Transcriptional Regulation of Heme Oxygenase-1 Gene Expression by
MAP Kinases of the JNK and p38 Pathways in Primary Cultures of Rat
Hepatocytes*
Thomas
Kietzmann
§,
Anatoly
Samoylenko
, and
Stephan
Immenschuh¶
From the
Institut für Biochemie und Molekulare
Zellbiologie, Georg-August-Universität Göttingen, D-37073
Göttingen, Germany and the ¶ Institut für Klinische
Chemie und Pathobiochemie, Justus-Liebig-Universität Giessen,
D-35392 Giessen, Germany
Received for publication, April 23, 2002, and in revised form, March 11, 2003
 |
ABSTRACT |
Heme oxygenase-1 (HO-1) gene
expression is induced by various oxidative stress stimuli including
sodium arsenite. Since mitogen-activated protein kinases (MAPKs)
are involved in stress signaling we investigated the role of arsenite
and MAPKs for HO-1 gene regulation in primary rat
hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA
expression. HO-1 protein and luciferase activity of reporter gene
constructs with
754 bp of the HO-1 promoter were induced
by overexpression of kinases of the JNK pathway and MKK3. By
contrast, overexpression of Raf-1 and ERK2 did not affect expression
whereas overexpression of p38
,
, and
decreased and p38
increased HO-1 expression. Electrophoretic mobility shift assays
(EMSA) revealed that a CRE/AP-1 element (
668/
654) bound c-Jun, a
target of the JNK pathway. Deletion or mutation of the CRE/AP-1
obliterated the JNK- and c-Jun-dependent up-regulation of
luciferase activity. EMSA also showed that an E-box (
47/
42) was
bound by a putative p38 target c-Max. Mutation of the E-box strongly
reduced MKK3, p38 isoform-, and c-Max-dependent effects on
luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates
HO-1 gene induction via activation of JNK/c-Jun
whereas p38 isoforms act through a different mechanism via the
E-box.
 |
INTRODUCTION |
The microsomal enzyme heme oxygenase
(HO,1 EC 1.14.99.3) catalyzes
the first and rate-limiting step of heme degradation producing carbon monoxide (CO), Fe2+, and biliverdin, which is
converted into bilirubin by biliverdin reductase (1). HO-1 is the
inducible isoform of HO and is identical to heat shock protein 32 (HSP32) (2), whereas HO-2 and HO-3 are constitutive isoforms (3). HO-1
expression is induced in response to its substrate heme by the second
messengers cAMP and cGMP (5), by various stress stimuli such as UV
light, heat-shock (6), sulfhydryl reactive reagents (7), heavy metals,
anoxia, hypoxia, hyperoxia (8, 9), as well as by inflammatory cytokines such as tumor necrosis factor
(TNF
), interleukin-1
(IL-1
), or interferon-
(IFN
) (10, 11). The up-regulation of HO-1 expression by these stimuli and their observed toxic effects in various
organs of HO-1-deficient mice (12) indicated that the enhancement of
HO-1 activity was an adaptive and protective response to cellular
stress (11, 13).
The transcriptional activation of HO-1 and other genes is mediated by a
network of signaling pathways, mostly by modulation of the activities
of transcription factors and target gene expression. A central position
in the signaling cascades regulating a number of cellular processes
such as cell growth, differentiation, stress responses, and apoptosis
occupy mitogen-activated protein kinases (MAPKs). Three major
subfamilies of MAPKs have been described: extracellular
signal-regulated kinases (ERK) (14), c-Jun N-terminal kinases (JNK)
(15), and p38 (16, 17). In general, the ERK pathway mediates cellular
responses to growth and differentiation factors including
platelet-derived growth factor, epidermal growth factor, and IL-5,
whereas JNK and p38 kinases are activated by stress-related stimuli,
such as heat shock, inflammation, hyperosmolarity, ultraviolet, and
irradiation (18).
Arsenic in the form of sodium arsenite, like other heavy metals, is
also a potent inducer of heme oxygenase (19); the mechanism of this
induction seems to be cell- and species-dependent (20-22). Arsenite is known to react with thiol groups of different proteins (e.g. receptors) and to be one of the inducers of ERK, JNK,
and p38 cascades (23, 24). This appears to be mediated by the activation of Shc due to tyrosine phosphorylation (25) and recruitment of the Grb2-Sos complex which can activate the p21 small GTPases of the
Rho family Ras and Rac. Ras, in turn, activates either the ERK pathway
or via action on PAK the JNK or p38 pathway. Rac activation can lead
again to PAK activation and subsequently to the activation of JNK and
p38 (26).
In liver cells the only detailed report so far regarding the role of
MAPKs in HO-1 gene regulation had demonstrated that
in the LMH chicken hepatoma cell line sodium
arsenite-dependent induction of HO-1 gene
expression was mediated by the ERK and p38 pathways (21). Despite the
extensive characterization of the MAPK signal transduction pathways and
the nearly universal induction of HO-1 expression by stress-related
stimuli, conflicting data have been reported as to the involvement of
MAPKs pathways in HO-1 gene activation in various tumor cell
lines (20, 21, 27-29). Moreover, little is known about HO-1
gene expression in response to the activation by arsenite and MAPKs in
primary cultured cells. Thus, it was the goal of the present study to
investigate the regulation of rat HO-1 gene expression by
MAPKs using primary rat hepatocyte cultures as a model system. In our
study basal levels of HO-1 mRNA expression as well as sodium
arsenite-induced HO-1 mRNA levels were down-regulated in the
presence of JNK inhibitor SP600125. It was demonstrated that the Ras
pathway via JNK induced HO-1 gene expression while the Raf
pathway via ERK was not involved in the regulation of HO-1
gene expression. The promoter sequence
668/
654 of the rat
HO-1 gene was shown to be responsible for HO-1 activation by
JNK and Jun. The E-box element (
47/
42) was involved in the
inhibition of HO-1 gene expression via an MKK3-independent mechanism by p38 and Max.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All biochemicals and enzymes were of analytical
grade and were purchased from commercial suppliers.
Cell Culture--
Cells from male Wistar rats (200-250 g) were
used for culture experiments. Hepatocytes were isolated by collagenase
perfusion (30). About 1 × 106 cells were cultured at
37 °C on 6-cm Falcon culture dishes under air/CO2 (19:5)
in medium 199 with Earle's salts containing bovine serum albumin (2 g/liter), NaHCO3 (20 mM), Hepes (10 mM), streptomycin sulfate (117 mg/liter), penicillin (60 mg/liter), insulin (1 nM), and dexamethasone (10 nM). Fetal calf serum (5%) was present in the initial
5 h after which cultures were incubated in serum-free medium for
another 18 h. Then, medium was changed again, and the cells were
further cultured in serum-free medium for 24 h. Hepatocytes were
treated with 5 µM sodium arsenite 8 h before
harvesting. The specific inhibitors of MEK PD98059 (New England
BioLabs) (5 µM) and U0126 (Cell Signaling) (10 µM), of JNK SP600125 (Biomol) (25 µM) and
of p38 SB203580 (Calbiochem) (20 µM) were added to the
culture medium also 8 h before harvesting.
Plasmid Constructs--
The luciferase reporter gene constructs
pHO-754 and pHO-754
A were previously described (5). The luciferase
reporter gene construct pHO-347 was generated by deletion of the
754/
347 fragment of HO-754 by ApaI and KpnI
followed by blunting of the remaining vector with Klenow enzyme
and ligation. The luciferase construct pHO-754Em was generated with the
QuickChangeTM XL site-directed mutagenesis kit (Stratagene)
using the oligonucleotide 5'-GGCTCAGCTGGGCGGCCACctctagACTCGAGTAC-3'.
The luciferase constructs p(CRE/AP-1)3-SV40Luc and
p(CRE/AP-1)6-SV40Luc were generated by ligation of the
oligonucleotide 5'-GATCCTGACTTCAGTCTGAATTCCTGACTTCAGTCTGACTTCAGTC-3' containing 3 CRE/AP-1 elements into the BglII site of pGl3
prom (pSV40Luc) (Promega). Expression vectors for MAP kinase signaling pathway components and transcription factors have been described: ERK2
(31), MEKK1 (32), JNK1 (33), MKK3, dominant-negative MKK3,
dominant-negative MKK4 (34), p38 isoforms, and dominant-negative p38
isoforms (16, 35-37), H-Ras (38), dominant-negative H-Ras (dnRas)
(38), Raf-1 (39), c-Jun (40), c-Myc (41).
RNA Isolation, Northern Blot Analysis, and
Hybridization--
Isolation of total RNA and Northern analysis were
performed as described (42). Digoxigenin (DIG)-labeled antisense RNAs served as hybridization probes; they were generated as described (43)
by in vitro transcription from pBS-HO-1 (800-bp cDNA
fragment) using T3 RNA polymerase or from pBS-
actin (550-bp
cDNA fragment) using T7 RNA polymerase and RNA labeling mixture
containing 3.5 mM 11-DIG-UTP, 6.5 mM UTP, 10 mM GTP, 10 mM CTP, 10 mM ATP.
Hybridizations and detections were carried out essentially as described
before (42). Blots were quantified with a videodensitometer (Biotech Fischer, Reiskirchen).
Western Blot Analysis--
Western blot analysis was carried out
as described (5). In brief, total protein from primary cultured
hepatocytes was prepared and the protein content was determined using
the Bradford method. 50 µg of protein were loaded onto a 10%
SDS-polyacrylamide gel and after electrophoresis blotted onto
nitrocellulose membranes. The primary antibodies against rat HO-1
(Stressgen), rat HO-2 (Stressgen), phospho-c-Jun (Ser63) (Cell
Signaling), phospho-HSP27 (Ser82) (Cell Signaling), phospho-p38 (Cell
Signaling), H-Ras (Santa Cruz), and c-Fos (Santa Cruz) were rabbit and
used at 1:1000 dilution. The primary monoclonal mouse antibody against
-actin (Sigma) was used at 1:5000 dilution, the primary mouse
antibody against phospho-p44/42 MAPK (Thr-202/Tyr-204) (Cell Signaling) was used at 1:2000 dilution, the primary mouse antibody against c-Jun
(Santa Cruz Biotechnology), the primary monoclonal mouse antibody
against HA tag (Cell Signaling) and primary monoclonal mouse antibody
against FLAG M2 (Sigma) were used at 1:1000 dilution. The secondary
antibodies were goat anti-rabbit IgG (Bio-Rad) and goat anti-mouse IgG
(Bio-Rad) and used at 1:2000 dilution. The ECL Western blotting system
(Amersham Biosciences) was used for detection. HO-1 was visible as a
32-kDa band, HO-2 as a 36-kDa band,
-actin as a 42-kDa band, H-Ras
as a 21-kDa band, c-Fos as a 55-kDa band, c-Jun as a 39-kDa band,
phospho-HSP27 as a 27-kDa band, phospho-ERK was seen as a double band
of 42 and 44 kDa. HA-MEKK1 was seen as 186-kDa band, HA-ERK as a 42-kDa
band, FLAG-Raf as a 68-kDa band, FLAG-MKK3 as a 40-kDa band, FLAG-JNK1
as a 56-kDa band, and FLAG-p38 as a 38-kDa band.
Cell Transfection and Luciferase Assay--
Freshly isolated rat
hepatocytes (about 1 × 106 cells per dish) were
transfected as described (5). In brief, rat hepatocyte cultures (about
1 × 106 cells per dish) were transiently transfected
with 2.5 µg of plasmid DNA containing 500 ng of the
Renilla luciferase construct pRL-SV40 (Promega) to control
transfection efficiency and 2 µg of the appropriate HO-1
promoter Firefly luciferase (FL) construct. In every culture experiment
two cultures were transfected per point. The DNA was transfected as a
calcium phosphate precipitate (5) for 5 h. After removal of the
medium cells were cultured under standard conditions without serum.
24 h later cells were treated with fresh medium and were cultured
for another 12 h. 12 h before harvesting the cells were
treated with PD98059 (5 µM), U0126 (10 µM),
SP600125 (25 µM), SB203580 (20 µM), or
LY294002 (Cell Signaling) (10 µM), as indicated. The Luc
activity of 20 µl of cell lysate was recorded in a luminometer
(Berthold) using the dual luciferase assay kit (Promega).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
Nuclear extracts were prepared essentially as
described (44). The sequences of the HO-1 oligonucleotides used for the
EMSA are 5'-TGTGTCAGAGCCATGTGTCCTGACTTCAGTCT-3' (spanning the
CRE/AP-1 (
664/
657), Fig. 4) and
5'-GGCTCAGCTGGGCGGCCACCACGTGACTCGAGTAC-3' (spanning the E-box
(
47/
42), Fig. 6). Equal amounts of complementary oligonucleotides
were annealed and labeled by 5' end-labeling with
[
-32P]ATP (Amersham Biosciences) and T4 polynucleotide
kinase (MBI). They were purified with the Nucleotide Removal kit
(Qiagen). Binding reactions were carried out in a total volume of
20 µl containing 50 mM KCl, 1 mM
MgCl2, 1 mM EDTA, 5% glycerol, 10 µg of
nuclear extract, 250 ng of poly(d(I-C)), and 5 mM DTE. For
competition analyses a 50-fold molar excess of unlabeled AP1 consensus
oligonucleotide (Promega) was added. After preincubation for 5 min at
room temperature, 1 µl of the labeled probe (104 cpm) was
added, and the incubation was continued for an additional 10 min. For
supershift analysis 1 µl of the ATF/CREB-1 (24H4B), c-JunC (epitope
corresponding to DNA binding domain), c-JunN (epitope mapping within
the N-terminal domain), c-Fos (K-25), Myc (C33), Max (C17) or SP-1
(PEP2-G) antibody (all obtained from Santa Cruz Biotechnology) as well
as a rabbit preimmune serum was added to the EMSA reaction, which was
then incubated at 4 °C for 2 h. The electrophoresis was then
performed with a 5% non-denaturing polyacrylamide gel in TBE buffer
(89 mM Tris, 89 mM boric acid, 5 mM
EDTA) at 200 V. After electrophoresis the gels were dried and exposed
to a phosphorimager screen.
 |
RESULTS |
Involvement of the JNK Pathway in the Sodium
Arsenite-dependent HO-1 mRNA Expression--
To
investigate whether the sodium arsenite-dependent HO-1
induction occurs via the activation of various MAPK signaling pathways primary hepatocytes were treated with sodium arsenite in the presence of specific inhibitors of the ERK, JNK, or p38 pathways. 5 µM sodium arsenite induced HO-1 mRNA expression in
primary rat hepatocytes after 8 h of treatment by about 8-fold
(Fig. 1, A and
B), in line with a previous study (45). The inhibitors of
the ERK pathway PD98059 and U0126 as well as the p38 pathway inhibitor
SB203580 did not change either basal or arsenite-enhanced HO-1 mRNA
levels. In contrast, the JNK inhibitor SP600125 attenuated the sodium arsenite-dependent HO-1 mRNA induction. Interestingly,
combination of the JNK and p38 inhibitors led to a more pronounced
inhibition of the arsenite-dependent HO-1 mRNA
induction pointing to the involvement of p38, which was overridden by
the JNK pathway. The effectiveness of the kinase inhibitors at the
concentrations used was demonstrated by Western blot analysis with
antibodies against phospho-ERK (for ERK pathway), phospho-Jun63 (for
JNK pathway), and phospho-HSP27 (for p38 pathway) (Fig. 1C).
These results indicated that kinases of the JNK and p38 pathways were
involved in the regulation of HO-1 expression by sodium arsenite. Since
the JNK and p38 pathways can be activated by MEKK1, the question of
whether overexpression of MEKK1 activates these pathways in hepatocytes was examined. Indeed, MEKK1 overexpression led to the activation of the
JNK and p38 pathway as demonstrated by the phosphorylation of cJun and
p38 (Fig. 1D).

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Fig. 1.
Involvement of MAP kinases in the induction
of rat HO-1 gene expression by sodium arsenite.
Primary hepatocytes were cultured for 42 h. 8 h before
harvesting, the cells were treated with 5 µM sodium
arsenite (NaAsO4) and with inhibitors of the MEK/ERK
pathway PD98059 (5 µM) and U0126 (10 µM),
of the JNK pathway SP600125 (25 µM) and of the p38
pathway SB203580 (20 µM). A, HO-1 mRNA
levels were measured by Northern blotting. For Northern analysis 20 µg of total RNA prepared from the cultured hepatocytes were
hybridized to digoxigenin-labeled HO-1 and -actin antisense RNA
probes (see "Experimental Procedures"). Autoradiographic signals
were obtained by chemiluminescence and scanned by videodensitometry.
The mRNA level in untreated hepatocytes was set equal to 100%.
Values are means ± S.E. of three independent culture experiments.
Statistics, Student's t test for paired values: *,
significant difference sodium arsenite versus control; **,
significant difference SP600125 and arsenite versus control
and arsenite, p 0.05. B, representative
Northern blots. C, inhibition of arsenite-induced
(A) phosphorylation of ERK, Jun, and HSP27 by specific inhibitors PD98059, U0126, SP600125, and
SB203580 detected by Western blotting with antibodies against
phospho-ERK, phospho-Jun, and phospho-HSP27 (see "Experimental
Procedures"). D, activation of the JNK and p38 pathway by
overexpression of MEKK1 as detected by Western blotting with antibodies
against phospho-Jun and phospho-p38 (see "Experimental
Procedures").
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Regulation of Transfected HO-1 Gene Promoter Constructs by
Overexpression of MAP Kinases in Primary Rat Hepatocytes--
To
further investigate the regulatory role of MAP kinases for
HO-1 gene expression primary cultured rat hepatocytes were
transfected with expression vectors for human H-Ras, Raf-1, MEKK1,
MKK3, ERK2, JNK1, and p38. Overexpression of these proteins was
confirmed by Western blot analysis. Hepatocytes transfected with H-Ras, MEKK1, JNK1, or MKK3 expression vectors showed enhanced HO-1 protein levels. By contrast, transfection with Raf-1 as well as ERK2 had no
effect on the HO-1 expression while transfection with p38 led to a
significant decrease of HO-1 protein levels (Fig.
2). The observed pattern appeared to be
specific for HO-1 since neither construct changed expression of HO-2 or
actin.

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Fig. 2.
Regulation of rat HO-1 protein expression by
overexpression of H-Ras and components of MAPK
pathways. Primary hepatocytes were transfected with 8 µg of Ras,
Raf, MKK3, ERK2, MEKK1, JNK1, or p38 expression vectors or the
control vector pCMV. After 5 h the medium was changed, and cells
were cultured for 43 h. A, HO-1 protein levels were
measured by Western blotting with an antibody against rat HO-1 (see
"Experimental Procedures"). Autoradiographic signals were obtained
by chemiluminescence and scanned by videodensitometry. The protein
level in hepatocytes transfected with the control pCMV vector was set
equal to 100%. Values are means ± S.E. of three independent
culture experiments. Statistics, Student's t test for
paired values: *, significant difference Ras versus control,
MKK3 versus control, MEKK1 versus control, JNK1
versus control, p38 versus control,
p 0.05. B, HO-1, HO-2, -actin, Ras,
HA-ERK2, HA-MEKK1, FLAG-Raf, FLAG-MKK3, FLAG-JNK1, FLAG-p38 proteins
were detected by Western blotting (see "Experimental
Procedures").
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To examine the molecular mechanism of HO-1 gene regulation
by MAPKs luciferase reporter gene constructs from the rat
HO-1 promoter (46) (pHO-754, pHO-750
A (5), and pHO-347)
were cotransfected with expression vectors encoding various components of the MAPK signaling pathways (Fig. 3).
Luciferase expression of pHO-754 was up-regulated by constitutive
active H-Ras, MEKK1, and MKK3 but not by Raf-1 or dominant-negative Ras
(dnRas). After cotransfection of both H-Ras and dnRas expression
vectors the stimulatory effect of H-Ras was attenuated by dnRas (Fig.
3). Cotransfection of MEKK1 and dominant-negative mutants of MKK3 and
MKK4 down-regulated HO-1 promoter activity. The
up-regulation of Luc activity of pHO-754
A and pHO-347, both of which
lack the previously described HO-1 CRE/AP-1 element (5), by
overexpressed MEKK1 was reduced compared with pHO-754 (Fig.
3A). Furthermore, the dominant-negative MKK3 still abolished
Luc activity whereas the dominant-negative MKK4 did not. The same
pattern was seen with pHO-347. This indicated that the JNK pathway
acted primarily through the CRE/AP-1 element, and the p38 pathway via
an element inside the first
347 bases of the HO-1
promoter. However, the remaining induction of pHO-347 by MEKK1 was not
attenuated by the inhibitors of either ERK, JNK, p38, or PI3K/PKB
because neither PD98059, U0126, SP600125, SB203580, nor the
phosphatidylinositol 3-kinase inhibitor LY294002 down-regulated
MEKK1-dependent enhancement of Luc activity. By contrast,
the inhibitor of the p38 pathway SB203580 led to an induction of Luc
activity.

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Fig. 3.
Induction of HO-1 promoter
controlled luciferase expression by overexpression of Ras and
MEKK1. A, hepatocytes were transiently cotransfected
with either Ras, dominant-negative Ras (dnRas), Raf, MEKK1,
MKK3, or dominant-negative MKK3 (MKK3dn) and
dominant-negative MKK4 (MKK4dn) expression
plasmids and luciferase (Luc) gene constructs driven by
wild-type 754 and 347 bp of the rat HO-1 promoter (pHO-754
and pHO-347) or the 754-bp promoter mutated at the previously described
(5) CRE/AP-1 site (pHO-754 A). In control experiments Luc constructs
were cotransfected with pCMV plasmid. In each experiment the fold
induction of Luc activity was determined relative to the pHO-754,
pHO-347, or pHO-754 A controls, which were set equal to 1. In
pHO-754 A the wild-type HO-1 sequence is shown on the upper strand,
deleted bases are indicated by and mutated bases are shown in
lowercase letters and indicated by *. Values represent
means ± S.E. of three independent experiments. Statistics,
Student's t test for paired values: *, significant
difference pHO-754+Ras versus pHO-754 control, pHO-754+MEKK1
versus pHO-754 control, pHO-754+MKK3 versus
pHO-754 control, pHO-754 A+Ras versus pHO-754 A control,
pHO-754 A+MEKK1 versus pHO-754 A control,
pHO-754 A+MKK3 versus pHO-754 A control, pHO-347+Ras
versus pHO-347 control, pHO-347+MEKK1 versus
pHO-347 control, pHO-347+MKK3 versus pHO-347 control,
p 0.05. B, hepatocytes were transiently
cotransfected with either MEKK1 expression plasmid or the empty control
vector and luciferase (Luc) gene construct pHO-347. 12 h before harvesting the cells were treated with PD98059 (5 µM), U0126 (10 µM), SP600125 (25 µM), SB203580 (20 µM), or LY294002 (10 µM). In each experiment the fold induction of Luc
activity was determined relative to the pHO-347 control, which was set
equal to 1. Values represent means ± S.E. of three independent
experiments. Statistics, Student's t test for paired
values: *, significant difference pHO-347+SB203580 versus
pHO-347 control, pHO-347+MEKK1+SB203580 versus
pHO-347+MEKK1.
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Because MEKK1 activates the JNK pathway, which is known to have the
transcription factor AP-1 as a nuclear target (47, 48) the regulation
of HO-1 gene promoter constructs was investigated in the
presence of overexpressed JNK1, and for comparison also ERK2. JNK1
enhanced Luc activity of pHO-754, which was abolished in the presence
of the JNK inhibitor SP600125. A JNK-dependent induction
was not observed with pHO-754
A or pHO-347. By contrast, ERK2 did not
affect Luc expression of the transfected HO-1 reporter gene constructs
(Fig. 4).

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Fig. 4.
Mutation of the CRE/AP1 site abolished the
JNK-dependent induction of HO-1
promoter-controlled luciferase expression. Hepatocytes were
transiently cotransfected with either ERK2 or JNK1 expression plasmids
and Luc gene constructs driven by wild-type 754 and 347 bp of the rat
HO-1 promoter (pHO-754 and pHO-347) or the 754-bp promoter
mutated at the CRE/AP-1 site (pHO-754 A). In control experiments Luc
constructs were cotransfected with pCMV plasmid. Tratment with the JNK
inhibitor SP600125 (25 µM) was performed as in Fig. 3. In
each experiment the fold induction of Luc activity was determined
relative to the pHO-754, pHO-347, or pHO-754 A controls, which were
set equal to 1. In pHO-754 A the wild-type HO-1 sequence is shown on
the upper strand, deleted bases are indicated by , and mutated bases
are shown in lowercase letters and are indicated by *. The
values represent means ± S.E. of three independent experiments.
Statistics, Student's t test for paired values: *,
significant difference pHO-754+JNK1 versus pHO-754 control,
p 0.05.
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Taken together, these results indicate that activation of the JNK
pathway may induce HO-1 gene expression through the HO-1 CRE/AP-1 site.
Role of the HO-1 CRE/AP-1 Element in the Activation of
HO-1 Promoter Luc Constructs by c-Jun--
Since the transcription
factor Jun is a substrate of JNK it was investigated whether c-Jun can
bind directly to the CRE/AP-1 element of the HO-1 promoter.
EMSA studies with nuclear extracts of cultured rat hepatocytes and an
oligonucleotide containing the HO-1 CRE/AP-1 site formed a strong
protein-DNA complex. The intensity of this complex was reduced by an
unlabeled AP-1 consensus oligonucleotide added in a 50-fold molar
excess to the binding reaction (Fig.
5).

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Fig. 5.
Binding of c-Jun to the CRE/AP-1 site of the
rat HO-1 promoter. A, The CRE (79) and
AP-1 (69, 70) consensus sequences and the rat HO-1 promoter
sequence 666/ 655 containing the CRE/AP1 site are shown. Bases
matching the consensus sequences are in bold. B,
EMSA: the 32P-labeled HO-1 CRE/AP1 oligonucleotide (see
"Experimental Procedures") was incubated with 10 µg of protein of
nuclear extracts from primary hepatocytes. In EMSA with antibodies the
nuclear extracts were preincubated with 1 µl of the JunC, JunN, Fos,
SP-1, ATF/CREB antibodies or a rabbit preimmune serum for 2 h at
4 °C before adding the labeled probe. For competition analyses a
50-fold molar excess of unlabeled AP1 consensus oligonucleotide
(Promega) was added. The DNA-protein binding was analyzed by
electrophoresis on 5% native polyacrylamide gels. C,
constitutive ATF/Jun-containing complex; S,
supershift.
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To investigate the presence of AP-1 and members of the ATF/CREB family
in this complex antibodies against c-Jun, c-Fos, and ATF/CREB were
included in the binding reactions. In the presence of the ATF/CREB
antibody a slightly supershifted complex was observed (Fig. 5). In
addition, the binding of the protein complex to the CRE/AP-1 nucleotide
was enhanced. Addition of antibodies against c-Fos, as well as against
the GC-box binding factor SP-1 or a rabbit preimmune serum, did not
result in a supershift or inhibition of complex formation. The Jun
antibody, which was raised against the highly conserved DNA binding
domain of c-Jun (c-JunC) abolished DNA-protein complex formation.
Addition of the c-JunN antibody generated against the N-terminal domain
formed a strong supershifted complex, indicating that the CRE/AP-1 site
was bound mostly by c-Jun.
To verify the functional relevance of c-Jun binding to the HO-1
CRE/AP-1 element the pHO-754 and pHO-754
A luciferase constructs were
cotransfected with expression vectors for c-Jun and c-Fos. Overexpression of both c-Jun and c-Fos was confirmed by Western blot
analysis. Overexpression of c-Jun, but not c-Fos, strongly induced
luciferase activity of pHO754 (Fig.
6A). No induction was observed
for the pHO-754
A construct in the presence of overexpressed c-Jun
and c-Fos. Similarly, c-Jun but not c-Fos induced luciferase activity
of SV40 promoter luciferase gene constructs containing 6 (p(CRE/AP-1)6-SV40Luc) or 3 (p(CRE/AP-1)3-SV40Luc) copies of the CRE/AP-1 element (Fig.
6B). Cotransfection of c-Jun did not induce luciferase
activity of the control pSV40Luc vector. Thus, it appears that c-Jun is
the major protein that interacts with the CRE/AP-1 element.

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Fig. 6.
Overexpression of c-Jun induced luciferase
expression controlled by either 754 bp of the HO-1
promoter or by 6 and 3 copies of the HO-1 CRE/AP1 element.
A, hepatocytes were transiently cotransfected with either
c-Jun, c-Fos, or both expression plasmids and Luc gene constructs
driven by the rat HO-1 promoter (pHO-754 and pHO-754 A) or
by the SV40 promoter regulated by 6 or 3 copies of the HO-1 CRE/AP1
elements (p(CRE/AP-1)6-SV40Luc and
p(CRE/AP-1)3-SV40Luc). In control experiments Luc
constructs were cotransfected with pCMV plasmid and pSV40Luc construct
was cotransfected with c-Jun, c-Fos, or both expression vectors. In
each experiment the fold induction of Luc activity was determined
relative to the pHO-754, pHO-754 A, p(CRE/AP-1)6-SV40Luc,
p(CRE/AP-1)3-SV40Luc, or pSV40Luc controls, which were set
equal to 1. In pHO-754 A the wild-type HO-1 sequence is shown on the
upper strand, deleted bases are indicated by , and mutated bases are
shown in lowercase letters and are indicated by *. The
values represent means ± S.E. of three independent experiments.
Statistics, Student's t test for paired values: *,
significant difference pHO-754+c-Fos versus pHO-754 control,
pHO-754+c-Jun+c-Fos versus pHO-754 control,
p(CRE/AP-1)6-SV40Luc+c-Jun versus
p(CRE/AP-1)6-SV40Luc control,
p(CRE/AP-1)6-SV40Luc+c-Jun+c-Fos versus
p(CRE/AP-1)6-SV40Luc control,
p(CRE/AP-1)3-SV40Luc+c-Jun versus
p(CRE/AP-1)3-SV40Luc control, p 0.05. B, overexpression of Jun and Fos proteins was detected by
Western blotting (see "Experimental Procedures").
|
|
Inhibition of HO-1-driven Luciferase Activity by p38 and c-Max Is
Abolished by Mutation of the HO-1 E-box Element--
Induction of HO-1
protein expression by overexpression of MKK3 and its repression by p38
suggested the presence of response elements for the p38 pathway within
the HO-1 promoter. Candidate transcription factors mediating
the activation by p38 are Myc and Max (49). These basic
helix-loop-helix leucine zipper (bHLHzip) proteins (50) are
known to bind to E-box elements one of which is also found in the rat
HO-1 promoter (
47/
42) (51). The potential binding of
nuclear proteins to oligonucleotide probes spanning the E-box sequence
of the rat HO-1 promoter was examined by EMSA studies. The
oligonucleotide
66/
32 containing the E-box formed two protein
complexes, which were no longer detectable with a mutated
oligonucleotide (Fig. 7). To investigate
the presence of bHLHzip factors in these complexes antibodies against
Myc and Max were included in the binding reaction. Addition of Max
antibody led to formation of a supershifted complex. Surprisingly,
incubation of the E-box oligonucleotide with an antibody against Myc
did not result in a supershift or inhibition of the complex indicating Max homodimer formation. To ensure specificity of the supershift mediated by the Max antibody the EMSA was also performed in the presence of an antibody against the GC-box binding factor SP-1. The
SP-1 antibody did not affect the complex formation with the E- box
oligonucleotide.

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|
Fig. 7.
Binding of Max to the E-box element of the
rat HO-1 promoter. A, E-box consensus
sequence and the rat HO-1 promoter sequence 50/ 39
containing the wild-type and the mutated E-box are shown. Bases
matching the consensus sequences are in bold, mutated bases
are shown in lowercase letters. B, EMSA: the
32P-labeled HO-1 wild-type (WT) and mutated
(M) E-box oligonucleotides were incubated with 10 µg of
protein of nuclear extracts. In EMSA with antibodies the nuclear
extracts were preincubated with 1 µl of the Myc, Max, or Sp1
antibodies for 2 h at 4 °C before adding the labeled probe. The
DNA-protein binding was analyzed by electrophoresis on 5% native
polyacrylamide gels. S, supershifted Max complex.
|
|
To investigate the functional role of the E-box in the regulation of
HO-1 gene expression by MAPK the wild-type pHO-347 and the
construct pHO-347Em, in which 3 of 6 bp of the E-box (
47/
42) were
mutated, were transfected along with expression vectors for MKK3,
different isoforms of wild-type p38 (p38
,
,
,
) or their respective dominant-negative forms (p38
,
,
,
, AF) and the p38 target Max. Overexpression of MKK3 enhanced
pHO-347-dependent Luc activity, and this enhancement was
reduced in the pHO-347Em-transfected cells. Transfection of pHO-347
with vectors for p38
, p38
, and p38
as well as Max inhibited
Luc activity whereas p38
enhanced Luc activity. The effects of the
different p38 isoforms were reversed by use of the respective
dominant-negative mutants p38
AF, p38
AF, p38
AF, p38
AF.
Furthermore, when pHO-347Em was used neither a repression nor an
induction of Luc activity was observed after cotransfection of the
vectors for the different p38 isoforms and their mutants (Fig.
8). These data indicate that the HO-1
E-box is responsible for the p38
, p38
, and
p38
-dependent down-regulation and the p38
-mediated
induction of HO-1 expression.

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[in a new window]
|
Fig. 8.
Mutation of the E-box abolished the p38 and
c-Max-dependent inhibition of HO-1
promoter-controlled luciferase expression. Hepatocytes were
transiently cotransfected with either p38 , p38 , p38 , p38
and the mutants, p38 AF, p38 AF, p38 AF, p38 AF, Max, or MKK3
expression plasmids and Luc gene constructs driven by the wild-type 347 bp of the rat HO-1 promoter (pHO-754) or the 347-bp promoter
mutated at the E-box site (pHO-347Em). In control experiments Luc
constructs were cotransfected with the empty control vectors. In
pHO-347Em the wild-type HO-1 sequence is shown on the upper strands,
and mutated bases are shown in lowercase letters and are
indicated by *. In each experiment the percent of Luc activity was
determined relative to the pHO-347 or pHO-347Em controls, which were
set equal to 100%. The values represent means ± S.E. of three
independent experiments. Statistics, Student's t test for
paired values: *, significant difference pHO-347+MKK3 versus
pHO-347 control, pHO-347+p38 versus pHO-347 control,
pHO-347+p38 versus pHO-347 control, pHO-347+p38
versus pHO-347 control, pHO-347+p38 versus
pHO-347 control, pHO-347+Max versus pHO-347 control,
p 0.05.
|
|
 |
DISCUSSION |
The present investigation has shown that kinases of the JNK
pathway are involved in the induction of rat HO-1 gene
expression by sodium arsenite in primary cultured rat hepatocytes. Rat
HO-1 gene expression is induced by Ras via MAPKs through the
JNK (MEKK1, JNK) pathway but not via MAPKs of the ERK (Raf, ERK2)
pathway. JNK activation was mediated via a CRE/AP-1 element
(
668/
654) which was bound mainly by c-Jun. Furthermore, different
roles of the p38 kinases could be demonstrated because MKK3 acted as an
inducer of HO-1, and the specific inhibitor of p38 pathway alone did
not influence HO-1 induction by sodium arsenite. The MKK3-dependent induction was partially mediated by p38
via an E-box element (
47/
42), which also appeared to be the target for direct inhibition of HO-1 expression by the p38 kinasese
,
,
and
. The target site for p38 was bound by Max. Overexpression of
Max, like the p38
,
, and
isoforms, inhibited HO-1 expression.
Cell-specific HO-1 Gene Activation by MAPKs--
HO-1
gene expression is induced by various stress stimuli that activate
MAPKs. Conversely, overexpression of HO-1 and HO-1-derived CO were
shown to exert anti-inflammatory and cytoprotective effects via MAPKs
as mediators (52, 53). The role of MAPKs have previously been
demonstrated in various cell culture systems (20, 21, 27-29, 54), and
contradictory results on the regulatory role of different MAPK pathways
for HO-1 gene expression were observed. Results obtained in
HeLa cells indicated that the induction of human HO-1 by NO was
mediated via ERK and p38 pathways but not via the SAPK/JNK pathway
(29). In another human cell line, HepG2, overexpression of MEKK1, a
kinase upstream of JNK, led to the induction of HO-1 protein levels
(28). On the other hand, it was reported that the induction of HO-1
mRNA expression by cadmium, arsenite, and hemin was not mediated
via MAPKs in Hela cells (20). In the same cells SB203580, an inhibitor
of p38 MAPK, had no effect on the cadmium-, arsenite-, and
hemin-dependent HO-1 mRNA induction.
In contrast, in the chicken hepatoma cell line LMH HO-1 gene
induction by sodium arsenite was mediated via ERK and p38 kinase pathways (21). Transfection of LMH cells with expression vectors for
wild-type and activated forms of the components of the ERK pathway and
the p38 cascade induced chicken HO-1 promoter-driven luciferase activity whereas dominant-negative forms of MAPKs blocked luciferase activity. Cotransfected expression vectors encoding wild-type and dominant-negative components of the JNK pathway (JNK,
MEKK1, MLK3) with chicken HO-1 promoter Luc reporter
genes did not affect Luc expression and its induction by
arsenite. However, data from our study show that induction of rat HO-1
expression by sodium arsenite was mediated via the JNK pathway (Fig. 1)
and that HO-1 can be induced via overexpression of JNK but not ERK2 (Figs. 2-4). Accordingly, inefficiency of the ERK pathway was
demonstrated in the cadmium-dependent activation of the
mouse HO-1 gene, thereby pointing for a role of the p38
cascade (27). A major reason for these regulatory discrepancies could
be species- or cell-specific variations that may affect the regulation
of HO-1 gene expression via MAPKs and different mechanisms
of arsenite-dependent HO-1 induction.
JNK-mediated HO-1 Gene Activation in Primary Rat
Hepatocytes--
Different regulation of the rat and chicken gene by
MAPKs could be caused by the lack of an AP-1 site at
668/
654 in the chicken HO-1 promoter, which mediates HO-1 gene activation
via the JNK and Jun pathway (Fig. 4). Although the rat and mouse
HO-1 gene 5'-flanking regions exhibit a much higher sequence
similarity than the rat and the chicken HO-1 gene
5'-flanking regions (46, 55, 56) different regulatory patterns were
observed. It was shown that the two distal enhancer regions E1 (
4 kb)
and E2 (
10 kb) containing several antioxidant response elements that
are regulatory sites for the transcription factor Nrf2 are
mainly responsible for stress-induced activation of mouse
HO-1 gene expression (22, 55, 57).
In contrast to the mouse gene, in the rat gene a number of apparently
inducer-specific regulatory elements are located in the proximal
promoter. These include the CRE/AP-1 site, which was shown in the
present study to be responsible for the induction of rat HO-1 via the
JNK pathway, a prostaglandin J2 element (
673/
668) and a
heat shock element (
278/
263) (5, 57-59). However, the
754 bp rat
HO-1 promoter does not contain a binding site for Nrf2. Accordingly, overexpression of Nrf2 did not enhance
rat HO-1 promoter activity in primary rat hepatocytes
although it was able to increase Luc activity from the Nrf2
responsive mouse HO-1 pE1-Luc gene (kindly provided by J. Alam) (data
not shown). However, this does not preclude a role for Nrf2 in
the regulation of the rat HO-1 gene, in particular, because
HO-1 induction is lost in Nrf2-deficient mice (60). Thus, there
could be an Nrf2 binding site in the yet unknown rat HO-1 far
upstream regulatory sequences, which in addition to the sequences
identified in this study might have a potential role in rat
HO-1 gene activation.
In the study with the mouse HO-1 gene, however,
dominant-negative mutants of JNK1 and JNK2 did not reduce basal and
cadmium-induced pE1-Luc activity suggesting that the
cadmium-dependent activation of the HO-1 gene
was not mediated via the JNK pathway (27). This result is in contrast
to the findings of this study and to the observation with rat liver in
which induction of HO-1 mRNA expression by the glutathione depletor
phorone was mediated by JNK1 and c-Jun (54). However, in that study
with rat liver the effects of MAPKs other than JNK1 and JNK2 on rat
HO-1 gene expression and the localization of the JNK target
site in the rat HO-1 promoter were not examined. That glutathione
depletion can regulate Ras/MAP kinases was previously demonstrated also
in rat pulmonary artery smooth muscle cells (61) and rat mesangial
cells (62). It is conceivable that the cascade identified in this study
via Ras/MEKK1/JNK/Jun is responsible for the rat HO-1 gene
activation by phorone.
The finding that the CRE/AP1 element is the target site for the
transcription factor c-Jun may also agree with previous studies showing
that cGMP/protein kinase G (PKG)-dependent HO-1
gene activation is mediated via this site (5). Since the PKG signal can
couple to the MEKK-1 pathway (63) it is conceivable that agents that stimulate the cGMP signaling like atrial natriuretic peptide (ANP) (64)
activate HO-1 gene expression via the PKG-MEKK1-JNK-Jun cascade. In addition, AP-1 has previously been demonstrated as a target
of the cGMP-dependent gene activation (5, 65, 66).
The most prominent AP-1 dimer is the c-Jun/c-Fos heterodimer, which
appears to be involved in the activation of a number of target genes
(67). However, AP-1 may consist also of heterodimers of c-Jun and a
member of the ATF/CREB family especially ATF-2 (68). The c-Jun/c-Fos
heterodimer binds to the TGANTCA consensus sequence whereas the
c-Jun/ATF heterodimer binds preferentially to the TGANNTCA consensus
site (69, 70). The rat HO-1 promoter CRE/AP-1 site is
homologous to the latter consensus sequence, which may be bound by
c-Jun/ATF proteins. This was supported by the data of EMSA studies in
which antibodies against c-Fos failed to produce a supershifted complex
(Fig. 5). Furthermore, overexpression of c-Jun induced HO-1
promoter activation whereas overexpression of c-Fos showed a negative
effect. This negative effect could be explained by the assumption that
overexpressed Fos dimerized with Jun competing with the ATF/CREB. Thus,
it appears that the HO-1 CRE/AP-1 site is a binding site for either Jun
homodimers or Jun/ATF heterodimers.
p38-mediated HO-1 Gene Regulation--
The p38 pathway has
previously been shown to activate the mouse and chicken HO-1
genes (21). In the mouse gene a stress response element in the upstream
E1 enhancer as target for Nrf2 was shown to be involved in
p38-dependent induction (27). Our data indicate that in rat
hepatocytes different p38 isoforms exert different patterns in the
regulation of HO-1 gene expression (Fig. 8). We observed a
p38
-dependent induction and inhibition of the HO-1 promoter activity by the other p38 isoforms. This
observation could explain why the p38 inhibitor SB203580 alone was not
effective on HO-1 expression. Furthermore, such different actions of
p38 isoforms have been observed in other cell types (71).
Interestingly, an inhibitory role for p38 has also been previously
described by others (28, 72). In particular, it was shown that the p38 pathway may suppress Ras proliferative signaling in NIH3T3 cells (72).
Furthermore, an inhibitory effect of p38 on gene expression was also
demonstrated (28, 73). Thereby, overexpression of wild-type p38 not
only inhibited basal expression of an antioxidant response element
(ARE)-dependent luciferase reporter gene construct but also
suppressed ARE-Luc activation by MEKK1 in HepG2 cells (28). In the same
study MEKK1 induced HO-1 expression (28).
In our study the E-box element (
47/
42) was identified as target for
p38-dependent induction and inhibition of HO-1
gene expression. The HO-1 Luc reporter gene construct containing a mutated E-box was not repressed by cotransfection of vectors encoding wild-type or mutated p38 (Fig. 8).
The E-box sequence (CACGTG) is known to be bound by members of the bHLH
leucine zipper (bHLHzip) protein family such as upstream stimulatory
factors (USF) or the Myc/Max dimer (74). Among bHLHzip proteins the
transcription factor Max (49) is known to be a target for p38 kinase.
It was further demonstrated that Myc in contrast to Max did not bind
the E-box element (
47/
42) of the rat HO-1 promoter (Fig.
7). Although this finding was surprising, Max not only forms
heterodimers with Myc, but also forms homodimers (75) and heterodimers
with other members of the bHLHzip family such as Mad1, Mad3, Mad4, and
Mnt (76, 77). It was shown that in contrast to the stimulatory effect
of Myc/Max heterodimers on transcription, Max homodimers, or
heterodimers between Max and members of the MAD family of bHLH-zip
proteins, repress transcription (78). In our study an inhibitory effect
of Max on HO-1-driven Luc activity was also observed. Mutation of
the HO-1 E-box abolished this effect. Thus, the E-box of the rat
HO-1 gene promoter is a target site for p38- and Max-mediated inhibition.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. J. Davis for the gift of
MKK3 and MKK4 dominant-negative mutants and Dr. Jiahuai Han for the
generous supply with the p38 vectors.
 |
FOOTNOTES |
*
This study was supported by the Deutsche
Forschungsgemeinschaft SFB 402 Teilprojekt A1 and GRK335 (to T. K.)
and SFB 402 Teilprojekt A8 and GRK335 (to S. I.).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: Institut für
Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany. Tel.: 49-551-395952; Fax: 49-551-395960; E-mail: tkietzm@gwdg.de.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M203929200
 |
ABBREVIATIONS |
The abbreviations used are:
HO, heme oxygenase;
bHLHzip, basic helix-loop-helix leucine zipper;
dnRas, dominant-negative H-Ras;
EMSA, electrophoretic mobility shift assay;
ERK, extracellular signal-regulated kinase;
FL, firefly luciferase;
JNK, c-Jun N-terminal kinase;
Luc, luciferase;
MAP, mitogen-activated
protein;
MAPK, MAP kinase;
p38AF, activated form of p38;
HA, hemagglutinin;
MEK, MAPK/ERK kinase;
MEKK, MEK kinase;
MKK, MAP kinase
kinase;
CRE, cAMP response element;
CREB, CRE-binding protein.
 |
REFERENCES |
1.
|
Tenhunen, R.,
Marver, H. S.,
and Schmid, R.
(1968)
Proc. Natl. Acad. Sci. U. S. A.
61,
748-755[Medline]
[Order article via Infotrieve]
|
2.
|
Maines, M. D.
(1988)
FASEB J.
2,
2557-2568[Abstract/Free Full Text]
|
3.
|
McCoubrey-WK, J.,
Huang, T. J.,
and Maines, M. D.
(1997)
Eur. J. Biochem.
247,
725-732[Abstract]
|
4.
|
Immenschuh, S.,
Kietzmann, T.,
Hinke, V.,
Wiederhold, M.,
Katz, N.,
and Muller, E. U.
(1998)
Mol. Pharmacol.
53,
483-491[Abstract/Free Full Text]
|
5.
|
Immenschuh, S.,
Hinke, V.,
Ohlmann, A.,
Gifhorn, K. S.,
Katz, N.,
Jungermann, K.,
and Kietzmann, T.
(1998)
Biochem. J.
334,
141-146[Medline]
[Order article via Infotrieve]
|
6.
|
Keyse, S. M.,
and Tyrrell, R. M.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
99-103[Abstract]
|
7.
|
Applegate, L. A.,
Luscher, P.,
and Tyrrell, R. M.
(1991)
Cancer Res.
51,
974-978[Abstract]
|
8.
|
Lee, P. J.,
Jiang, B. H.,
Chin, B. Y.,
Iyer, N. V.,
Alam, J.,
Semenza, G. L.,
and Choi, A. M.
(1997)
J. Biol. Chem.
272,
5375-5381[Abstract/Free Full Text]
|
9.
|
Lee, P. J.,
Camhi, S. L.,
Chin, B. Y.,
Alam, J.,
and Choi, A. M.
(2000)
Am. J. Physiol. Lung Cell Mol. Physiol.
279,
L175-L182[Abstract/Free Full Text]
|
10.
|
Rizzardini, M.,
Terao, M.,
Falciani, F.,
and Cantoni, L.
(1993)
Biochem. J.
290,
343-347[Medline]
[Order article via Infotrieve]
|
11.
|
Choi, A. M.,
and Alam, J.
(1996)
Am. J. Respir. Cell Mol. Biol.
15,
9-19[Abstract]
|
12.
|
Poss, K. D.,
and Tonegawa, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10919-10924[Abstract/Free Full Text]
|
13.
|
Maines, M. D.
(1997)
Annu. Rev. Pharmacol. Toxicol.
37,
517-554[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Gomez, N.,
and Cohen, P.
(1991)
Nature
353,
170-173[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Davis, R. J.
(1999)
Biochem. Soc. Symp.
64,
1-12[Medline]
[Order article via Infotrieve]
|
16.
|
Han, J.,
Lee, J. D.,
Bibbs, L.,
and Ulevitch, R. J.
(1994)
Science
265,
808-811[Medline]
[Order article via Infotrieve]
|
17.
|
Nebreda, A. R.,
and Porras, A.
(2000)
Trends. Biochem. Sci.
25,
257-260[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Kyriakis, J.,
and Avruch, J.
(2001)
Physiol. Rev.
81,
807-869[Abstract/Free Full Text]
|
19.
|
Sardana, M. K.,
Drummond, G. S.,
Sassa, S.,
and Kappas, A.
(1981)
Pharmacology
23,
247-253[Medline]
[Order article via Infotrieve]
|
20.
|
Masuya, Y.,
Hioki, K.,
Tokunaga, R.,
and Taketani, S.
(1998)
J. Biochem. (Tokyo)
124,
628-633[Abstract]
|
21.
|
Elbirt, K. K.,
Whitmarsh, A. J.,
Davis, R. J.,
and Bonkovsky, H. L.
(1998)
J. Biol. Chem.
273,
8922-8931[Abstract/Free Full Text]
|
22.
|
Alam, J.,
Stewart, D.,
Touchard, C.,
Boinapally, S.,
Choi, A. M.,
and Cook, J. L.
(1999)
J. Biol. Chem.
274,
26071-26078[Abstract/Free Full Text]
|
23.
|
Ludwig, S.,
Hoffmeyer, A.,
Goebeler, M.,
Kilian, K.,
Hafner, H.,
Neufeld, B.,
Han, J.,
and Rapp, U. R.
(1998)
J. Biol. Chem.
273,
1917-1922[Abstract/Free Full Text]
|
24.
|
Liu, Y.,
Guyton, K. Z.,
Gorospe, M.,
Xu, Q.,
Lee, J. C.,
and Holbrook, N. J.
(1996)
Free Radic. Biol. Med.
21,
771-781[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Chen, W.,
Martindale, J. L.,
Holbrook, N. J.,
and Liu, Y.
(1998)
Mol. Cell. Biol.
18,
5178-5188[Abstract/Free Full Text]
|
26.
|
Takai, Y.,
Sasaki, T.,
and Matozaki, T.
(2001)
Physiol. Rev.
81,
153-208[Abstract/Free Full Text]
|
27.
|
Alam, J.,
Wicks, C.,
Stewart, D.,
Gong, P.,
Touchard, C.,
Otterbein, S.,
Choi, A. M.,
Burow, M. E.,
and Tou, J.
(2000)
J. Biol. Chem.
275,
27694-27702[Abstract/Free Full Text]
|
28.
|
Yu, R.,
Chen, C.,
Mo, Y. Y.,
Hebbar, V.,
Owuor, E. D.,
Tan, T. H.,
and Kong, A.-N. T.
(2000)
J. Biol. Chem.
275,
39907-39913[Abstract/Free Full Text]
|
29.
|
Chen, K.,
and Maines, M. D.
(2000)
Cell Mol. Biol. Noisy. le. grand.
46,
609-617[Medline]
[Order article via Infotrieve]
|
30.
|
Immenschuh, S.,
Iwahara, S.,
Satoh, H.,
Nell, C.,
Katz, N.,
and Muller, E. U.
(1995)
Biochemistry
34,
13407-13411[Medline]
[Order article via Infotrieve]
|
31.
|
Whitmarsh, A. J.,
Yang, S. H.,
Su, M. S.,
Sharrocks, A. D.,
and Davis, R. J.
(1997)
Mol. Cell. Biol.
17,
2360-2371[Abstract]
|
32.
|
Xu, S.,
Robbins, D. J.,
Christerson, L. B.,
English, J. M.,
Vanderbilt, C. A.,
and Cobb, M. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5291-5295[Abstract/Free Full Text]
|
33.
|
Derijard, B.,
Hibi, M.,
Wu, I. H.,
Barrett, T.,
Su, B.,
Deng, T.,
Karin, M.,
and Davis, R. J.
(1994)
Cell
76,
1025-1037[Medline]
[Order article via Infotrieve]
|
34.
|
Raingeaud, J.,
Whitmarsh, A. J.,
Barrett, T.,
Derijard, B.,
and Davis, R. J.
(1996)
Mol. Cell. Biol.
16,
1247-1255[Abstract]
|
35.
|
Han, J.,
Lee, J. D.,
Jiang, Y.,
Li, Z.,
Feng, L.,
and Ulevitch, R. J.
(1996)
J. Biol. Chem.
271,
2886-2891[Abstract/Free Full Text]
|
36.
|
Jiang, Y.,
Chen, C.,
Li, Z.,
Guo, W.,
Gegner, J. A.,
Lin, S.,
and Han, J.
(1996)
J. Biol. Chem.
271,
17920-17926[Abstract/Free Full Text]
|
37.
|
Jiang, Y.,
Gram, H.,
Zhao, M.,
New, L.,
Gu, J.,
Feng, L.,
Di Padova, F.,
Ulevitch, R. J.,
and Han, J.
(1997)
J. Biol. Chem.
272,
30122-30128[Abstract/Free Full Text]
|
38.
|
Feig, L. A.,
and Cooper, G. M.
(1988)
Mol. Cell. Biol.
8,
3235-3243[Medline]
[Order article via Infotrieve]
|
39.
|
Dent, P.,
Reardon, D. B.,
Morrison, D. K.,
and Sturgill, T. W.
(1995)
Mol. Cell. Biol.
15,
4125-4135[Abstract]
|
40.
|
Schonthal, A.,
Buscher, M.,
Angel, P.,
Rahmsdorf, H. J.,
Ponta, H.,
Hattori, K.,
Chiu, R.,
Karin, M.,
and Herrlich, P.
(1989)
Oncogene
4,
629-636[Medline]
[Order article via Infotrieve]
|
41.
|
Kretzner, L.,
Blackwood, E. M.,
and Eisenman, R. N.
(1992)
Nature
359,
426-429[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Kietzmann, T.,
Roth, U.,
Freimann, S.,
and Jungermann, K.
(1997)
Biochem. J.
321,
17-20[Medline]
[Order article via Infotrieve]
|
43.
|
Kietzmann, T.,
Hirsch, E. K.,
Kahl, G. F.,
and Jungermann, K.
(1999)
Mol. Pharmacol.
56,
46-53[Abstract/Free Full Text]
|
44.
|
Kietzmann, T.,
Roth, U.,
and Jungermann, K.
(1999)
Blood
94,
4177-4185[Abstract/Free Full Text]
|
45.
|
Jacobs, J. M.,
Nichols, C. E.,
Andrew, A. S.,
Marek, D. E.,
Wood, S. G.,
Sinclair, P. R.,
Wrighton, S. A.,
Kostrubsky, V. E.,
and Sinclair, J. F.
(1999)
Toxicol. Appl. Pharmacol.
157,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Muller, R. M.,
Taguchi, H.,
and Shibahara, S.
(1987)
J. Biol. Chem.
262,
6795-6802[Abstract/Free Full Text]
|
47.
|
Tibbles, L. A.,
and Woodgett, J. R.
(1999)
Cell Mol. Life Sci.
55,
1230-1254[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Vuong, H.,
Patterson, T.,
Shapiro, P.,
Kalvakolanu, D. V.,
Wu, R.,
Ma, W. Y.,
Dong, Z.,
Kleeberger, S. R.,
and Reddy, S. P.
(2001)
J. Biol. Chem.
275,
32250-32259[Abstract/Free Full Text]
|
49.
|
Zervos, A. S.,
Faccio, L.,
Gatto, J. P.,
Kyriakis, J. M.,
and Brent, R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10531-10534[Abstract]
|
50.
|
Murre, C.,
Bain, G.,
van, D. M.,
Engel, I.,
Furnari, B. A.,
Massari, M. E.,
Matthews, J. R.,
Quong, M. W.,
Rivera, R. R.,
and Stuiver, M. H.
(1994)
Biochim. Biophys. Acta
1218,
129-135[Medline]
[Order article via Infotrieve]
|
51.
|
Maeshima, H.,
Sato, M.,
Ishikawa, K.,
Katagata, Y.,
and Yoshida, T.
(1996)
Nucleic Acids Res.
24,
2959-2965[Abstract/Free Full Text]
|
52.
|
Takeda, A.,
Perry, G.,
Abraham, N. G.,
Dwyer, B. E.,
Kutty, R. K.,
Laitinen, J. T.,
Petersen, R. B.,
and Smith, M. A.
(2000)
J. Biol. Chem.
275,
5395-5399[Abstract/Free Full Text]
|
53.
|
Otterbein, L. E.,
Bach, F. H.,
Alam, J.,
Soares, M.,
Tao, L. H.,
Wysk, M.,
Davis, R. J.,
Flavell, R. A.,
and Choi, A. M.
(2000)
Nat. Med.
6,
422-428[CrossRef][Medline]
[Order article via Infotrieve]
|
54.
|
Oguro, T.,
Hayashi, M.,
Nakajo, S.,
Numazawa, S.,
and Yoshida, T.
(1998)
J. Pharmacol. Exp. Ther.
287,
773-778[Abstract/Free Full Text]
|
55.
|
Alam, J.,
Cai, J.,
and Smith, A.
(1994)
J. Biol. Chem.
269,
1001-1009[Abstract/Free Full Text]
|
56.
|
Lu, T. H.,
Lambrecht, R. W.,
Pepe, J.,
Shan, Y.,
Kim, T.,
and Bonkovsky, H. L.
(1998)
Gene (Amst.)
207,
177-186[CrossRef][Medline]
[Order article via Infotrieve]
|
57.
|
Alam, J.,
Camhi, S.,
and Choi, A. M.
(1995)
J. Biol. Chem.
270,
11977-11984[Abstract/Free Full Text]
|
58.
|
Okinaga, S.,
and Shibahara, S.
(1993)
Eur. J. Biochem.
212,
167-175[Abstract]
|
59.
|
Koizumi, T.,
Odani, N.,
Okuyama, T.,
Ichikawa, A.,
and Negishi, M.
(1995)
J. Biol. Chem.
270,
21779-21784[Abstract/Free Full Text]
|
60.
|
Ishii, T.,
Itoh, K.,
Takahashi, S.,
Sato, H.,
Yanagawa, T.,
Katoh, Y.,
Bannai, S.,
and Yamamoto, M.
(2000)
J. Biol. Chem.
275,
16023-16029[Abstract/Free Full Text]
|
61.
|
Lander, H. M.,
Tauras, J. M.,
Ogiste, J. S.,
Hori, O.,
Moss, R. A.,
and Schmidt, A. M.
(1997)
J. Biol. Chem.
272,
17810-17814[Abstract/Free Full Text]
|
62.
|
Sandau, K. B.,
Callsen, D.,
and Brune, B.
(1999)
Mol. Pharmacol.
56,
744-751[Abstract/Free Full Text]
|
63.
|
Soh, J. W.,
Mao, Y.,
Liu, L.,
Thompson, W. J.,
Pamukcu, R.,
and Weinstein, I. B.
(2001)
J. Biol. Chem.
276,
16406-16410[Abstract/Free Full Text]
|
64.
|
Silberbach, M.,
and Roberts, C. T. J.
(2001)
Cell. Signal.
13,
221-231[CrossRef][Medline]
[Order article via Infotrieve]
|
65.
|
Gudi, T.,
Huvar, I.,
Meinecke, M.,
Lohmann, S. M.,
Boss, G. R.,
and Pilz, R. B.
(1996)
J. Biol. Chem.
271,
4597-4600[Abstract/Free Full Text]
|
66.
|
Pilz, R. B.,
Suhasini, M.,
Idriss, S.,
Meinkoth, J. L.,
and Boss, G. R.
(1995)
FASEB J.
9,
552-558[Abstract/Free Full Text]
|
67.
|
Karin, M.,
Liu, Z.,
and Zandi, E.
(1997)
Curr. Opin. Cell Biol.
9,
240-246[CrossRef][Medline]
[Order article via Infotrieve]
|
68.
|
Shaulian, E.,
and Karin, M.
(2001)
Oncogene
20,
2390-2400[CrossRef][Medline]
[Order article via Infotrieve]
|
69.
|
Ivashkiv, L. B.,
Liou, H. C.,
Kara, C. J.,
Lamph, W. W.,
Verma, I. M.,
and Glimcher, L. H.
(1990)
Mol. Cell. Biol.
10,
1609-1621[Medline]
[Order article via Infotrieve]
|
70.
|
Hai, T.,
and Curran, T.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3720-3724[Abstract]
|
71.
|
Pramanik, R.,
Qi, X.,
Borowicz, S.,
Choubey, D.,
Schultz, R. M.,
Han, J.,
and Chen, G.
(2003)
J. Biol. Chem.
278,
4831-4839[Abstract/Free Full Text]
|
72.
|
Chen, G.,
Hitomi, M.,
Han, J.,
and Stacey, D. W.
(2000)
J. Biol. Chem.
275,
38973-38980[Abstract/Free Full Text]
|
73.
|
Lavoie, J. N.,
L'Allemain, G.,
Brunet, A.,
Muller, R.,
and Pouyssegur, J.
(1996)
J. Biol. Chem.
271,
20608-20616[Abstract/Free Full Text]
|
74.
|
Sawadogo, M.,
and Roeder, R. G.
(1985)
Cell
43,
165-175[Medline]
[Order article via Infotrieve]
|
75.
|
Tchan, M.,
Choy, K.,
Mackay, J.,
Lyons, A.,
Bains, N.,
and Weiss, A.
(2001)
J. Biol. Chem.
275,
37454-374561[Abstract/Free Full Text]
|
76.
|
Hurlin, P. J.,
Queva, C.,
Koskinen, P. J.,
Steingrimsson, E.,
Ayer, D. E.,
Copeland, N. G.,
Jenkins, N. A.,
and Eisenman, R. N.
(1995)
EMBO J.
14,
5646-5659[Abstract]
|
77.
|
Meroni, G.,
Reymond, A.,
Alcalay, M.,
Borsani, G.,
Tanigami, A.,
Tonlorenzi, R.,
Nigro, C. L.,
Messali, S.,
Zollo, M.,
Ledbetter, D. H.,
Brent, R.,
Ballabio, A.,
and Carrozzo, R.
(1997)
EMBO J.
16,
2892-2906[Abstract/Free Full Text]
|
78.
|
Zhang, H.,
Fan, S.,
and Prochownik, E. V.
(1997)
J. Biol. Chem.
272,
17416-17424[Abstract/Free Full Text]
|
79.
|
Hoeffler, J. P.,
Meyer, T.,
Yun, Y.,
Jameson, J. L.,
and Habener, J. F.
(1988)
Science
242,
1430-1433[Medline]
[Order article via Infotrieve]
|
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