From the Greenebaum Cancer Center, Department of
Microbiology and Immunology, Molecular and Cellular Biology Program,
University of Maryland School of Medicine, Baltimore, Maryland 21201, the ¶ University of Maryland School of Pharmacy, Baltimore,
Maryland 21201, the
Department of Environmental Health
Sciences, Johns Hopkins University School of Public Health, Baltimore,
Maryland 21205, the
Section of
Hematology and Oncology, University of Illinois, Chicago, Illinois
60607, and the §§ Department of Pathology
and Immunology, Washington University School of Medicine,
St. Louis, Missouri 63110
Received for publication, June 6, 2000, and in revised form, September 14, 2000
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ABSTRACT |
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Interferons (IFNs) regulate the expression
of a number of cellular genes by activating the JAK-STAT pathway. We
have recently discovered that CCAAAT/enhancer-binding
protein- Interferons (IFNs)1
regulate the antiviral, antitumor, and immune responses in vertebrates
by inducing the transcription of a number of IFN-stimulated genes
(ISGs). Induction of ISGs occurs primarily due to the activation of the
JAK-STAT pathway (1, 2). Type I (IFN- Induction of ISGs by IFN- IFN- C/EBP- In this investigation, we show that IFN- Reagents--
Recombinant murine IFN- Cell Culture and Plasmids--
The RAW murine macrophage cell
line (RAW264.7) was grown in RPMI 1640 medium supplemented with 5%
fetal bovine serum. The murine ISGF3
The murine ISGF3 Gene Expression Analyses--
Northern and Western blot
analyses, transfection, ERK Activation and in Vitro Kinase Assays--
After stimulation
with the indicated reagents, cell extracts were prepared using a lysis
buffer supplemented with protease and phosphatase inhibitors as
described (32). ERK2-, JNK1-, p38-, or Raf-1-specific antibodies (0.4 µg) conjugated to protein A-Sepharose (Amersham Pharmacia Biotech)
were used to immunoprecipitate the cognate proteins from ~200 µg of
total cell protein at 4 °C for 2 h. Immunoprecipitates were
washed extensively with 25 mM HEPES (pH 7.4), 25 mM MgCl2, 1 mM dithiothreitol, and
0.2 mM sodium orthovanadate. ERK2, JNK1, p38, and Raf-1
kinase activities were determined using 2.5 µg of myelin basic
protein (MBP), glutathione S-transferase-Jun-(1-79),
glutathione S-transferase-activating transcription factor-2,
and wild-type MKK1 coupled to kinase-inactive ERK2, respectively.
Kinase reactions were stopped with SDS-PAGE loading buffer after
incubation at 30 °C for 30 min in the presence of
[ IFN-
Since staurosporine is a general protein kinase inhibitor and recent
reports indicated that MAPKs are activated during IFN treatment (39,
40), we examined the effect of different inhibitors of the MAPK
pathways on IFN-
The effect of MKK1 inhibitors on endogenous p48 (ISGF3 Inhibitors of MKK1 Block C/EBP- MKK1 Is Necessary for IFN-
MKK1 is downstream of Raf-1, a protein kinase (43, 44). In response to
growth factors, Ras is first activated, followed by Raf. Therefore, we
have studied the effects of dominant-negative Ras and Raf on
GATE-driven gene expression (Fig. 3B). Cells were transfected with expression vectors carrying the constitutively active
or dominant-negative mutants of Ras or c-Raf along with the P4
reporter. Coexpression of constitutively active Ras and Raf did not
enhance IFN-stimulated luciferase gene expression compared with the
vector controls. More importantly, neither dominant-negative Ras nor
Raf inhibited gene expression. Instead, dominant-negative c-Raf
slightly augmented IFN- MKK1 Augments C/EBP-
The importance of MKK1 in IFN- ERK1 and ERK2 Are Activated by IFN-
ERK1/2 activation was also confirmed by immunoprecipitation with
isoform-specific antibodies, followed by Western blotting with
phospho-ERK-specific antibodies (data not shown). Finally, the
functional activity of ERK was monitored in an in vitro
kinase activity using myelin basic protein as substrate (Fig.
4F). Although a slight ERK activity was seen at 30 min and
1 h, maximal enzymatic activity was found after 2 h of IFN
treatment. The differential kinetics of ERK activation suggests that
IFN- Raf-1 Activity Is Not Necessary for IFN-
We next examined whether the absence of Raf-1 activity in
IFN- ERK1 and ERK2 Mutants, but Not p38 Mutants, Block IFN-
To rule out the role of other MAPKs in IFN-stimulated gene expression
through GATE, gene expression was measured in the presence of a
dominant-negative p38 kinase. Cells were transfected with the P4
reporter along with the pCMV5 vector or the same vector carrying a
mutant cDNA of p38 MAPK. This mutant lacks the critical phosphorylation sites (TGY IFN-
Based on these results, we next determined whether the stimulatory
effect of IFN- ERK1/2 Activation Is Dependent on STAT1--
Since STAT1 and ERKs
were both required for stimulating C/EBP-
IFN- JAK1 Is Not Critical for IFN-
Based on the above results, we next determined whether ERK1 and ERK2
were activated in U4A cells. Western blot analysis was performed using
a phospho-ERK-specific antibody. ERK1/2 activation was weaker (2-fold)
in U4A cells and delayed (Fig. 9C) compared with 2fTGH
cells. The kinetics of ERK activation in U4A cells was similar to that
in RAW cells. ERK enzymatic activity (MBP phosphorylation) also
correlated with these data (data not shown). Interestingly, IL-6, which
was active in the U3A cells, also caused a similarly delayed and weaker
ERK activation. The increase in ERK activity was quantified in
independent samples and is presented in Fig. 9D. The
white and black bars show the ppERK1 and ppERK2 activation data, respectively.
The JAK1-independent activation of ERKs by IFN- ERK Phosphorylation Site of C/EBP-
A similar result was obtained with a human C/EBP-
To further prove that C/EBP- Given the spectrum of IFN action, it is quite unlikely that only a
specific set of genes regulate the antimicrobial, antitumor, antiviral,
and immunomodulatory effects. It is conceivable that coordinate
regulation of genes involved in each biological effect of IFN- In this study, we have identified an IFN- The relevance of IFN- ERK activation by IFN- Since IFN- We have noted a clear but weaker activation of ERK in
JAK1 A previous study in HeLa cells has shown that Raf-1, but not Ras, is
activated by IFN- (C/EBP-
) induces gene transcription through a novel IFN
response element called the
-IFN-activated transcriptional element
(Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe
a new IFN-
-stimulated pathway that operates C/EBP-
-regulated gene
expression independent of JAK1. We show that ERKs are activated by
IFN-
to stimulate C/EBP-
-dependent expression.
Sustained ERK activation directly correlated with
C/EBP-
dependent gene expression in response to IFN-
.
Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-
-stimulated gene induction through the
-IFN-activated
transcriptional element. Ras and Raf activation was not required for
this process. Furthermore, Raf-1 phosphorylation negatively correlated
with its activity. Interestingly, C/EBP-
-induced gene expression
required STAT1, but not JAK1. A C/EBP-
mutant lacking the ERK
phosphorylation site failed to promote IFN-stimulated gene expression.
Thus, our data link C/EBP-
to IFN-
signaling through ERKs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) and type II (IFN-
)
IFNs bind to distinct cell-surface receptors and activate the signals
that up-regulate the expression of ISGs (2). Upon binding to their
receptor, IFN-
/
induce the tyrosine phosphorylation of the
cytoplasmic tails of receptor using the JAKs Tyk2 and JAK1. JAKs
undergo tyrosyl phosphorylation prior to inducing the receptor
phosphorylation. Activated JAKs phosphorylate the STAT2 and STAT1
proteins at critical tyrosines. The STAT2-STAT1 dimer dissociates from
the receptor and forms a heteromeric complex with a DNA-binding
protein, p48 or IFN regulatory factor-9 or ISGF3
(3). This
complex, known as ISGF3, binds to the IFN-stimulated response element
of the ISGs and induces gene expression (1, 2). Ligand-activated IFN-
receptor recruits the Janus kinases JAK1 and JAK2, which selectively stimulate the phosphorylation of STAT1 (4). STAT1 dimers
then rapidly migrate to the nucleus and induce the expression of ISGs
that contain a
-IFN-activated site (1, 2). STAT activation does not
require new protein synthesis and is short-lived, lasting no longer
than 30 min after ligand/receptor engagement. It does not persist over
longer periods of time, despite the presence of IFN in the
extracellular environment (1, 2). Such deregulation may occur due to
the action of tyrosine phosphatases (5, 6) or degradation of STATs by
proteasome (7). The SOCS-1 (suppressor of
cytokine signaling-1) protein has been shown to
be critical for inhibiting IFN-
responses in vivo
(8).
is far more complex than that of
IFN-
/
, largely due to the facts that the temporal control of these genes is variable and that, in some cases, blockade of protein synthesis prevents their expression (9). These data suggest that
IFN-
may activate different transactivating factors in a JAK-STAT-dependent or -independent manner. Evidence for
these pathways is only beginning to accumulate. For example, the
repression of the c-myc gene by IFN-
occurs via both
STAT1-dependent and -independent mechanisms (10). A number
of transcription factors such as IFN regulatory factor-1 (11), IFN
consensus sequence binding protein (12), class II transactivator
(13, 14), and RF-X (15) activate specific sets of ISGs in an
IFN-
-dependent manner.
augments IFN-
/
-induced gene expression by up-regulating
the gene encoding the p48 subunit of ISGF3 (16, 17). A central role for
p48 in IFN-regulated pathways is highlighted by several observations.
Certain oncogenic viruses down-regulate p48 expression to evade the
action of IFNs (18), and its activity is inhibited in some human tumor
cell lines (19). IFN-
-induced expression of the p48 gene is rather
slow (12-18 h) and is inhibited by the protein synthesis inhibitor
cycloheximide (16, 17, 20), indicating the involvement of a novel
regulatory element and its cognate factors. Promoter analysis revealed
that the p48 gene promoter has no
-IFN-activated site, but instead
contains a unique IFN-
response element termed GATE (20). Although
GATE is partially homologous to the IFN-stimulated response
element, factors that bind to the IFN-stimulated response element do
not bind to GATE. The activity of GATE-binding factors is modulated by
IFN-
and is inhibited by cycloheximide and staurosporine (20). These
observations suggest that IFN-
regulates not only the synthesis of
GATE-binding factors, but also their post-translational modifications. We have recently identified one of these as the transcription factor
CCAAT/enhancer-binding protein-
(C/EBP-
) (21), a regulator of
acute-phase responses and cell differentiation (22, 23). Although
overexpression of C/EBP-
alone elevates basal gene expression, treatment with IFN-
further augments gene expression through GATE
(21). Our recent study demonstrated for the first time a role for
C/EBP-
in IFN-
-regulated gene expression. Since the JAK-STAT
pathway is rapidly activated and deactivated after IFN-
treatment
(1, 2), it was unclear which kinases could regulate the function of
C/EBP-
under these conditions.
binds to the consensus sequence TTNNGNAAT (14, 15). The
nucleotide sequence of GATE is as following:
5'-CCCGAGGAGAATTGAAACTTAGGG-3'. Six of the nine nucleotides
in this region of GATE are homologous to the consensus
C/EBP-
-binding site. Mutation of the AAACTT nucleotides of wild-type
GATE resulted in a loss of C/EBP-
binding. In addition, a shorter
GATE (GAGGAGGAATTGAAACTT) that encompassed the
C/EBP-
-binding site (20) or the C/EBP-binding site of GATE alone did
not support gene induction by IFN-
(data not shown). Furthermore,
the majority (80%) of the IFN-
-induced response was lost upon
mutation of the C/EBP-
-binding site. Thus, a full-length GATE
including the C/EBP-
-binding site is critical for IFN-
induction.
In addition, a C/EBP-
mutant lacking the transactivation domain
blunted GATE-dependent gene expression (21). These data together indicate that C/EBP-
is an important component of
GATE-dependent gene expression.
stimulates the
transcriptional activity of C/EBP-
through activation of ERK1 and ERK2 MAPKs, but independently of c-Raf and c-Ras. Pharmacological and
gene-specific dominant-negative inhibitors of MKK1, ERK1, and ERK2
block gene expression. Surprisingly, STAT1, but not JAK1, is required
to activate the C/EBP-
-dependent IFN-
response. Together, our study identifies a new IFN-
signaling pathway that couples the ERKs (MAPKs) to C/EBP-
-dependent gene regulation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Roche Molecular
Biochemicals); human IFN-
(Pestka Biomedical Labs); IL-6 (Genzyme
Corp.); staurosporine (Sigma); PD98059, SB202190, and SB202474
(Calbiochem); and U0126 (Promega) were used in this study. Antibodies
specific for phospho-ERK1/2 (Sigma) and ERK1, ERK2, JNK1, p38, Raf-1,
C/EBP-
, tubulin, and actin (Santa Cruz Biotechnology) were used as
recommended by the manufacturers.
promoter construct P4 has been
described previously (20). Human 2fTGH (parental), U3A
(STAT1-deficient), and U4A (JAK1-deficient) cell lines (24, 25) were a
gift from George Stark (Cleveland Clinic Lerner Research Institute).
They were cultured in Dulbecco's modified Eagle's medium with 5%
newborn calf serum and hygromycin (100 µg/ml) to retain the mutant
characteristics. The isogenic mouse fibroblasts WTSIM (wild-type), SKIM
(STAT1
/
), WTJIM (wild-type), and JKIM
(JAK1
/
) have been described elsewhere (26,
27). The SCC mouse fibroblast cell line expressing the wild-type or
mutant human IFN-
receptor has been described (28). These cell lines
express human chromosome 21, which provides the accessory factor of the
receptor, and permit human IFN-
-induced actions in the mouse background.
(p48) promoter construct P4 has been described in
our previous study (20). In this construct, a 74-base pair element of
the murine p48 gene promoter encompassing GATE was cloned upstream of
the SV40 early promoter. This construct, like the wild-type full-length
promoter, consistently responds to IFN-
and C/EBP-
(21) when
transfected into multiple cell types. Mutation of the GATE sequence in
this construct caused a loss of IFN-
response and C/EBP-
binding
(21). C/EBP-
cDNA cloned into a mammalian expression vector was
provided by Richard Hanson (Case Western Reserve University, Cleveland,
OH). Wild-type and mutant (Mut1 and Mut2) murine C/EBP-
(C/EBP-related protein-2) proteins were provided by Peter
Johnson (National Cancer Institute-Frederick Cancer Research and
Development Center, Frederick, MD) (29). Wild-type and point mutant
(T235A) human C/EBP-
(NF-IL6) proteins cloned into the pCMV vector
were a gift from S. Akira (30). Human STAT1 cDNA cloned under the
control of a constitutive enhancer of the pCXN2 vector has been
described (31). Wild-type ERK2 cDNA cloned into the pCMV vector has
been described earlier (32). Catalytically inactive dominant-negative
mutants of ERK1 (K71R) and ERK2 (K52R) cloned into the pCEP4 vector
(33) were provided by Melanie Cobb (University of Texas Southwestern
Medical Center, Dallas, TX). The wild-type, constitutively active, and
kinase-inactive MKK1 cDNAs cloned into pCMV were described
previously (32, 34). The p38 kinase AGF mutant (35) was provided by
Roger Davis (Howard Hughes Medical Institute, University of
Massachusetts, Worcester, MA). Kinase-inactive MKK1 has a K97M
mutation, and the constitutively active MKK1 mutant has a deletion in
the N-terminal region from amino acids 44 to 51 and serines 218 and 222 mutated to glutamate and aspartate, respectively (32, 34). Wild-type
Ras and dominant-negative Ras (N-17) constructs were described
elsewhere (36). Constitutively active c-Raf (Raf-BXB) and
dominant-negative c-Raf were described previously (37). The
authenticity of these mutants has been confirmed using a luciferase
reporter gene driven by the AP-1 response element (AP-1RE). The
AP-1RE-Luc plasmid was provided by Robert Freund (University of
Maryland, Baltimore, MD). In this construct, two copies of the
consensus AP-1RE were inserted upstream of the SV40 early promoter in
the pGL3 promoter vector (Promega).
-galactosidase and luciferase assays, and
SDS-PAGE analyses were performed as described in our earlier work (20).
The total amount of transfected DNA (1.5 µg) was kept constant by
adding pBluescript SK DNA where required. In general, 0.4 µg of
luciferase and 0.1 µg of C/EBP-
expression vector were
used. A
-actin promoter-driven
-galactosidase reporter
(0.2 µg) was used as an internal control for normalizing variations
in transfection efficiency as described in our previous study (20).
Transfection assays were repeated at least three times. Luciferase and
-galactosidase assays were essentially similar to those described in
common laboratory manuals (38).
-32P]ATP. Proteins in the kinase reactions were
separated by SDS-PAGE and Western-blotted, and substrate
phosphorylation was determined by PhosphorImager analysis. For
analyzing active ERK1/2, cell lysates were prepared as described above,
separated by SDS-PAGE, and immunoblotted using the
phospho-ERK1/2-specific antibody. Total ERK was determined in these
samples by using antibodies specific for ERK2 or ERK1. Intensity of the
activated ERK bands was quantified using a Molecular Dynamics laser
densitometer. Data from at least three independent samples are
presented in the bar graphs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced Gene Expression through GATE Is Inhibited by
Protein Kinase Inhibitors--
To understand the role of protein
kinases in IFN-
-stimulated gene expression through GATE, we studied
the influence of staurosporine, a protein kinase inhibitor. The RAW
macrophage cell line was transfected with the P4 reporter gene, in
which a 74-base pair element from the p48 promoter drives the
expression of the luciferase gene. Cells were treated with IFN-
in
the presence or absence of staurosporine. Although IFN-
alone
strongly up-regulated GATE-dependent gene expression, it
was strongly blocked by staurosporine (Fig.
1A). Since C/EBP-
binds to
GATE and induces gene expression (21), we examined whether
staurosporine also blocks C/EBP-
-regulated gene expression. As
expected, C/EBP-
strongly induced luciferase gene expression upon
cotransfection with the P4 reporter compared with the
vector-transfected cells (compare the scales of Fig. 1, A
and B). Treatment of cells with IFN-
further enhanced
C/EBP-induced expression. Staurosporine treatment not only inhibited
IFN-
-augmented gene expression, but also yielded lower luciferase
activity than that obtained with C/EBP-
alone. These data suggest
that C/EBP-
undergoes phosphorylation in response to IFN-
treatment.
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Fig. 1.
Effect of protein kinase inhibitors on
IFN- -induced gene expression. In
A, RAW cells were transfected with the P4-luciferase
construct (0.4 µg) and treated with murine IFN-
(1000 units/ml).
Luciferase activity was measured as described under
"Materials and Methods," using 30 µg of total cell
protein, 30 h post-transfection. Each bar represents
the mean ± S.E. of triplicate measurements. Where indicated,
cells were treated with 500 nM U0126 for 30 min
prior to treatment with IFN-
. B is similar to
A, except that a C/EBP-
expression vector (0.1 µg) was
cotransfected with the P4-luciferase plasmid (0.4 µg). C
shows the effect of MAPK kinase inhibitors on IFN-
-induced
luciferase gene expression in the P4 construct. Prior to stimulation
with IFN-
, the cells were exposed to the indicated inhibitors (25 µM) for 30 min. Me2SO (DMSO), the
solvent in which the drugs were reconstituted, was used as a control.
D shows the effect of U0126 on IFN-
-induced gene
expression. Transfection was performed, and luciferase activity was
determined after treatment with the indicated agents for 16 h as
described for A. C, untreated control;
, IFN-
(1000 units/ml). Shown in E
and F is the inhibition of IFN-
-stimulated
C/EBP-
-dependent gene expression by inhibitors of the
MAPK pathway. The P4 reporter (0.4 µg) was cotransfected with an
expression vector for C/EBP-
(0.1 µg) and then treated with the
indicated agents. E shows the effects of PD98059
(PD) and SB202474 (SB) on luciferase gene
expression. Cells were treated as described above. Equal amounts of
cell extract (50 µg) from triplicates were assayed for luciferase
activity. F shows the effect of U0126 on gene induction.
Transfection was performed as described for A.
-induced gene expression through GATE. Three
inhibitors, PD98059 (an MKK1-specific inhibitor), SB202474 (a negative
control inhibitor for MKK1), and SB202190 (a p38 MAPK-specific
inhibitor), were used in this study. RAW cells were transfected with
the P4 reporter gene and then treated with a 25 µM
concentration of each inhibitor prepared in Me2SO prior to
IFN-
treatment. PD98059, but not the negative control inhibitor
SB202474, strongly blocked gene expression (Fig. 1C). SB202190 was marginally inhibitory at this dose, but this low-level inhibition is probably not specific since p38 MAPK is not activated under these conditions (see below). PD98059 inhibits activation of MKK1
and suppresses the phosphorylation of ERKs (41). We also studied the
effect of a synthetic compound, U0126 (42), that specifically blocks
the activated MKK1 function (i.e. ERK activation) on
IFN-
-induced gene expression. U0126 inhibited IFN-
-induced gene
expression in a concentration-dependent manner (Fig.
1D). Treatment with Me2SO (vehicle) had no
effect on gene induction by IFN-
. Thus, MKK1 activation appears to
be necessary for IFN-
-induced gene expression.
) gene
expression was also studied. RAW cells were treated with IFN-
in the
presence or absence of either SB202190 or PD98059 for 16 h.
Poly(A)+ RNA was extracted, Northern-blotted, and probed
with p48 cDNA (Fig. 2A).
IFN-
strongly induced the expression of p48 mRNA (compare lanes 1 and 2). However, PD98059, but not
SB202190, strongly inhibited IFN-
-induced gene expression (compare
lanes 3 and 4). A 6-8-fold reduction of p48
mRNA expression occurred. Neither inhibitor alone significantly
altered p48 mRNA levels (lanes 5 and 6).
Although the blot shows a marginal increase in the p48 mRNA signal
with the inhibitors alone, this is not a reproducible effect. A
reprobing of these blots with 32P-labeled
-actin
cDNA revealed the presence of a comparable amount of RNA in all
lanes (Fig. 2B). Thus, a reporter driven by p48 promoter
elements and endogenous p48 behave in similar manner in the presence of
the MKK inhibitor PD98059.
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Fig. 2.
RAW cells were exposed to the indicated agents for
16 h, and poly(A)+ RNA was isolated in each case.
A, RNA (2 µg) was Northern-blotted, and the blots were
hybridized to 32P-labeled p48 cDNA. The blots were
washed stringently and autoradiographed overnight. Various treatments
are indicated above the gels. The plus and minus
signs indicate the presence or absence of the indicated reagent
during treatment, respectively. B, the stripped blot in
A was reprobed with 32P-labeled -actin
cDNA. Autoradiography was performed for 30 min.
-induced Gene
Expression--
Based on above results, we next examined the effect of
MKK1 inhibitors on C/EBP-
-dependent gene expression.
Cells were transfected with the P4 reporter and C/EBP-
expression
vector and then treated with IFN-
in the absence or presence of the
indicated inhibitors. C/EBP-
itself induced gene expression, and
IFN-
, as expected, stimulated it very strongly (Fig. 1E).
PD98059, but not SB202474, strongly inhibited
C/EBP-
-dependent gene expression stimulated by IFN-
.
To ascertain the specificity of such inhibition, a similar experiment
was performed using U0126. As expected, U0126 inhibited IFN-
-stimulated expression in a concentration-dependent
manner (Fig. 1F).
-induced Transcription through
GATE--
To directly demonstrate the role of MKK1 (MEK1), the effect
of MKK1 coexpression on GATE-dependent gene expression was
determined. Three different MKK1 expression vectors that carry the
wild-type, constitutively active, or non-catalytic mutant MKK1
cDNA were employed in these experiments. A control transfection
with the pCMV4 vector was also performed. Overexpression of the
wild-type MKK1 cDNA alone slightly induced the luciferase gene
compared with the vector control (Fig.
3A). Treatment with IFN-
further stimulated the activity. The constitutively active MKK1 mutant elevated basal expression and augmented IFN-
-induced
expression to significantly higher levels compared with the wild-type
MKK1 control. Thus, MKK1 may drive gene expression to some extent, using the basal levels of endogenous C/EBP-
. More importantly, the
non-catalytic MKK1 mutant suppressed basal and IFN-
-induced expression. These data indicate that MKK1 plays a critical role in
IFN-
-induced C/EBP-
-mediated signaling pathways.
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Fig. 3.
MKK1, but not Raf-1 or Ras, is necessary for
IFN- -induced gene expression through
GATE. In A, the P4 reporter was cotransfected with MKK1
variant expression vectors cloned into the pCMV vector. MKK1
wt, wild-type MKK1; MKK1 CA, constitutively active
MKK1; MKK1 Mut, catalytically inactive mutant MKK1.
C, untreated control;
, IFN-
(1000 units/ml). Shown in B is the effect of Ras and c-Raf on
IFN-stimulated gene expression through GATE. RAW cells were transfected
as described for A, except that expression vectors for
constitutively active Ras (N-14), a dominant-negative Ras
(Ras-DN; N-17), constitutively active c-Raf, or its
dominant-negative mutant (Raf-DN) were used as activator
plasmids, and luciferase activity was measured. In C, Ras
(N-17) and c-Raf mutants were cotransfected along with AP-1RE-Luc into
RAW cells. Where indicated, the cells were treated with EGF (40 ng/ml)
for 16 h, and luciferase activity was assayed as described under
"Materials and Methods." C, untreated control;
E, EGF. In D and E is shown the effect
of MKK1 mutants on IFN-
-induced gene expression through GATE.
D, MKK1 is required for stimulating
C/EBP-
-dependent gene expression by IFN-
. The P4
reporter (0.4 µg) was cotransfected with expression vectors for
C/EBP-
(0.1 µg) and the indicated MKK1 variant gene (0.4 µg).
Note the differences in the y axes of A and
D. E, U0126 blocks the stimulatory
effects of a constitutively active mutant of MKK1 on C/EBP-
. RAW
cells were transfected with the P4 reporter and the expression vectors
of C/EBP-
and constitutively active MKK1. Where indicated (+), the
cells were treated with 10 µM U0126 prior to IFN-
treatment.
-induced gene expression. To test the
authenticity of the dominant-negative mutants, they were cotransfected with AP-1RE-Luc, in which the luciferase reporter gene is controlled by
the consensus AP-1RE. As expected, EGF induced the expression of the
luciferase reporter. However, in the presence of mutant Ras or Raf,
EGF-induced luciferase expression was suppressed significantly (Fig.
3C). These mutants also blocked EGF-induced ERK
phosphorylation in transient transfection
assays.2 Taken together,
these data indicate that Ras and Raf are not critical for
IFN-
-induced GATE-driven gene expression.
-mediated Transcription--
The effect of
MKK1 on C/EBP-mediated transcriptional induction was examined next. RAW
cells were transfected with the P4 reporter and a C/EBP-
expression
vector. Along with these plasmids, wild-type, constitutively active, or
non-catalytic mutant MKK1 was transfected, and luciferase activity was
measured (Fig. 3D). C/EBP-
alone induced a low level of
transcription, which was augmented by IFN-
. Although IFN-
further
enhanced luciferase gene expression in the presence of wild-type or
constitutively active MKK1, it failed to exert a similar effect in the
presence of catalytically inactive MKK1. On the contrary,
C/EBP-
-dependent gene expression was repressed in the
presence of catalytically inactive MKK1. These data indicate that MKK1
is critical for regulating C/EBP-
-dependent gene
expression stimulated by IFN-
.
signaling was further confirmed by
use of U0126. Cells transfected with plasmids for P4-luciferase, constitutively active MKK1, and C/EBP-
were stimulated with IFN-
in the presence or absence of U0126 (Fig. 3E).
C/EBP-
-dependent gene expression was strongly induced in
the presence of IFN-
and constitutively active mutant MKK1, whereas
U0126 completely ablated such gene induction. These data suggest that
C/EBP-
is a downstream target of MKK1 signaling.
--
Since inhibition of
MKK1 led to the suppression of IFN-
-induced
C/EBP-
-dependent gene expression, we next determined
whether ERK1 (p44 MAPK) and ERK2 (p42 MAPK), the downstream substrates of MKK1 (43, 44), were also activated by IFN-
(Fig.
4). In these experiments, IL-6 was used
as a positive control because it is a known activator of C/EBP-
(45)
and because it stimulates gene expression through GATE (46). RAW cells
were treated with IFN-
, and ERK phosphorylation was monitored on
Western blots by probing an antibody that specifically detects the
diphosphorylated (activated) form of ERK. Indeed, both ERK1 and ERK2
both were activated in response to IFN-
(Fig. 4A). ERK1
activation by IFN-
was slower (relative to IL-6) and occurred
maximally only after 2 h of treatment, although slight activation
was seen earlier. In contrast, IL-6 rapidly induced ERK phosphorylation
within 30 min and dropped 2 h after treatment. IFN-
-stimulated
ERK1/2 activation persisted over longer periods of time (Fig.
4C) and could be observed up to 8 h. Such a pattern of
ERK activation correlates well with induced C/EBP-
synthesis (Fig.
4G). As observed earlier (21), C/EBP-
protein was
increased in cells after IFN-
treatment. On these blots, a slower
moving band that may represent the phosphorylated form of C/EBP-
could also be seen. These data are consistent with the slower induction
of the p48 gene by IFN-
.
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Fig. 4.
ERK1 and ERK2 are activated by
IFN- . RAW cells were treated with IFN-
and IL-6 where indicated for defined lengths of time, and cell extracts
were prepared as described under "Materials and Methods." In
A, equal amounts of whole cell lysate from each sample were
separated on SDS-polyacrylamide gels and Western-blotted. ERK
activation was determined using phospho-ERK-specific antibodies.
+Ve Control,
12-O-tetradecanoylphorbol-13-acetate-treated cell extracts.
B shows the Western blot data obtained after stripping and
reprobing of the blot shown in A with ERK2-specific
antibodies. C shows the sustained ERK activation by IFN-
over longer periods of time. Western blotting was performed as
described for A. In D is shown a reprobing of the
blot in C with antibodies specific for ERK1 and ERK2. Note a
higher amount of ERK1 and ERK2 (2-fold) at 0 min (lane 1).
E is a Western blot of immunoprecipitated ERK2. The
positions of IgG and ERK2 are indicated. F shows the IFN-
stimulation of the enzymatic activity of ERK2. ERK2 was
immunoprecipitated from cell lysates after treatment with IFN-
for
the indicated times and incubated with MBP in the presence of
[
-32P]ATP. The reaction products were separated by
SDS-PAGE and transferred to Western blots. 32P
incorporation into the MBP band was quantified using a PhosphorImager.
The -fold increase in MBP phosphorylation relative to the 0-min control
is shown below F. G and H show the
Western blots with C/EBP-
- and actin-specific antibodies,
respectively. The numbers above the lanes indicate time (in
minutes) after IFN-
treatment.
employs a signaling cascade distinct from that of IL-6 and that
the delayed ERK activation seems to be a ligand-dependent function.
-mediated ERK
Activation--
In the growth factor signaling pathways, Raf-1
activation precedes MKK1 phosphorylation (43, 44). Therefore, we
examined whether MKK1 activation was dependent on Raf-1 in response to IFN-
. Raf-1 was first immunoprecipitated from IFN-
-stimulated cell extracts and then incubated with [
-32P]ATP and
bacterially produced MKK1. After the reaction proteins were separated
by SDS-PAGE, they were transferred to a polyvinylidene difluoride membrane. Incorporation of radioactivity into MKK1 was
monitored using a PhosphorImager. No significant Raf-1-induced phosphorylation of MKK1 occurred under these conditions (Fig. 5A), although a comparable
amount of Raf was present in all the reactions (Fig. 5B).
Quantification of the radioactivity in the MKK1 band showed a lower
level of MKK1 phosphorylation by Raf-1 from IFN-
-treated cells (Fig.
5C).
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Fig. 5.
IFN- induces the
phosphorylation of Raf-1, but not its kinase activity. Raf-1 was
immunoprecipitated from IFN-
-treated RAW cells (B) and
incubated with bacterially produced MKK1 in the presence of
[
-32P]ATP for 30 min. MKK1 was visualized by
autoradiography of the Western-blotted reaction components
(A). Incorporation of 32P into MKK1 was
quantified using a PhosphorImager (C). Time (in minutes) of
IFN-
treatment is shown. D-F, IFN-
-induced
hyperphosphorylation of Raf-1 does not activate kinase function.
D shows immunoprecipitated Raf-1. The positions of Raf-1 and
IgG are indicated on the right. Note the slow moving Raf-1 band in
IFN-treated (120 min) cell extracts, but not in control cell extracts.
Immunoprecipitated Raf-1 was incubated with bacterially produced
wild-type MKK1 and kinase-dead ERK2 (ERK2-KD) in the
presence of [
-32P]ATP for 30 min. Following
termination of reactions, the samples were separated by SDS-PAGE,
Western-blotted, and autoradiographed. E shows the regions
corresponding to MKK1 and kinase-dead ERK2. The quantitative
representation of 32P in MKK1 (white bars) and
kinase-dead ERK2 (black bars) bands is shown in
F. C, untreated control;
, IFN-
(1000 units/ml). G-I, Raf-1 and ERK activation in EGF (40 ng/ml) for 30 min. In G, lysates prepared from untreated
(U) and EGF-treated (E) cells were
immunoprecipitated using Raf-1-specific antibodies as described for
D. Numbers below the gel indicate -fold increases
in Raf-1 kinase activity on its substrate MKK1. H and
I are Western blots for ppERK1/2- and ERK1/2-specific
antibodies in the same samples, respectively.
-treated cells was due to a lack of its phosphorylation. To demonstrate this more clearly, Raf-1 was immunoprecipitated from untreated and IFN-
-treated cells, and the samples were separated by
SDS-PAGE. Raf-1 from IFN-
-treated cells (Fig. 5D) had a
slower mobility compared with Raf-1 from untreated cells. Since Raf-1 phosphorylation may reflect its kinase function (43, 44), we monitored
its activity in a coupled assay. Immunoprecipitated Raf-1 was incubated
with [
-32P]ATP, bacterially produced wild-type MKK1,
and catalytically inactive ERK2 proteins. In this assay, ERK2 will be
phosphorylated only if MKK1 is activated by Raf-1. Fig. 5E
shows the radioactive phosphate incorporation into the substrates. No
increase in MKK1 or ERK2 phosphorylation was observed when incubated
with Raf-1 from IFN-
-treated cells compared with Raf-1 from
untreated control cells. Quantification of 32P in these
bands revealed no significant difference in MKK1 and ERK2
phosphorylation between control and IFN-
-treated cells (Fig. 5F, white bars). Thus, Raf-1 phosphorylation in
response to IFN-
does not correspond to an increase in its enzymatic
activity. In contrast, EGF induced both Raf-1 phosphorylation (as seen
by its slower mobility on the gel) and its kinase activity compared with the untreated cells (Fig. 5G). In these cells, EGF
induced ERK1/2 phosphorylation as expected (Fig. 5H). The
hypermobility of Raf-1 in response to IFN-
and EGF appeared to be
primarily due to phosphorylation since phosphatase treatment resulted
in a loss of such mobility (data not shown).
-induced
Transcription--
The functional relevance of ERKs in IFN-
-induced
transcription was examined next. RAW cells were transfected with the P4 reporter and an expression vector carrying wild-type ERK2. ERK2 alone
enhanced the basal level of transcription. Treatment with IFN-
strongly stimulated reporter gene expression (Fig.
6A). To further prove the
importance of ERKs in the regulation of GATE-driven gene expression,
dominant-negative ERK1 and ERK2 mutants were transfected along with the
P4 reporter construct. Cells were treated with IFN-
, and luciferase
activity was determined. ERK1 and ERK2 mutants alone caused only a
marginal reduction in luciferase activity compared with
vector-transfected IFN-
-treated cells. However, in the presence of
both mutants, IFN-
-induced expression was abrogated (Fig.
6B). The dominant-negative effects of ERK mutants were
confirmed using AP-1RE-driven reporter plasmids with either EGF (data
not shown) or phorbol 12-myristate 13-acetate as the stimulus (62).
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Fig. 6.
ERKs regulate gene expression through
GATE. A, ERK2 stimulates gene transcription through
GATE. RAW cells were transfected with the P4 reporter and the indicated
expression vector. ERK2, wild-type ERK2 cDNA in pCMV.
Luciferase activity was determined as described in the legend to Fig. 1
after IFN- treatment for 18 h. B, mutant ERKs block
IFN-
-stimulated gene expression through GATE. The P4 reporter was
transfected with an expression vector carrying dominant-negative mutant
ERK1 or ERK2 or both. The total amount of DNA transfected was kept
constant by adding the pCEP4 vector where necessary. Vector,
pCEP4; E1 mut, ERK1 mutant (K71R); E2 mut, ERK2
mutant (K52R). Cells were treated with IFN-
in all cases.
C, dominant-negative p38 does not inhibit
GATE-dependent gene expression. RAW cells were transfected
with the P4 reporter and the p38 AGF mutant cloned into the pCMV5
vector where indicated. Luciferase activity was determined as described
for A. C, untreated control;
,
IFN-
(1000 units/ml).
AGF) and has been confirmed previously to
exert a dominant-negative effect when overexpressed in the cells (40).
IFN-
activated luciferase expression to a comparable level in the
absence or presence of dominant-negative p38 (Fig. 6C).
Thus, ERK blockade is sufficient for inhibiting IFN-
-induced gene
expression through GATE, whereas p38 kinase does not play a role in
this process.
-stimulated C/EBP-
-dependent Gene Expression
Requires STAT1--
Since STAT1 is essential for driving
IFN-
-induced responses (24), the role of STAT1 in IFN-
-stimulated
C/EBP-dependent transcription was determined using 2fTGH
(parental) and U3A (STAT1-deficient) cells (24). Cells were transfected
with the P4 reporter in the presence or absence of C/EBP-
. C/EBP-
alone enhanced the transcription of the luciferase reporter compared
with the vector (white bars) in 2fTGH cells (Fig.
7A). Treatment of cells with
IFN-
strongly augmented transcription in C/EBP-transfected cells
(black bars). Such augmentation of gene expression was above
the levels observed in vector-transfected IFN-treated cells. A similar
experiment conducted with U3A cells revealed two important pieces of
information. 1) IFN-
did not stimulate reporter gene expression; and
2) C/EBP-
stimulated gene expression in the absence of STAT1 to a
level comparable to that in the parental cell line, 2fTGH. However, no
hyperactivation of gene expression occurred in the presence of IFN-
.
Since U3A cells were derived by chemical mutagenesis, it is possible
that mutations in genes other than STAT1 could cause a defective
C/EBP-
response. Therefore, we have performed the same experiment
with fibroblasts derived from STAT1 knockout mice (26). Whereas
wild-type fibroblasts (WTSIM) showed enhanced C/EBP-
-dependent gene expression after IFN-
treatment, STAT1
/
cells (SKIM) did not.
However, basal transcription was elevated in the presence of C/EBP-
in these cells (Fig. 7B).
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Fig. 7.
STAT1 is required for
IFN- -stimulated
C/EBP-
-dependent gene expression
through GATE. A and B, IFN-
fails to
induce C/EBP-
-dependent gene expression in
STAT1-deficient cells: human U3A cells and mouse SKIM fibroblasts,
respectively. The corresponding wild-type cells, 2fTGH (human) and
WTSIM (mouse), were employed as controls in these experiments. Cells
were transfected with the indicated effector plasmid (0.1 µg) along
with the P4 reporter (0.4 µg). C, STAT1 restores
IFN-
-stimulated gene expression via C/EBP-
in U3A cells. Cells
were transfected with the P4 reporter and the C/EBP-
expression
vector and, in addition, either the control expression vector pCXN2
(Vector; white bars) or the same vector carrying
STAT1 cDNA (STAT1; black bars), where
indicated. Luciferase activity was measured as described in
Fig. 1. C, untreated control;
, IFN-
(1000 units/ml).
on C/EBP-
-dependent gene expression
could be rescued by transfecting an expression vector for STAT1 (Fig. 7C). The P4 reporter was cotransfected with C/EBP-
in the
presence or absence of STAT1 in U3A cells. The cells were then treated with IFN-
, and luciferase expression was determined. As expected, a
basal level of C/EBP-
-dependent gene expression was
observed in these cells, which was not stimulated further by IFN-
.
However, in the presence of STAT1, IFN-
strongly induced
C/EBP-dependent gene expression.
-dependent gene
transcription, we examined whether ERK activation occurred in
STAT1-deficient cells after IFN-
treatment. 2fTGH cells were used as
a positive control in these experiments. U3A cells were exposed to
IFN-
or IL-6 for various lengths of time, and ERK1/2 activation was
monitored on Western blots using a phospho-ERK-specific antibody.
Several differences were noted in the ERK activation profiles of 2fTGH
and U3A cells (Fig. 8A). In
2fTGH cells, ERK1 and ERK2 were activated by 30 min by IL-6, but not by
IFN-
. At least 1 h of IFN-
treatment was required for
activating ERK1 and ERK2. However, in both cases, the activation was
turned off by 2 h. Since U3A and 2fTGH cells expressed similar
levels of ERKs, these differences may not be due to different levels of ERK protein in the cells (Fig. 8B). In contrast, no ERK1 or
ERK2 activation was noted in U3A cells in response to IFN-
. However, IL-6 stimulated ERK in U3A cells (2-2.5-fold) compared with the untreated controls. Thus, ERK1 and ERK2 are activated in response to
IFN-
in other cell types with a characteristic lag compared with
IL-6 treatment. However, the length of the lag in
IFN-
-dependent ERK activation is cell
type-dependent. These data suggest that STAT1 is required
for ERK activation by IFN-
, but not by IL-6. A slightly
higher basal ERK activity was seen in U3A cells. This may be due
to fact that these cells were derived after multiple rounds of chemical
mutagenesis and were grown in different medium from that used for
growing RAW cells. In addition, these cells grow faster than RAW cells.
The consistency of ERK activation was measured in different samples and
quantified by laser densitometry. These data are shown in Fig. 8
(C (for ppERK1) and D (for ppERK2)). As shown,
IL-6 was able to activate ERK1 independent of STAT1. IFN-
did
not activate ERKs significantly in the absence of STAT1.
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Fig. 8.
IFN- fails to
activate ERK activation in STAT1-deficient cells. U3A and 2fTGH
cells were treated with 1000 units/ml IFN-
or IL-6 for the indicated
times. In A, ERK activation was measured by Western blotting
using phospho-ERK-specific antibodies. In B, the same blot
was stripped and then probed with ERK2-specific antibodies.
C and D show the quantification of the ppERK1 and
ppERK2 bands, respectively. Each bar indicates the mean
intensity in arbitrary units ± S.E. of triplicates. E
shows the IFN-
-stimulated ERK activation in wild-type (WTSIM) and
STAT1
/
mouse fibroblasts. Note the presence
of a higher amount of ERK2 protein (middle panel) in
lanes 4-6 (STAT1
/
) than in
lanes 1-3 (WTSIM). The lower panel shows the
reprobing of the blot with anti-tubulin antibodies.
-dependent ERK activation was also examined in
wild-type (WTSIM) and STAT1
/
(SKIM) mouse
fibroblasts (Fig. 8E, upper panel). IFN-
readily activated both ERKs in the wild-type cells, but not in the
STAT1
/
cells. No ERK2 phosphorylation was
detectable in the SKIM cells even after loading a high amount of ERK2
protein (Fig. 8E, middle panel). Anti-tubulin
antibody probing demonstrated the presence of a comparable amount of
protein in the control and treated lanes of each cell line (Fig.
8E, lower blot).
-induced C/EBP-
-mediated Gene
Expression--
Since STAT1 was necessary for inducing
C/EBP-
-dependent gene expression, we investigated
whether JAK1 was also critical for enhancing
C/EBP-
-dependent gene expression. To examine this
aspect, U4A (JAK1-deficient) cells (25) were transfected with the P4 reporter and C/EBP-
and then treated with IFN-
(Fig.
9A). As expected, C/EBP-
strongly elevated basal gene expression compared with the vector
transfection. Treatment of C/EBP-
-transfected cells with IFN-
caused a strong up-regulation of luciferase expression, as in 2fTGH
cells. Similar results were obtained using fibroblasts (27) derived
from wild-type (WTJIM) and JAK1
/
(JKIM)
mice (Fig. 9B). Although the vector alone did not stimulate GATE-dependent gene expression in JKIM cells, transfection
of C/EBP-
not only elevated basal expression, but also enhanced gene
expression in the presence of IFN-
. This is in contrast to the U3A
and SKIM cells (Fig. 8), where the reporter gene was not induced by
IFN-
in the absence of STAT1. These data suggest that JAK1 is
dispensable for the "activation" of C/EBP-
by IFN-
.
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Fig. 9.
JAK1-independent stimulation of
C/EBP- -dependent gene expression
by IFN-
. In A and
B, JAK1-deficient U4A (human) or JKIM (mouse) fibroblasts
were transfected with the P4 reporter along with the C/EBP-
expression vector where indicated, and luciferase expression was
measured after IFN-
treatment as in the legend to Fig. 7. A parallel
transfection was also performed in 2fTGH (human) and WTJIM (mouse)
cells. C shows IFN-
- or IL-6-induced ERK activation
profiles in U4A cells. D shows the quantification of ppERK1
(white bars) and ppERK2 (black bars) in these
cells. In E is shown the effect of IFN-
receptor
mutations on ERK activation. The SCC mouse fibroblast cell line
expressing the wild-type or mutant human IFN-
receptor (28) was
exposed to human IFN-
(1000 units/ml) for the indicated times. Cell
extracts were Western-blotted using specific antibodies directed
against ppERK1 and ppERK2 (upper panel), ERK2 (middle
panel), and tubulin (lower panel). The cells expressed
wild-type hGR or the hGR-LPKS
A mutant (does not bind JAK1).
F shows the quantification of ppERK1 (white bars)
and ppERK2 (black bars) in hGR- and hGR-LPKS
A-expressing
cells. C, untreated control;
, IFN-
(1000 units/ml).
was also determined
in the SCC murine fibroblast cell line expressing the wild-type human
IFN-
receptor (hGR) or a mutant hGR that cannot bind JAK1
(hGR-LPKS
A). These cells express the ligand-binding
-chain and a
portion of the human chromosome 21 that expresses the
-chain of the
receptor (28). Cells were treated with human IFN-
for various
lengths of time, and ERK activation was measured (Fig. 9E).
IFN-
activated ERKs similarly in cells expressing the wild-type or
mutant receptor. In these experiments, we noticed a high activation of
ERK2 over ERK1 due to the fact that these cells expressed lower amounts
of ERK1. ERK activation by IFN-
was slow (2 h), as in RAW cells.
ERK1/2 activation by IFN-
was quantified in independent samples
(Fig. 9F).
Is Critical for Stimulating
Gene Expression in the Presence of IFN-
--
To determine directly
the role of ERKs in the phosphorylation of C/EBP-
, we employed two
independent mutants of C/EBP lacking the ERK phosphorylation consensus
sites (29, 30). In the first set of experiments, two mutants of murine
C/EBP-
lacking the serine and threonine residues proximal to the
basic leucine zipper domain were employed (Fig.
10A). Although these
residues are at different positions (owing to different sizes of the
protein) in the mouse and human C/EBP-
proteins, the sequence is
highly conserved between the species. C/EBP-
Mut1, a mutant with
alanines in place of serines distal to the amino terminus of the basic leucine zipper domain (29), responded normally to IFN-
. However, Mut2, in which the ERK phosphorylation consensus sequence GTPS is
converted to alanines, did not stimulate transcription upon IFN-
treatment (Fig. 10B). A slight inhibition of basal
transcription was noted in the presence of IFN-
.
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Fig. 10.
ERK phosphorylation consensus site of
C/EBP- is critical for stimulating
IFN-
-activated transcription through
GATE. A, domain structure of mouse C/EBP-
.
AD, transactivation domain; RD, regulatory
domain; Basic, domain rich in basic amino acids;
LZ, leucine zipper. The amino acid sequences of the
wild-type protein (Wt) and the corresponding changes in
mutants Mut1 and Mut2 are indicated. B, comparison of the
transcriptional activities of wild-type and mutant C/EBP-
proteins
in GATE-driven gene expression in response to IFN-
. C,
the threonine residue in the ERK consensus GTPS motif is critical for
IFN-stimulated gene expression. Transfection with the P4 reporter was
carried out in the presence of wild-type (black bars) or T-A
mutant (T-A mut; white bars) C/EBP-
expression
vectors in RAW cells. D, constitutively active MKK1 does not
augment C/EBP-
-induced gene expression in the presence of T-A mutant
C/EBP-
. RAW cells were transfected with the P4 reporter and an
expression vector for constitutively active MKK1. In addition, either
the wild-type or T-A mutant C/EBP-
expression vector was also
included where indicated. Luciferase expression was measured after
IFN-
treatment. C, untreated control;
,
IFN-
(1000 units/ml).
mutant
(T235A) in which the threonine residue in GTPS was converted to alanine (30). Although wild-type C/EBP-
strongly induced reporter gene expression upon IFN-
treatment, its corresponding mutant C/EBP-
lacking the critical threonine residue (T-A mutant) did not
(Fig. 10C). These data suggest that ERK phosphorylation at threonine of C/EBP-
is essential for gene expression. This mutant inhibited basal gene expression.
is a downstream target of the MKK1-ERK
pathway, RAW cells were transfected with wild-type or T235A mutant
C/EBP-
along with constitutively active mutant MKK1. Cells were
treated with IFN-
, and luciferase activity was measured, as in Fig.
10B. A high basal and IFN-
-induced expression was
observed in cells transfected with wild-type C/EBP-
(Fig.
10D). However, mutant C/EBP-
failed to respond to
IFN-
. A low basal expression was noted in the cells transfected with
mutant C/EBP-
, which was not induced further by IFN-
. These data
suggest that C/EBP-
is a downstream target of MKK1-activated ERKs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
requires the activation of distinct transcription factors and kinases.
Previously, we identified a novel IFN-
response element known as
GATE that did not seem to bind any known IFN-stimulated transcription
factors (20). We have recently discovered (21) that the transcription
factor C/EBP-
binds to GATE (22, 23, 47) and stimulates gene
expression. Treatment of C/EBP-
-transfected cells with IFN-
further robustly stimulated such expression. These data suggest that
C/EBP-
undergoes a post-translational modification(s) in the
presence of IFN-
. C/EBP-
(NF-IL6, liver-activating protein, C/EBP-related protein-2, and nuclear factor M) is a
member of the basic leucine zipper family of transcription factors (22, 23, 47) that responds to a number of extracellular stimuli, including
IL-6, IL-1, tumor necrosis factor-
, and lipopolysaccharide (22, 23).
Consistent with the spectrum of its activities, defects in carbohydrate
metabolism, lipid storage, Th1 immune responses, macrophage phagosome,
and female sterility were observed in
C/EBP-
/
mice (47, 48). That IFN-
is a
critical regulator of macrophage responses (49, 50) and that C/EBP-
regulates IFN-
-stimulated gene expression (21) add a new dimension
to the biology of this cytokine. To date, the ISGF3
gene is the only
ISG known to respond to C/EBP-
, although there could be others.
Thus, the understanding of IFN action through C/EBP-
may be
physiologically relevant.
-regulated signaling
pathway in which IFN-
-activated ERK1 and ERK2 regulate
C/EBP-
-dependent gene stimulatory effects. The temporal
activation of ERKs correlated with the presence of C/EBP-
protein
(Fig. 4G) and the previously described slower induction of
the ISGF3
gene (20). That activated ERK is necessary for
IFN-
-induced gene expression was confirmed using inactive ERK1 and
ERK2 mutants. Although neither mutant alone blocked gene expression,
together they strongly repressed IFN-
-induced gene expression (Fig.
6). It is important to note that both ERKs were phosphorylated in
response to IFN-
(Fig. 4). Therefore, even if ERK1 is blocked by the
corresponding mutant, ERK2 will still be active in cells transfected
with kinase-inactive ERK1. The converse is true in cells
transfected with the kinase-inactive ERK1 gene. The reason for such
redundancy is unclear at this stage. It is likely that
IFN-
-activated ERK1 and ERK2 differentially control specific sets of genes.
-dependent ERK activation to
C/EBP-
was supported by several observations. 1) Wild-type human or
murine C/EBP-
strongly induced gene expression through the p48
promoter in the presence of IFN-
. 2) C/EBP-
mutants lacking the
threonine residue in the ERK consensus sequence GTPS did not respond to IFN-
(Fig. 10, B and C). 3) The MKK1 inhibitor
U0126 blocked IFN-
-stimulated C/EBP-
-dependent gene
expression. 4) Constitutively active MKK1 augmented
C/EBP-
-dependent IFN-
-driven transcription (Fig. 3). However, it failed to activate a similar response in the presence of
mutant C/EBP-
(Fig. 10D). This latter observation
indicates that without a functional target, i.e. C/EBP-
,
ERK signals cannot drive gene expression through GATE. Previously, a
peptide derived from this region of C/EBP-
has been shown to serve
as a substrate for ERKs (30). p38 kinase is not phosphorylated
(data not shown), and a dominant-negative p38 kinase did not block gene
expression induced by IFN-
(Fig. 6). These results indicate the
specificity of MKK1-ERK for up-regulating gene expression through GATE.
Interestingly, the stimulatory effect of constitutively active MKK1 on
GATE-driven gene expression was further induced by IFN-
. This result
indicates that constitutively active MKK1 is still dependent on other
IFN-
-stimulated modifications or factors for inducing
GATE-dependent gene expression. Furthermore, shorter GATE
sequences (20) or those containing the minimal C/EBP-binding site of
GATE alone (data not shown) did not permit the induction of luciferase
by IFN-
. The fact that a minor fraction (20%) of IFN-
induction
occurs in the GATE construct with a mutant C/EBP-
-binding site (data
not shown) suggests that IFN-
-stimulated ERKs may also modulate
other factors, in addition to C/EBP-
.
differs from that by IL-6 (Fig.
4A). This is evidenced by the following. 1) STAT1 was
required for IFN-
(but not IL-6)-dependent ERK
activation (Fig. 8, A, C, and D). 2)
ERK activation was rapid in response to IL-6 in RAW and 2fTGH cells,
but slow in response to IFN-
(Figs. 4A and
8A). 3) IL-6 did not activate ERKs over a longer period of
time (Figs. 4A and 8A). That said, IL-6 and
IFN-
act similarly in other respects. IL-6 and IFN-
caused a
weaker but delayed ERK activation in JAK1-deficient cells (Fig.
9C). Slower activation of ERKs by IFN-
also suggests that
additional IFN-
-controlled factors are necessary for ERK activation.
Indeed, ERK activation by IFN-
requires STAT1, but not JAK1. Thus, a
STAT1-regulated factor is critical for C/EBP-
to sense the
IFN-
-induced transcriptional stimulus (Figs. 7 and 8). This factor
could be a signaling enzyme or a coactivator of C/EBP-dependent gene expression. In an analogous manner,
serine (but not tyrosine)-phosphorylated STAT1 has been shown to be
critical for the steady-state expression of certain caspase genes (51). Consistent with this, we have isolated another factor that interacts with GATE.3 It is unlikely
that STAT1 directly associates with C/EBP-
because electrophoretic
mobility shift analysis did not reveal the presence of STAT1 in
GATE-bound complexes (20). STAT1 did not co-immunoprecipitate with
C/EBP-
in preliminary studies (data not shown).
activates ERKs slowly, this study cannot rule out a
possibility that other intermediate factors may play a role in this
process. For example, a recent study (52) has shown that the induction
of cyclooxygenase-2 mRNA in normal human epidermal keratinocytes cells by IFN-
in part involves transforming
growth factor-
. In this case, both p38 MAPK and ERKs appear to be
activated by transforming growth factor-
, as suggested by a chemical
inhibition of these kinases and loss of cyclooxygenase-2 mRNA
induction. In contrast, p38 MAPK is not required for
C/EBP-dependent gene expression. These data suggest that
induction of the p48 gene is different from that of the
cyclooxygenase-2 gene. Future studies are required to analyze the
involvement of any secondary factors. Our study used mutant fibroblasts
and fibrosarcoma cell lines to define the mechanisms of control of
C/EBP-
-dependent gene expression by IFN-
. However,
there could be some tissue-specific differences between fibroblasts and
macrophages with respect to magnitude and duration of such responses.
Since the p48 gene is induced by IFN-
in most cell types, we believe
that these data are generally applicable to different cell types.
/
cells. Notably, ERK activation was
delayed in JAK1
/
cells in response to
IFN-
and IL-6 compared with their wild-type counterpart (Fig.
9C). Thus, JAK1 may enhance the kinetics of ERK activation
by IFN-
, but is not critical for it. That JAK1 is not required for
ERK activation is also supported by the data obtained with IFN-
receptor mutants that cannot bind JAK1 (Fig. 9E). Similarly,
in a previous study, kinase-inactive JAK1 was shown to sustain gene
expression, but not the antiviral state (53). We have shown earlier
that the endogenous p48 gene is not induced by IFN-
in U4A and U3A
cells (20). Consistent with this, another study has also shown that
IFN-
-induced p48 gene induction was suppressed in cells derived from
STAT1
/
mice (54). Furthermore, the
IFN-
-induced expression of p48 and C/EBP-
genes was not detected
in mouse fibroblast SKIM and JKIM cells, although it could be readily
seen in wild-type cells (data not shown). This is because the
GATE-binding factors are synthesized and/or modified in response to
IFN-
. Consistent with the properties of a GATE-binding factor, the
expression and gene-inductive effects of C/EBP-
are induced by
IFN-
(21) and are dependent on JAK1 and STAT1 (data not shown). In
the experiment shown in Fig. 9, C/EBP-
was constitutively expressed
in JAK1
/
cells, thus "short-circuiting"
its induction by IFN-
. Once C/EBP-
protein is available, its
functional activity becomes independent of JAK1, but dependent on
IFN-
. Thus, only one of the two steps involved in C/EBP-
-mediated
gene expression is JAK1-dependent.
(55). In contrast to that study, we found that
Ras, Raf-1, and JAK1 are not required for IFN-
-induced C/EBP-
-mediated gene expression through GATE. In contrast, the Ras
and Raf mutants were able to repress EGF-induced AP-1RE-driven transcription (Fig. 3C). This difference could be due to
different cell types used in our study. However, unlike the previous
study, we show here a functional target for the IFN-
-activated ERKs. Although IFN-
-dependent Raf-1 phosphorylation occurs in
RAW cells, it appears to have an inhibitory effect on Raf-1 activity.
Raf-1 did not activate its target MKK1 or the subsequent ERK
phosphorylation (Fig. 5). Consistent with these results,
dominant-negative Ras and Raf-1 slightly promoted
GATE-dependent gene expression (Fig. 3B) instead
of inhibiting it. In response to a number of extracellular stimuli,
Raf-1 is phosphorylated at multiple serine and tyrosine residues (43,
44). Raf-1 phosphorylation does not always mean a positive regulation
of its kinase function. A number of recent studies have shown that Raf
phosphorylation negatively modulates its kinase function (56-60). In
the proliferative signal transduction pathways, recruitment of
activated Raf to the plasma membrane transiently stimulates its kinase
activity (43, 44). Phosphorylation of Raf-1 decreases its affinity for
the plasma membrane. Kinetically, Raf-1 phosphorylation correlates with
a loss of function rather than a gain of function (57). IFN-
seems
to inhibit the proliferative signals by inducing Raf phosphorylation.
The negative effect of IFN-
on Raf-1 reflects its intrinsic nature.
Raf-1 promotes cell growth and is an oncogene (59). In contrast, IFNs
inhibit cell growth in a number of cell types (61). In agreement with
these properties, we have observed a consistently lower Raf-1 kinase activity in IFN-
-treated cells. In contrast, EGF enhanced Raf-1 kinase activity (Fig. 5). C/EBP-
, like-IFN-
, induces
differentiation in a number of cells (23, 47). Since differentiation
results in growth arrest, it is important to switch off the
proliferative signals. This may be more critical for macrophages since
their differentiation and action require C/EBP-
and IFN-
(48,
49). All together, our study identified a new IFN-
-regulated
signaling pathway in which ERKs couple the signals emanating from the
IFN-
receptor to a versatile transcription factor.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank several colleagues who provided the cell lines and plasmids used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Cancer Institute Grants CA 78282 and CA 71401 (to D. V. K.), CA 73381 and CA77816 (to L. C. P.), and CA 43059 (to R. D. S.) from the National Institutes of Health.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.
§ These authors contributed equally to this work and should be considered as first authors.
** Supported by National Institutes of Health Grant HL 58122.
¶¶ To whom correspondence should be addressed: Tel.: 410-328-1396; Fax: 410-328-1397; E-mail: dkalvako@umaryland.edu.
Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M004885200
2 P. S. Shapiro, unpublished observations.
3 J. Hu, S. K. Roy, P. S. Shapiro, S. R. Rodig, S. P. M. Reddy, L. C. Platanias, R. D. Schreiber, and D. V. Kalvakolanu, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IFNs, interferons;
ISG, interferon-stimulated gene;
ISGF, interferon-stimulated gene
factor;
JAK, Janus kinase;
STAT, signal transducer and activator of
transcription;
GATE, -interferon-activated transcriptional element;
C/EBP-
, CCAAT/enhancer-binding protein-
;
ERK, extracellular
signal-regulated kinase;
ppERK, diphosphorylated extracellular
signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
MKK, mitogen-activated protein kinase kinase;
IL, interleukin;
AP-1RE, activator protein-1 response element;
PAGE, polyacrylamide gel
electrophoresis;
MBP, myelin basic protein;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
EGF, epidermal growth factor;
hGR, human
-interferon
receptor.
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