells (9). The present results
argue that MEK7 activates both JNK and p38 in keratinocytes, and that
the p38 activation appears to be more substantial. To our knowledge,
this is among the first reports suggesting that p38 MAPK is a MEK7
target. A recent report indicates that MEK7, along with MEK3, 4 and 6, can activate p38
in 293T cells (36). The outcome of our experiments
suggest that MEK7-dependent regulation of downstream kinase
activation may be tissue- or perhaps cell-type specific with respect to
MEK7 targets.
An additional point of interest in the present study, is the
observation that JNK appears to suppress hINV promoter
activity. This is consistent with a recent report indicating that JNK
inhibits expression of the SPRR1B gene, another gene that,
like involucrin, is regulated in a
differentiation-dependent manner in squamous epithelia
(37). Our results suggest that coincident regulation by MEK7 of p38
and JNK activity may function as a mechanism to determine the overall
level of hINV gene expression, with p38
functioning to
increase and JNK functioning to decrease expression (Fig.
4B). This is likely to be part of a more complex mechanism of signal integration that defines the impact of p38 versus
the JNK signals.
 |
ACKNOWLEDGEMENT |
We thank the Skin Diseases Research Center of
Northeast Ohio for the use of their facilities.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of Health
(to R. L. E.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, Rm. E532, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4970. Tel.:
216-368-5530; Fax: 216-368-5586; E-mail: rle2@po.cwru.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.C000862200
 |
ABBREVIATIONS |
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
JNK, c-Jun N-terminal kinase;
hINV, involucrin;
RT-PCR, reverse transcriptase-polymerase chain reaction;
bp, base pair;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
wt, wild-type;
dn, dominant-negative;
ca, constitutively active;
KSFM, keratinocyte serum-free medium;
MEK, MAPK kinase;
PKC, protein kinase
C.
 |
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