From the
Interferon-
These results suggest a model involving the
inhibition of IFN-
Interferon-
In this
report, using DNA mobility shift assays (EMSA) and transient DNA
transfection assays, we have examined in Jurkat T cells the effect of
the glucocorticoid hormone dexamethasone on the transcriptional
activity of the human IFN-
The
double-stranded probes were end-labeled using Klenow fragment (Life
Technologies, Inc.) and [
The following
double-stranded oligomers were used as labeled probes or cold
competitors: IFN-
CREB (wt) and CREB (mut)
consensus oligonucleotides were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
To prepare the
NF(P)
To construct the
plasmids(-108-36)CAT (wild-type sequence) and
(-108-36)
The plasmid
The plasmid pHIV-CAT, containing a 727-bp
HindIII-XhoI insert encoding the human
immunodeficiency virus, type 1 long terminal repeat linked to
the CAT gene
(22) , was obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, National Institutes
of Health. The deletion mutant of human c-Jun (TAM-67) has been
described previously
(16) . It was cloned as an EcoRI
fragment and then blunted and ligated into the NotI site of
the pCMV
The deletion
mutant of human c-Jun (c-Jun/1-286), carrying the truncation of
the leucine-zipper domain, was constructed as described previously
(23) and inserted into the NotI site of the
pCMV
As shown in Fig. 1, the
In order to determine whether AP-1 related
binding elements were present in the IFN-
Interestingly,
these two identified binding sites showing homology with the
AP-1
Fig. 3
shows the DNA binding pattern obtained using
nuclear extracts from unstimulated and PMA/ionomycin-treated Jurkat
cells in the presence of a labeled probe spanning nucleotides from
-96 to -75 (, the distal element of the
IFN-
Interestingly, a cold
oligonucleotide encompassing the distal element of the IFN-
In Fig. 5, a
supershift analysis in the presence of anti-Jun or anti-Fos antibodies
is shown; both of the antibodies specific for Jun and Fos family
members were able to compete for the binding when used in the presence
of nuclear extracts from PMA/ionomycin-stimulated Jurkat cells, while a
nonrelated antibody was not able to modify the binding capability or
the migration of the complexes in EMSA. The figure also shows a
comparison between canonical AP-1 and CREB-ATF binding sequences with
the two ``essential'' IFN-
Fig. 8A,
shows that the transcriptional activity of the (-108-36)CAT
reporter was enhanced by PMA/ionomycin, and the treatment with
dexamethasone was able to significantly inhibit this activation. On the
contrary, the mutations of the two AP-1
All of these data taken together suggest that
the two AP-1
Physiological activation of T cells requires the interaction
of antigen with the T cell receptor and the cooperation of a second
signal provided by an antigen presenting cell, a process that can also
be mimicked by treating the cells with PMA and calcium ionophore. The
typical hallmark of this phenomena involves several distinct steps
that, in the case of CD4
In this report, we focused our interest on the
inhibition of IFN-
The AP-1
complex, composed of Jun and Fos family proteins
(52) , is
intimately involved in T cell activation. The activity of this complex
is regulated both at the level of jun/fos gene
transcription and by post-translational modifications of these proteins
(52-56). For example, activation of the IL-2 promoter in T cells
has been shown to be regulated by the cooperative interaction of
several transcriptional factors, such as AP-1, NF-
Recent data have also shown that the Jun/Fos transcription factors
are also able to cross-heterodimerize with CREB-ATF DNA binding factors
through the ``leucine zipper''
motif
(32, 33, 63) . These resulting heterodimers
have been shown to display distinct DNA binding specificities from the
parental homo/heterodimers
(32, 33) .
We have
demonstrated here that the IFN-
The similar down-regulation observed in
cotransfection assays, utilizing a GR expression vector and
dexamethasone treatment, or two different dominant negative mutants of
c-Jun (TAM-67 and c-Jun/1-286), correlates well with the presence
of AP-1
As a negative regulation involving
direct protein-protein interference between AP-1 and GR has been
reported by several investigators in different
systems
(12, 13, 14, 15, 64) , we
propose here a similar model involving a functional impairment mediated
by GR on the ``essential'' AP-1
The inhibitory mechanism of the c-Jun dominant
negative mutant TAM-67 has been only partially characterized. This
inhibitor is in fact able to form stable complexes with Jun and Fos
proteins, and these complexes bind DNA with the same affinity as the
normal Jun and Fos heterodimers (17). In addition, recent data obtained
by analysis of different TAM-67 chimeric proteins suggest a model
involving a ``quenching mechanism'' rather than a
``blocking mechanism,'' through dimerization of TAM-67 with
wild-type Jun and Fos proteins, this interfering with the normal AP-1
transcriptional activation
(17) .
A different mechanism could
be evoked for the negative mutant c-Jun/1-286, that bears a
selective deletion of the leucine zipper domain; in this case, a direct
interference involving a ``squelching mechanism'' via the
transactivation domain on the IFN-
In conclusion, these results further emphasize the transcriptional
control complexity of the IFN-
We thank Dr. Christopher B. Wilson for providing
plasmids pIFN-538,339,108, Dr. Ronald M. Evans for providing the human
GR expression vector pRShGR
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IFN-
) is an immunoregulatory cytokine
expressed in large granular lymphocytes and T cells. However, the
molecular mechanisms underlying IFN-
gene transcription have not
been fully defined. Here, we analyze the mechanisms responsible for the
inhibition of IFN-
promoter activity by the glucocorticoid hormone
dexamethasone. Cotransfection assays performed in Jurkat T cells
demonstrated that the activity of the initial 108 base pairs of the
IFN-
promoter was down-regulated in the presence of dexamethasone.
Furthermore, utilizing electrophoretic mobility shift analysis, we
identified activator protein 1 AP-1-cAMP response element binding
protein-activating transcription factor (CREB-ATF) binding elements
situated in positions of the IFN-
promoter previously identified
as essential for promoter activity. Moreover, dominant negative mutants
of the c-Jun proto-oncogene were able to mimic the same down-regulatory
effect exerted by dexamethasone, and mutations that abolished the
binding of the AP-1
CREB-ATF factors were able to block the
glucocorticoid effect.
AP-1
CREB-ATF DNA binding complexes as one
of the mechanisms involved in the negative regulatory action of
glucocorticoids on IFN-
gene expression and support the relevance
of AP-1
CREB-ATF binding factors during the transcriptional
activation of the IFN-
promoter in T cells.
(IFN-
)
(
)
is an
immunoregulatory cytokine involved in modulating nearly all phases of
immune and inflammatory responses
(1, 2) . Furthermore,
this cytokine is relevant as a therapeutic agent for immunodeficiency
states, infections, and neoplastic states and has been evoked in the
pathogenesis of such disorders
(1) . IFN-
expression in
vivo seems to be strictly regulated, as the production of
messenger RNA has been detected predominantly in activated T cells and
large granular lymphocytes
(1, 2) . Inhibition of
IFN-
production has been reported to be caused by different
agents, such as cyclosporin A, corticosteroids, prostaglandins,
etc.
(1) . Glucocorticoids in particular, already shown to play a
major role in the treatment of different autoimmune, allergic, and
inflammatory diseases, are able to affect the growth, the
differentiation, and the function of monocytes and lymphocytes and to
strongly regulate the production of different cytokines, including
IFN-
(3, 4, 5, 6, 7, 8) .
Previous investigations have already shown that a synthetic
glucocorticoid hormone dexamethasone inhibits the induction of
IFN-
mRNA in normal human lymphocytes
(4, 5) ; in
addition, nuclear transcription of the IL-2 gene in human T cells has
been shown to be inhibited by the same
agent
(4, 5, 7, 8, 9, 10, 11) .
With regard to the IL-2 gene, these results suggested that inhibition
of transcription is the result of a negative interference with nuclear
transcriptional factors AP-1 and
NF-AT
(7, 8, 9, 10, 11) , which
have been demonstrated to be of crucial importance for the activity of
this cytokine promoter. Moreover, a functional antagonism that involves
direct protein-protein interactions between AP-1 and GR previously has
been reported by several investigators in different
systems
(12, 13, 14, 15) .
promoter. In this context, we also have
analyzed the relevance of the AP-1 complex binding factors during the T
cell activation by using two different inhibitors of the AP-1-mediated
transactivation, dominant negative mutants of the c-Jun proto-oncogene
(TAM-67 and c-Jun/1-286)
(16, 17) . Our data
indicate that the PMA/ionomycin-stimulated IFN-
promoter activity
is significantly down-regulated by dexamethasone after cotransfection
with a human GR expression vector. Additionally, AP-1
CREB-ATF
binding sequences present in the IFN-
promoter, at positions
previously demonstrated to be essential for the full promoter activity,
are involved in this inhibition. Moreover, dominant negative mutants of
the c-Jun proto-oncogene are able to mimic the inhibitory action
exerted by the GR on this promoter. These data strongly suggest a model
involving inhibition of the IFN-
AP-1
CREB-ATF complexes, as
one of the possible mechanisms of action for the GR-mediated negative
regulation on the IFN-
gene and support the relevance of AP-1 and
CREB-ATF binding factors on the activation of the IFN-
promoter in
T cells.
Cell Lines and Reagents
Jurkat cells
(CD4 human lymphoblastoid T cell line) were cultured
in complete RPMI 1640 medium, supplemented with 10% fetal calf serum, 2
mM glutamine, and 100 units/ml penicillin-streptomycin.
Antibodies against c-Jun (a rabbit affinity-purified polyclonal
antibody corresponding to the highly conserved residues 247-263
within the C-terminal DNA binding domain of the c-Jun protein) and
c-Fos (a mouse monoclonal antibody raised against a peptide
corresponding to c-Fos amino acid residues 128-152) transcription
factors were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). PMA was purchased from Sigma, and ionomycin was purchased from
Calbiochem (La Jolla, CA).
Nuclear Extraction
Nuclear proteins were prepared
as follows
(18) . The cellular pellet was resuspended in
10-20 times its volume in buffer A (lysis buffer): 50 mM
KCl, 0.5% Nonidet P-40, 25 mM Hepes buffer (pH 7.8), 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20
µg/ml aprotinin, 100 µM dithiothreitol and
subsequently incubated 4 min on ice. Cells were collected by
centrifugation at 2500 rpm, and the supernatant was decanted. The
nuclei were washed in buffer A without Nonidet P-40, collected at 2500
rpm, and resuspended in buffer B (extraction buffer): 500 mM
KCl, 25 mM Hepes (pH 7.8), 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM dithiothreitol for 5 min on ice. The
samples were subsequently frozen and thawed (twice) utilizing dry ice
and a 37 °C water bath, rotated 20 min at 4 °C, and centrifuged
at 14,000 rpm for 20 min. The clear supernatant was collected, and the
proteins were dialyzed for 2 h (4 °C) against buffer C (dialysis
buffer): 50 mM KCl, 25 mM Hepes (pH 7.8), 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 20 µg/ml aprotinin, and 100 µM
dithiothreitol. The amount of nuclear proteins obtained were quantified
utilizing a commercial reagent (BCA, Pierce).
Electrophoretic Mobility Shift Assay
The nuclear
proteins (5 µg) were incubated with radiolabeled DNA probes in a
20-µl reaction mixture containing 20 mM Tris (pH 7.5), 60
mM KCl, 2 mM EDTA, 0.5 mM dithiothreitol, 2
µg of poly(dI-dC), and 4% Ficoll. In some cases, the indicated
amount of double-stranded oligomer was added as a cold competitor, and
the mixture was incubated at room temperature for 10 min prior to
adding the DNA probe. Nucleoprotein complexes were resolved by
electrophoresis on 5% nondenaturing polyacrylamide gels in 0.5
Tris borate-EDTA buffer at 12 V/cm for 2 h at room temperature. Dried
gels were exposed to Kodak XAR-5 film (Eastman Kodak Co.) at -70
°C with intensifying screens. Oligonucleotides were synthesized by
the phosphoramitide method on a DNA/RNA synthesizer (Applied
Biosystems, model 394). Complementary strands were denaturated at 85
°C for 5 min and annealed at room temperature.
-
P]dCTP (Amersham
Corp.); approximately 1 ng of labeled DNA was used in a standard EMSA
reaction. In supershift analysis, the antisera were added to the
binding reaction, and the mixture was incubated for 30 min at room
temperature prior to adding the labeled DNA probe.
/-66 to -47,
5`-gatcTTGTGAAAATACGTAATCC-3`; IFN-
/-96 to -75,
5`-gatcGCCTATCTGTCACCATCTCATC-3`; IFN-
/-66 to -47
(
-mutant), 5`-gatcTTGTGAAAATcgcTAATCC-3`;
TRE-(h-Collagenase gene, -73 to -42),
5`-agctATGAGTCAGACACCTCTGGCTTTCTGGAAGG-3`; AP-1 (hIL-2prox.),
5`-agctGAAATTCCAAAGAGTCATCAGA-3`; OCT (hIL-2prox.),
5`-agctTAATATGTAAAACATT-3`; CREB(wt),
5`-AGAGATTGCCTGACGTCAGAGAGCTAG-3`; CREB(mut.),
5`-AGAGATTGCCTGtgGTCAGAGAGCTAG-3`.
Plasmid Constructions
The different deletions of
the IFN- promoter pIFN-538, pIFN-339, pIFN-108
(19) were
kindly provided by Dr. Christopher B. Wilson (Department of Pediatrics
and Immunology, University of Washington); human GR expression vector
pRShGR
(20) was kindly provided by Dr. R. M. Evans (The
Salk Institute, La Jolla, CA).
-tkCAT, three copies of the human IL-4 promoter NF(P)
binding site (nucleotides -88 to -64) were subcloned into
the HindIII-BamHI sites upstream to the thymidine
kinase (tk) promoter in the pBLCAT2 parental vector. The plasmids
3x(-66/-47)CAT, and 3x(-96/-75)CAT, contain
three copies of the regions IFN-
/-66 to -47 bp and
IFN-
/-96 to -75 bp, subcloned into the
HindIII-BamHI sites upstream to the thymidine kinase
promoter in the pBLCAT2 parental vector.
`CAT (containing the mutations for the two
AP-1
CREB-ATF binding sites) the appropriate DNA fragments were
synthesized and subcloned into the HindIII-BamHI
sites upstream to the thymidine kinase promoter in the pBLCAT2 parental
vector.
AP1-2xGRE-tk-CAT, containing two GREs
in front of the thymidine kinase promoter
(21) , was kindly
provided by Dr. A. Gulino (University ``La Sapienza'' Rome,
Italy).
-gal expression vector (Clontech Laboratories, Inc., Palo
Alto, CA) replacing the
-galactosidase gene.
-gal expression vector replacing the
-galactosidase gene.
DNA Transfections
Transfections of Jurkat cells
were carried out by the DEAE-dextran method
(24) . For each
treatment, 5 10
cells (harvested in log phase of
growth) were incubated with the indicated amounts of plasmid DNA in the
presence of 350 µg/ml DEAE-dextran in RPMI 1640, 50 mM
Tris-Cl (pH 7.5) for 70 min at 37 °C. To decrease variations in
transfection efficiency, cells were transfected in single batches,
which were then separated into different drug treatment groups, and
empty expression vector DNA was added as needed to maintain a constant
total DNA amount in each cotransfection series. Cells were then washed
with RPMI 1640, 50 mM Tris-Cl (pH 7.5) and replated in
duplicate, in complete medium. After 24 h, cells were treated with
different combinations of stimuli, and, after an additional 24 h, cells
were harvested and washed in phosphate-buffered saline. Protein
extracts were prepared for the
-galactosidase assay and CAT assay,
by 3 cycles of rapid freezing and thawing followed by centrifugation at
14,000 rpm (4 °C) for 15 min. Protein concentration was quantified
utilizing a commercial reagent (BCA, Pierce).
The -Galactosidase Assay
-galactosidase
assay was carried out according to the published procedure
(25) .
Enzyme activity was determined spectrophotometrically at 570 nm by the
hydrolysis of chlorophenol red
-D-galactopyranoside.
Duplicate
-galactosidase assays were normalized based on protein
amount loaded at each point, and generally had variations of less than
10%. Results are expressed as percent of activity, relative to the
control PMA/ionomycin-activable
-galactosidase expression
in each cotransfection series, without dexamethasone in the case of the
GR action, or cotransfected with the empty vector in the case of the
dominant negative mutants of c-Jun (TAM-67 and c-Jun/1-286).
CAT Assay
CAT assay was carried out according to
the published procedure
(26) by incubating different amounts of
cell lysate protein for 12 h at 37 °C so that the assay was within
the linear range. Acetylated and unacetylated
[C]chloramphenicol were separated by thin layer
chromatography and quantified by a radioactivity scanner (Ambis Inc.,
San Diego Ca.).
PMA/Ionomycin Activation of Different Deletions of the
IFN-
As for the IL-2 gene, dexamethasone has been shown to
inhibit the induction of IFN- Promoter Is Down-regulated by Dexamethasone in Jurkat
Cells
messenger RNA in T
cells
(4, 5) . Although a mechanism for the IL-2 gene
inhibition by dexamethasone has been reported
(9, 11) ,
it is not clear if the down-regulation of the IFN-
gene expression
is caused by a direct effect on the promoter activity or is mediated by
other mechanisms, such as regulation of RNA stability, etc. In order to
determine whether one of the possible mechanisms of dexamethasone
inhibition could be the direct interference with the transcriptional
activity of the human IFN-
promoter, we transiently cotransfected
Jurkat cells with different
-galactosidase vectors in which the
reporter gene transcription was directed by progressive deletions of
the human IFN-
promoter, together with an expression vector for
the human GR.
-galactosidase
activity driven by the promoter fragments -538 to +64
(pIFN-538) and the deletions -339 to +64 (pIFN-339) and
-108 to +64 (pIFN-108) were all significantly inhibited by
treatment with dexamethasone. These data indicate the sensitivity of
the promoter to GR/dexamethasone and demonstrate that GR-sensitive
element(s) were present in the promoter fragment -108 to +64
bp of the IFN-
gene. The inhibition was dependent on the presence
of the GR expression vector, since treatment with dexamethasone alone
was not able to exert any detectable effect on the IFN-
promoter
activity in the Jurkat cells used in these studies (data not shown). In
this context, as shown in Fig. 2A, a CAT reporter driven
by two copies of a GRE, was strongly activated after dexamethasone
treatment only in the presence of the cotransfected GR expression
vector. These experiments indicate that Jurkat cells used in these
studies were normally resistant to glucocorticoids and required
cotransfection of a GR expression vector for the dexamethasone-induced
inhibition of the IFN-
promoter. Similar observations have been
previously reported where the dexamethasone-induced inhibition of the
human IL-2 promoter was dependent upon the presence of a functional
cotransfected GR, in experiments utilizing murine T cell lines (BFS and
EL-4) and Jurkat cells
(7) .
Figure 1:
Effect of dexamethasone (Dex)
on different IFN- promoter deletions. 5
10
Jurkat T cells were cotransfected with 10 µg of the indicated
reporter gene vector plus 2 µg of GR expression vector as described
under ``Experimental Procedures.'' 24 h after transfection,
cells were stimulated with 10 ng/ml PMA and 1 µg/ml ionomycin in
the presence or in the absence of 1 µM dexamethasone.
After a further 24 h, cells were harvested, and protein extracts were
prepared for the
-galactosidase assay. The percentage of
activation, relative to the controls in the absence of dexamethasone
(considered as 100%), represents the mean value (X ±
S.E.) from at least four individual experiments.
-galactosidase
activities with PMA/ionomycin treatment for each construct were,
respectively, (units/µg of protein) 0.1
10
± 0.016 (pIFN-538), 0.068
10
± 0.009 (pIFN-340), 0.088
10
± 0.013 (pIFN-108).
Figure 2:
Effect of dexamethasone (Dex) on
the expression of AP1-2xGRE-tk-CAT (A) and pHIV-CAT
(B). 5
10
Jurkat T cells were
cotransfected with 10 µg of the indicated reporter gene vector plus
2 µg of GR expression vector as described under ``Experimental
Procedures.'' 24 h after transfection, cells were stimulated with
1 µM dexamethasone (A), or 10 ng/ml PMA and 1
µg/ml ionomycin in the presence or in the absence of 1
µM dexamethasone (B). After a further 24 h, cells
were harvested and protein extracts were prepared for the CAT
assay.
As a further control for the
specificity of GR/dexamethasone action on the PMA/ionomycin-mediated
gene activation, a CAT reporter driven by the human immunodeficiency
virus, type 1 long terminal repeat was used; as shown in
Fig. 2B, the PMA/ionomycin inducibility of this reporter
was not modified by the presence of the GR expression vector and
dexamethasone treatment. These data are in agreement with previous
observations by Vacca et al.(7) where the
PMA/ionomycin-mediated activation of different promoters (SV40 early
promoter, Rous sarcoma virus and human T cell lymphotrophic virus long
terminal repeats) was unaffected by GR/dexamethasone.
AP-1
Several recent reports have clearly
demonstrated that inhibition of the IL-2 promoter by GR in T cells
involves a functional impairment of the NF-AT and the proximal AP-1
binding sites
(9, 10, 11) . Moreover, AP-1
recently has been found to be a component of NF-AT
(27) , thus
indicating the AP-1 complex as the primary target for GR-mediated IL-2
promoter inhibition.
CREB-ATF Binding Elements Are Present in the
IFN-
Promoter
promoter, a sequence
homology search was done by comparison of the -538/+64
IFN-
fragment sequence, with the canonical consensus sequences for
AP-1 and CREB-ATF DNA binding factors (28). Based on this analysis
() and on the observation that a GR-mediated negative
response was identified at the level of the -108 to +64 bp
promoter fragment, two different noncanonical sequences were identified
to form specific AP-1
CREB-ATF-related DNA-bound protein complexes
in band-shift assays using nuclear extracts prepared from Jurkat cells
(Figs. 3-5) and fresh T cells (data not shown).
CREB-ATF DNA consensus sequences, were situated at positions
(-96 to -75) and (-66 to -47) (),
previously demonstrated by Penix and co-workers
(19) to be
critical for the full transcriptional activation of this promoter and
named ``distal'' and ``proximal'' essential
elements.
promoter). Three specific and constitutively expressed
DNA-protein(s) complexes were detected and designated here as complex
a, b, and c (lane1). A
slower migrating complex, designated here as AP-1, was induced
after the PMA/ionomycin stimulation (lane6) and
specifically competed either by a molar excess of a cold competitor
specific for CREB-ATF or by the proximal AP-1 site of the human IL-2
promoter (lanes8 and 10) but not by a
mutated version of the CREB-ATF oligonucleotide (lane9) or a nonrelated cold competitor specific for the SP-1
binding factor (data not shown). Interestingly, the complex a was partially competed by the cold oligonucleotide specific for
CREB-ATF (lanes3 and 8), while both the
mutant CREB-ATF oligonucleotide and the IL-2/AP-1 cold oligonucleotide
failed to compete in the same manner (lanes4 and
10). Complexes b and c were observed to be
specifically competed by a typical DNA binding sequence for the GATA
binding factors
(29, 30, 31) , present in the
human T cell receptor
-chain promoter
(31) (data not
shown).
Figure 3:
Electrophoretic mobility shift assay of
the distal IFN- promoter region -96 to -75 bp. EMSA
was performed using the indicated
P-labeled
oligonucleotide as a probe in the presence of nuclear extracts from
unstimulated or PMA/ionomycin-treated Jurkat cells. Lanes1-5, untreated cells; lanes6-10, 4 h of PMA/ionomycin
treatment.
A more complex binding pattern was observed with a
oligonucleotide encompassing the proximal DNA binding element of the
IFN- promoter (nucleotides -66 to -47) (Fig. 4)
(). Several specific DNA binding complexes were present
using nuclear extracts from unstimulated Jurkat cells in EMSA (lane1), and the stimulation with PMA/ionomycin was able to
induce the binding of other complex(es) over the basal DNA binding
activity (lane2). Interestingly, a cold
oligonucleotide specific for CREB-ATF binding factors was able to
totally compete all of these complexes (lane6), with
the exception of one designated here as complex 1. This
complex was recognized to contain the Oct-1 binding protein by specific
competition with a cold oligonucleotide encompassing the proximal
octamer binding site of the human IL-2 promoter (lane8) and by supershift assay using an antibody able to
specifically recognize only the Oct-1 DNA binding factor (data not
shown). When a cold competitor specific for the proximal AP-1 binding
site of the human IL-2 promoter was used in this EMSA, only the
PMA/ionomycin-induced complex(es) were specifically competed (lane5). These data suggest that the overlapping DNA binding
activity observed with stimulated Jurkat cell nuclear extracts was
caused by induction of AP-1 complex-related binding proteins
(designated here as complex 2), already known to be able to
bind with different affinities to the CREB-ATF DNA binding
sequences
(32, 33) .
Figure 4:
Electrophoretic mobility shift assay of
the proximal IFN- promoter region -66 to -47 bp. EMSA
was performed using the indicated
P-labeled
oligonucleotide as a probe in the presence of nuclear extracts from
unstimulated or PMA/ionomycin-treated Jurkat cells. Lane1, untreated cells; lanes2-9, 4
h of PMA/ionomycin treatment.
The constitutively present
complexes not competed by the AP-1 oligonucleotide were probably other
CREB-ATF-related DNA-binding proteins that have a more specific
affinity for this particular noncanonical CREB-ATF consensus sequence
than the AP-1 binding sequence.
promoter (nucleotides -96 to -75) was able to compete for
the overlapping PMA/ionomycin-induced AP-1 related binding but not the
constitutively present CREB-ATF-related DNA binding activity (lane9).
The IFN-
GR has been shown to inhibit the transcriptional activity
of AP-1 cis-acting elements by interfering with Jun and Fos proteins,
two normal components of the AP-1 complex. Since both the sequence
homology search and the EMSAs strongly indicated that AP-1-related
proteins were specific components of the induced complexes described
above, we wanted to determine whether Jun/Fos proteins were actually
present in these PMA/ionomycin-induced bands.
Promoter AP-1
CREB-ATF-like Motifs
Bind Complexes Containing Jun and Fos Proteins in Jurkat
Cells
promoter binding
elements
(28) . The position indicated here as AP-1 shows the induced complexes specifically competed by antibodies
for Jun and Fos, overlapping with other CREB-ATF family members in the
case of the proximal element of the IFN-
promoter (nucleotides
-66 to -47). A more detailed mutational analysis and
characterization of the proteins that form the CREB-ATF-related
complexes in this proximal regulatory element are the subject of a
separate report.
(
)
Cotransfection of Dominant Negative Mutants of c-Jun (TAM-67
and c-Jun/1-286) Is Able to Down-regulate the Activity of the
IFN-
Promoter-GR has been demonstrated to down-regulate a
number of different gene promoters by interaction with transcriptional
factors, including AP-1 family members, Rel-A, CREB family members,
Oct-2A, and
GATA-1
(12, 13, 14, 15, 34, 35, 36, 37) .
In addition, other mechanisms of GR-mediated transcriptional repression
include specific binding to different GR noncanonical sequences, as
negative GR response elements
(38) or interaction with GR
binding sites overlapping important regulatory elements such as the
TATA box region
(39, 40, 41) .
Figure 5:
Supershift analysis of the DNA-protein
complexes binding to the IFN- promoter regions -96 to
-47 and -66 to -47 bp. EMSA was performed using the
indicated
P-labeled oligonucleotides as probes in the
presence of nuclear extracts from PMA/ionomycin-treated Jurkat cells. 1
µg of purified anti-c-Jun, anti-c-Fos, or nonspecific antibody was
added to the reaction where indicated, as described under
``Experimental Procedures.'' Lanes14-15 and 17-18 contain the two different probes
indicated above, plus the antibody for c-Jun (14, 17) or c-Fos (15, 18)
without nuclear extracts as controls.
As a further
test of the hypothesis that the identified AP-1CREB-ATF complexes
play an important role during the activation of the IFN-
promoter
and are potential targets for GR negative regulation of the IFN-
transcription, we utilized two different dominant negative mutants of
the c-Jun proto-oncogene (TAM-67 and c-Jun/1-286), potent
inhibitors of AP-1 mediated transactivation, in cotransfection
experiments
(16, 17, 23) . In particular, TAM-67
already has been shown to selectively inhibit the IL-2 promoter by
directly interfering with the NF-AT complex activity in Jurkat T cells,
but not with other DNA binding regions including the NF-IL2A or the
NF-
B sites
(42) . Cotransfection assays, in the presence of
the different IFN-
promoter
-galactosidase reporters
described above showed that both TAM-67 and c-Jun/1-286 were able
to mimic the same kind of suppression observed in Jurkat cells by
cotransfection of the GR expression vector followed by dexamethasone
stimulation (Fig. 6), with all the progressive deletions
significantly inhibited. These data indicate that selective impairment
of the c-Jun-mediated transactivation is able to inhibit the activation
triggered by PMA/ionomycin stimulation of the IFN-
promoter.
Figure 6:
5 10
Jurkat T cells
were cotransfected with 10 µg of the indicated reporter gene vector
plus 5 µg of Tam-67 expression vector or c-Jun/1-286
expression vector as described under ``Experimental
Procedures.`` In the case of the control samples, the same amount
of empty expression vector DNA was added. Cells were treated 24 h later
with 10 ng/ml PMA and 1 µg/ml ionomycin, and protein extracts were
prepared for the
-galactosidase assay as described before. The
percentage of activation, relative to the controls transfected with
empty vector (considered as 100%), represents the mean value (X ± S.E.) from at least four individual experiments.
-galactosidase activities with PMA/ionomycin treatment for each
construct were, respectively (units/µg of protein), 0.085
10
± 0.009 (pIFN-538), 0.064
10
± 0.007 (pIFN-340), 0.084
10
± 0.007
(pIFN-108).
In
order to test the specificity of these two dominant negative mutants,
we cotransfected (using a CAT-reporter/dominant negative vector ratio
of 1:1) TAM-67 or c-Jun/1-286 together with a CAT vector in which
the reporter gene transcription was directed by three copies of the
human NF(P) regulatory element, present in the promoter of the IL-4
gene
(43) . The PMA/ionomycin induction of this reporter was not
significantly inhibited by TAM-67 and c-Jun/1-286 expression
(data not shown), indicating the selective specificity for the AP-1
inhibition of these vectors. The Proximal and Distal AP-1CREB-ATF Binding Sites in the
-108-bp Fragment of the IFN-
Promoter Are Responsive to the
Inhibitory Action of GR/Dexamethasone-In order to better
characterize the mechanism by which GR/dexamethasone are able to
negatively interfere with the IFN-
promoter-activation process, we
studied in cotransfection assay the behavior of multimers of the two
identified AP-1
CREB-ATF binding sites in the presence of the GR
expression vector and different stimulations. As shown in
Fig. 7A, both the CAT reporters studied,
3x(-66/-47)CAT and 3x(-96/-75)CAT, were
responsive to the PMA/ionomycin treatment in Jurkat cells, and
dexamethasone was able to significantly inhibit this activity, while
the normal basal induction of the parental pBL2CAT vector was
unchanged.
Figure 7:
A, effect of dexamethasone
treatment on the thymidine kinase promoter-CAT activity driven by
multimers of the IFN- proximal and distal elements. 5
10
Jurkat T cells were cotransfected with 10 µg of the
indicated reporter gene vector plus 2 µg of GR expression vector as
described under ``Experimental Procedures.'' 24 h after
transfection, cells were stimulated with 10 ng/ml PMA and 1 µg/ml
ionomycin in the presence or in the absence of 1 µM
dexamethasone. After a further 24 h, cells were harvested, and protein
extracts were prepared for the CAT assay. Results are expressed as the
average (X ± S.E. of at least three individual
experiments) -fold induction of the CAT activity measured in
PMA/ionomycin-treated samples versus untreated, in the absence
(Control) or in the presence (+Dex) of 1
µM dexamethasone. B, sequence of the
oligonucleotides used as multimers in the vectors
3x(-66/-47)CAT and
3x(-96/-75)CAT.
The essential role of these two AP-1CREB-ATF
binding sites during the normal activation process, and the correlation
with the negative action exerted by GR/dexamethasone on the IFN-
promoter, was further investigated by using mutations able to
selectively abolish the binding of these complexes on the promoter. The
mutation able to eliminate the binding activity of the distal
AP-1
CREB-ATF element, present in the promoter segment from
nucleotides -96 to -75 (named here as
`) has been
already described by Penix et al.(19) (Fig. 8B). The mutation able to block the
binding of AP-1
CREB-ATF binding proteins, at the level of the
proximal site present in the promoter segment from nucleotides
-66 to -47, is shown in Fig. 8B and 9. Only
a single DNA-binding complex, designated as 1 and
corresponding to the transcriptional factor Oct-1 described above, was
able to bind this mutated sequence (named here as
) in EMSA, both
in unstimulated and PMA/ionomycin-stimulated Jurkat cells nuclear
extracts (Fig. 9).
Figure 8:
A, mutations of the IFN- proximal and
distal elements are able to abolish the transactivating capability of
the promoter fragment (-108 to -36) and dexamethasone
(Dex) induced negative regulation. Results are expressed as
the average (X ± S.E. of at least three individual
experiments) -fold induction of the CAT activity measured in
PMA/ionomycin-treated samples versus untreated, in the absence
(Control) or in the presence (+Dex) of 1
µM dexamethasone. B, mutations introduced in the
(-108-36)
`CAT vector.
Figure 9:
EMSA was performed using the wild-type
IFN- proximal region (-66 to -47 bp) or the
-mutant oligonucleotides as labeled probes, in the presence of
nuclear extracts from unstimulated or PMA/ionomycin-treated Jurkat
cells. Lanes1 and 3, untreated cells;
lanes2 and 4, 4 h of PMA/ionomycin
treatment.
The simultaneous site mutation, or
deletion, of these two binding elements is able to dramatically reduce
the -galactosidase activity of the IFN-
promoter reporters
described above to levels not suitable for studying negative
regulations in transfection assays (data not shown and Ref. 19). Thus
the wild-type IFN-
promoter region encompassing the two essential
regulatory elements (nucleotides -108 to -36), and the
double mutant for these elements, were subcloned into the
HindIII-BamHI sites upstream to the thymidine kinase
promoter in the pBLCAT2 parental vector.
CREB-ATF binding elements
in the(-108-36)
`CAT reporter, strongly reduced the
activation level after PMA/ionomycin, and the treatment with
dexamethasone was not able to modulate the residual CAT activity driven
by the mutated promoter fragment and the thymidine kinase promoter of
the parental vector.
CREB-ATF binding sites identified in the first 108 bp
of the IFN-
promoter, function normally as enhancers during the
activation process through a mechanism mediated by the cooperation of
AP-1
CREB-ATF proteins and represent sensitive elements for the
GR/dexamethasone-induced down-regulation of the promoter.
Th1 cell lines, include
induction of immediate early genes including c-jun,
c-fos, CD25 and induction of cytokine genes such as
interleukin-2 and IFN-
(44, 45). With regard to the IFN-
gene, the molecular mechanisms involved in its transcriptional
activation have been only partially characterized. Along with other
investigators, our laboratory has reported the presence of positive and
negative cis-acting promoter elements in a region 500 bp 5` to the
transcription start
site
(1, 46, 47, 48, 49, 50) .
Moreover, recent results obtained using reporter vectors driven by the
first 538 or 108 bp of the IFN-
promoter appeared to faithfully
mirror the endogenous gene's requirements for specific induction
by exogenous stimuli and suppression by cyclosporin A
(19) . Two
essential highly conserved elements within the first 108 bp of the
transcription initiation site in the IFN-
genomic DNA have been
demonstrated to sufficiently confer activation-specific promoter
function in T cells
(19) , and recently our laboratory also has
shown that the proximal region spanning nucleotides -71 to
-49 bp of the IFN-
promoter appears to be differently
methylated in murine CD4
TH1 and TH2 T cell clones,
and this difference correlates with the transcriptional activity of the
gene
(51) .
promoter activity by the glucocorticoid hormone
dexamethasone. To address the molecular mechanism involved in this
transcriptional repression, we have investigated the relevance of
AP-1
CREB-ATF DNA binding complexes in this inhibition and in the
overall regulation of IFN-
gene transcription.
B, NF-AT,
NF-IL-2A
(44) . In particular, the AP-1 complex, although
directly involved at a functionally relevant AP-1 site in the IL-2
promoter
(57) , also participates in the formation of the NF-AT
and NF-IL-2A regulatory
elements
(28, 58, 59, 60) . Moreover the
AP-1 binding complex also may be a target for T cell clonal
anergy
(61) , and, in different experimental systems, its
activity can be strongly inhibited by direct interference with hormone
receptors such as the GR or retinoid
receptors
(7, 8, 9, 10, 11, 12, 13, 14, 15, 60, 62) .
promoter contains different
AP-1
CREB-ATF binding elements, in positions previously shown to
be critical for its full transcriptional activity. Noteworthy, at the
position corresponding to the proximal regulatory element (nucleotides
from -66 to -47), we detected in EMSA the simultaneous
presence of a basal CREB-ATF-related DNA binding activity and an
inducible AP-1-related binding activity ( Fig. 4and
Fig. 5
), thus suggesting a further level of possible
interaction/regulation between these two families of DNA-binding
proteins in this context.
CREB-ATF binding sequences in the promoter and highlights
the relevance of these binding complexes for the transcriptional
regulation of this cytokine.
CREB-ATF binding
complexes of the IFN-
promoter. In support of this model, specific
mutation of these binding elements correlated with a significant
inhibition of the promoter activation and with the loss of the
glucocorticoid-mediated down-regulation. A similar model has also been
proposed for the IL-2 gene promoter, where the GR interferes with the
activity of NF-AT and proximal AP-1 binding sites, two regulatory
elements shown to be necessary for the transcriptional activation of
this gene
(9) .
AP-1
CREB-ATF complexes
might be involved
(65) , since this deletion mutant is not able
to bind DNA or to form dimers with wild-type Jun and Fos proteins via
the leucine zipper domain. The fact that both of the mutants of c-Jun
were effective in suppressing the IFN-
promoter transcriptional
activity underlines the relevance of the dimerization of c-Jun with Fos
and/or CREB-ATF family proteins (i.e. a quenching mechanism
proposed for TAM-67), and suggests the requirement of ``accessory
factors'' that do not bind directly to the DNA during the
activation process (i.e. a squelching mechanism proposed for
c-Jun/1-286)
(16, 17, 23, 65) .
gene, and demonstrate the
sensitivity of the IFN-
promoter to the suppressive action caused
by glucocorticoid hormone treatment during T cell activation.
Table:
Sequence homology between IFN-
AP-1
CREB-ATF binding sites and canonical consensus sequences for
AP-1 and CREB-ATF
, Dr. Alberto Gulino for providing the
AP1-2xGRE-tk-CAT vector, Dr. John R. Ortaldo and Dr. Kathrin
Muegge for critical comments regarding this manuscript, and Joyce
Vincent for editorial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.