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Participates in the Activation of Cyclic AMP-Responsive Element-Binding Protein and Its Subsequent Binding to the -180 Site of the IL-2 Promoter in Normal Human T Lymphocytes1
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20814
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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phosphorylates CREB, which subsequently binds to the
-180 site and associates with the transcriptional coactivator p300.
Rottlerin, a specific PKC- inhibitor, diminished p-CREB protein
levels when normal T cells were treated with it. Rottlerin also
prevented the formation of p-CREB/p300 complexes and the DNA-CREB
protein binding. Cotransfection of fresh normal T cells with luciferase
reporter construct driven by two tandem -180 sites and a PKC-
construct caused a significant increase in the transcription of the
reporter gene, indicating that this site is functional and regulated by
PKC-. Cotransfection of T cells with a luciferase construct driven
by the -575/+57 region of the IL-2 promoter/enhancer and a PKC-
construct caused a similar increase in the reporter gene transcription,
which was significantly limited when two bases within the -180 site
were mutated. These findings show that CREB plays a major role in the
transcriptional regulation of IL-2 and that a major pathway for the
activation of CREB and its subsequent binding to the IL-2
promoter/enhancer in normal T cells is mediated by
PKC-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, NF-AT, and Oct (2, 3). The -180 site
(-164–189 bp) of the IL-2 promoter/enhancer had been proposed to be a
distal AP-1 binding site (4), but recently, it was
reported that in anergic cells this site binds heterodimers of
cAMP-responsive element-binding protein/cAMP-responsive element
modulator (CREB/CREM)3
and not AP-1 (5). CREB, CREM, inducible cAMP early repressor (ICER), and activating transcription factor-1 are members of the cAMP-responsive NFs and exhibit a high degree of sequence homology. One common feature is the basic domain/leucine zipper motifs, which bind an 8-bp regulatory palindromic DNA sequence (cAMP-responsive elements). These NFs are activated following phosphorylation by several kinases in response to different signaling routes, including protein kinase A (PKA), protein kinase C (PKC), ribosomal S6 kinase pp90rsk, mitogen- and stress-activated kinase, mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2), and Ca2+/calmodulin-dependent kinase IV. Phosphorylation of Ser133 in CREB and Ser117 in CREM acts as a molecular switch, because it dictates the ability of these factors to interact with the ubiquitously expressed coactivators CREB-binding protein (CBP) and p300 that form a bridge with the basal transcriptional machinery (6, 7, 8).
CREB and CREM consist of the transcriptional activation domain (Q1, P-box, Q2) and the DNA-binding/dimerization domain (bZip region). Both the CREB and CREM genes encode multiple isoforms. CREB isoforms act as transcription activators, whereas CREM isoforms can act either as activators or repressors. CREM isoforms containing only the P-box or the Q2 domain act as repressors. ICER is produced by alternative promoter usage within the CREM gene and acts only as a repressor (6, 7, 8).
Stimulation of T cells through the TCR leads to immediate
phosphorylation and activation of several cytoplasmic protein tyrosine
kinases. Subsequently, phospholipase C is activated and hydrolyzes
inositol phospholipids into inositol polyphosphates and diacylglycerol
(DAG). Inositol polyphosphate leads to elevation of intracellular
Ca2+, which acts synergistically with DAG to
activate multiple kinases, including PKC (9, 10). PKC
isoforms are serine/threonine-specific protein kinases that can be
divided into three subclasses (11): the conventional PKCs
(PKC-
, PKC-
I and II, PKC-
) are activated by DAG,
phosphatidylserine, and Ca2+; the novel PKCs
(PKC-
, PKC-
, PKC-, PKC-
, and PKC-µ), which are activated
by DAG and phosphatidylserine, but not Ca2+; and
the atypical PKCs (PKC-
, PKC-i, and PKC-
), which only
respond to phosphatidylserine.
Expression patterns and differences in their potential substrate
specificity suggest that each isoenzyme may be involved in specific
regulatory processes (12, 13, 14). High protein levels of
PKC- are present in muscle cells, hemopoietic cells, and T but not B
lymphocytes (15, 16). Recent studies show that PKC-
(17) is the only isoform to translocate to the site of
contact between T cells and APCs (11), which occurs only
upon exposure to Ag. Furthermore, it has been shown that AP-1
(18) and NF-B (19, 20) are activated
through PKC- and subsequently bind to the IL-2 promoter/enhancer
(21).
Mice expressing a dominant-negative form of CREB have defective
thymocyte proliferation and IL-2 production (22),
suggesting that CREB is crucial in the transcription of the IL-2 gene.
In this study, we investigated whether CREB is important in the
transcription of the IL-2 gene in normal human T cells. Our studies
show that the -180 site of the IL-2 promoter/enhancer binds p-CREB,
and that PKC- is involved in its phosphorylation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heparinized peripheral venous blood was obtained from the study subjects. PBMC were separated from RBC on Lymphoprep gradient (Nycomed Pharma, Oslo, Norway), and T cells were separated subsequently by magnetic depletion of non-T cells, as recommended by the manufacturer (MACS Pan T cell isolation kit; Miltenyi Biotec, Auburn, CA). Briefly, non-T cells (B cells, monocytes, NK cells, dendritic cells, early erythroid cells, platelets, and basophils) from PBMC were indirectly magnetically labeled using a cocktail of hapten-conjugated CD11b, CD16, CD19, CD36, and CD56 Abs, and MACS microbeads coupled to an anti-hapten mAb. The magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of MidiMACS. The purified T cells were >95% positive for CD3, as tested using flow cytometry. Where mentioned, stimulation of T cells was performed using anti-CD3 (OKT3) (10 µg/ml) and anti-CD28 (2.5 µg/ml) Abs, or 10 ng/ml PMA and 0.5 µg/ml ionomycin.
Antibodies
Anti-phospho-CREB (rabbit polyclonal IgG), anti-p300 (rabbit polyclonal IgG), and murine anti-human CREB-binding protein (rabbit polyclonal IgG, CBP-NT) Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-CREM (rabbit polyclonal IgG), anti-CREB (rabbit monoclonal IgG), anti-actin (goat polyclonal IgG), as well as the goat anti-rabbit and goat anti-mouse HRP-conjugated mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Inhibitors
Calphostin C (0.05 µM) and rottlerin (30 µM) were used for
the inhibition of PKC and PKC-, respectively (20, 23, 24, 25). KT5720 (5 µM) was used for the inhibition of PKA
pathway (26); SB 203580 (10 µM) and PD 98059 (50 µM)
for the inhibition of MAPKAP kinase-2 and mitogen-activated
protein/extracellular signal-regulated kinase kinase (MEK),
respectively (27, 28); and W7 (15 µM) for the inhibition
of calmodulin (29). All inhibitors were purchased from
Calbiochem (La Jolla, CA). Where mentioned, freshly isolated normal T
cells were incubated with the above concentrations of the inhibitors
for 30 min at 37°C (95% O2, 5%
CO2), followed by stimulation with PMA alone, PMA
and ionomycin, or anti-CD3 and anti-CD28 Abs for 6
h.
Preparation of nuclear extracts
At least five million T cells were used for preparation of extracts for each experimental point. T cells following treatment with the appropriate stimulus were washed twice in PBS and resuspended in 300 µl buffer A (10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) with mixture of protease inhibitors (1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 mM NaF, and 1 mM Na3VO4) at 4°C. Cells were incubated on ice for 15 min, and after adding 25 µl of 10% Nonidet P-40 were vigorously vortexed for 10 s and centrifuged to homogenate at 13,000 rpm at 4°C for 30 s. The supernatant cytoplasmic extract was transferred in a new tube, and the nuclear pellet was resuspended in 30 µl buffer B (20 mM HEPES-KOH (pH7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA) with protease inhibitors (1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin). The resuspended nuclear pellet was incubated, rocking for 15 min at 4°C, and then was spun at 13,000 rpm in a microcentrifuge to remove insoluble material. The extracts were frozen at -80°C until they were assayed. The protein content of the extracts was determined using the Bio-Rad (Hercules, CA) protein assay.
EMSAs
The dsDNA probe of the -180 site on the IL-2 promoter used was 5'-catccattcagtcagtctttgggggt-3'. The M1 and M2 oligonucleotides represent the -180 site dsDNA probes in which a base pair was mutated: M1, 5'- catccataaagtcagtctttgggggt-3', and M2, 5'-catccattcagtcaccctttgggggt-3'. GATA dsDNA probe was used as control in the cold-inhibition experiments: 5'-attcttatctaattcctatcttgattgg-3'. The probes were synthesized by Life Technologies (Grant Island, NY). Nuclear extracts (2 µg) were incubated with 30,000 cpm end-labeled probe, 4 µl buffer (Hi-Density 5x Tris-borate-EDTA sample buffer; Novex, San Diego, CA), 1 µl KCl 1 M, and 1 µg of poly(dG)·poly(dC) (Sigma, St. Louis, MO) as nonspecific competitor for 30 min at room temperature in total volume 20 µl. Gel electrophoresis was then run on 6% DNA retardation gels (Novex) in 0.5x Tris-borate-EDTA buffer. The gel was then dried under vacuum on blotting paper, and the protein-DNA complexes were visualized using phosphor imager (Bio-Rad).
The comparison concerning band density was made for each individual gel, and quantitation of each of the bands was determined using the QuantityOne software (Bio-Rad). In all the assays performed, the background was determined for each individual lane and subtracted from the band density.
Supershift analysis
Nuclear and cytoplasmic extracts were preincubated with 3 µg of the indicated Abs for 15 min at room temperature before the addition of the labeled probe. Supershift analysis was then completed as described above.
Immunoblotting and immunoprecipitation
Nuclear proteins (10 µg/lane) were resolved by 10% Tris-glycine SDS gel (Novex) electrophoresis at 125 V. Resolved proteins from the gel were transferred on Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA; Sigma) in transfer buffer at 14 V overnight at 4°C. The membrane was blocked for 2 h with 5% BSA in PBS and 0.05% Tween 20 and incubated in primary Ab. For the detection of CREB, the blot was first incubated with anti-p-CREB Ab for 2 h at room temperature. The membrane was then washed for 15 min in PBS, by changing the buffer every 5 min, and subsequently was incubated with anti-rabbit Ab HRP conjugated. Secondary HRP-conjugated Ab incubation was performed at 1/1000 dilution at room temperature for 1 h. Following the washing of the membrane with PBS and 0.05% Tween 20 for 1 h by changing the buffer four times and PBS for another 15 min, detection of the bands of interest was performed with the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Membranes were stripped after the first blotting in ImmunoPure IgG elution buffer (Pierce, Rockford, IL) for 1 h shaking at room temperature, reblocked, and reblotted with anti-p-CREB Ab, as described above, to detect the p-CREB protein levels. To evaluate equal loading of the lanes with protein, membranes were restripped, reblocked, and reblotted with anti-actin goat polyclonal Ab.
To immunoprecipitate (30) p300, 50 µg of nuclear or cytoplasmic protein extract, obtained as described earlier, was incubated with 2 µg of p300 polyclonal Ab at 4°C shaking for 1 h. A total of 25 µl of 50% slurry of protein A/G plus agarose (Santa Cruz) was added to capture immune complexes and incubated for 1 h at 4°C on a rotator. Agarose-bound immune complexes were collected, washed four times with lysis buffer (20 mM HEPES-KOH (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA) with protease inhibitors (1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin), and the pellet was boiled for 3 min with 50 µl Laemmli sample buffer to dissociate the agarose beads from the immune complexes. Agarose was discarded, and the supernatants containing the immune complexes were brought to a final concentration of 5% with 2-ME. Electrophoretic protein fractionation of equal sample volumes (25 µl of sample per lane) on 10% Tris-glycine SDS gels (Novex) was followed by transfer of the membranes on Immobilon polyvinylidene difluoride membrane (Millipore; Sigma) and immunoblotting with rabbit polyclonal anti-p-CREB Ab. Detection was performed as described above. Densitometric measurements were performed using QuantityOne software (Bio-Rad).
Transfection and luciferase assays
Freshly isolated normal T cells were left overnight in medium
containing 10% FCS and PHA (1 µg/ml) at 37°C. We used PHA because
we have found that it permits maximal transfection efficacy compared
with cells transfected in the absence of PHA. However, our transfection
experiments described in this work were performed without PHA, and the
patterns of luciferase activity were similar (31, 32).
Luciferase reporter plasmid (pGL2; Promega,
Madison, WI) driven by either two -180 sites on the IL-2 promoter, or
the whole IL-2 promoter freed from pIL2 CAT (kind
gift of Dr. A. Rao, Harvard University, Cambridge, MA), plasmids
expressing constitutively active PKC-, , and isoforms (kind
gift of Dr. G. Baier (University of Innsbruck, Innsbruck, Austria), Dr.
A. Altman (La Jolla Institute of Allergy and Immunology, San Diego,
CA), and Dr. W. C. Greene (University of California, San
Francisco, CA), and plasmid expressing CREM (kind gift of Dr. P.
Sassone-Corsi (Centre National de la Recherche Scientifique,
Strasbourg, France)) were used for transfection experiments.
Normal T cells (5 x 106) were transiently
transfected by electroporation (Gene pulser II; Bio-Rad) at 250 mV, 950
µF in 0.25 ml of complete medium (RPMI, 10% FCS). The total amount
of plasmids used in each sample was 8 µg. After 18 h, T cells
were stimulated with PMA and ionomycin for 6 h, as we have found
in preliminary experiments to be the optimal time for greater
luciferase activity. Cytoplasmic extracts were prepared using a
luciferase assay kit (Promega). Briefly, cells were resuspended in
lysis solution (Tropix, Bedford, MA) with DTT (0.01 M) and incubated at
room temperature for 15 min. After a brief centrifugation, 30 µl of
the supernatant was used with 100 µl of luciferase assay reagent.
Luminescence was measured immediately for 30 s using a
luminometer. Transfection efficiency was established in all samples by
cotransfection of a plasmid encoding -galactosidase (2 µg for each
sample). The luciferase activity was normalized using the
-galactosidase readings. Jurkat cells (5 x
106 for each sample) were transfected with the
same constructs as described above. After transfection, Jurkat cells
were rested overnight, followed by stimulation with PMA and ionomycin
for 6 h. Cells were collected, and luciferase activity was
examined, as described earlier.
Data analysis
Analysis of the OD of the CREB/CREM band was performed using QuantityOne software (Bio-Rad) after background subtraction from each band. Data were evaluated for statistical significance by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The site on the IL-2 promoter/enhancer that lies between NF-B
and CD28 response element binding sites (-164 to -189 bp, known as
-180 site) has been proposed to be a distal AP-1 binding site because
of the homology that shares with the consensus AP-1 sequence;
nevertheless, this binding was never clearly shown (4).
PKC- has been shown to be involved in the activation of
transcription factors that bind to the -150 (proximal AP-1 binding
site) (18, 33) and -200 (NF-B binding site) binding
sites (19, 20). To understand the role of the
transcription factor that binds to the -180 site on the IL-2
promoter/enhancer in normal human T cells in the transcriptional
regulation of the IL-2 gene, and determine whether PKC- is involved
in the activation of this transcription factor, we studied the
DNA-protein interactions using nuclear extracts from freshly isolated
normal T cells and an oligonucleotide that spans from -164 to -189 bp
(-180 site) on the human IL-2 promoter/enhancer.
The -180 site is located on a minor groove of the DNA
(5), and this may have been the reason that no definite
binding had been previously detected by using poly(dI · dC) as
nonspecific competitor in shift assay experiments (4, 38).
We first examined the binding of nuclear extracts from normal T cells
stimulated with PMA for 6 h to the -180 site oligonucleotide
incubated either with poly(dG)·poly(dC) or poly(dI · dC) as
nonspecific competitor. The binding observed when using
poly(dG)·poly(dC) could not be detected when using poly(dI · dC)
(Fig. 1
). Therefore, all subsequent shift
assay experiments were performed with poly(dG)·poly(dC) as
nonspecific competitor.
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and 3),
whereas nuclear extracts from stimulated T cells bound to it.
Specifically, nuclear extracts from T cells stimulated with
anti-CD3 and anti-CD28 Abs displayed significant binding that
reached maximal values at 6 h and decreased thereafter (Fig. 2A). Similar results were obtained when we stimulated the T
cells with PMA and ionomycin, to bypass membrane-mediated signaling
events (Fig. 2B). This band decreased by 80% in the
presence of 10-fold and disappeared in the presence of 100-fold molar
excess of unlabeled oligonucleotide, indicating that the binding was
specific (Fig. 2C).
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E)
and after 6 h when T cells were stimulated with PMA (Fig. 2F) or forskolin (not shown) alone. As above, the presence
of 100-fold molar excess of unlabeled oligonucleotide or the presence
of anti-p-CREB Ab diminished the binding (data not shown).
To determine the composition of the shifted bands, we used Abs directed
against p-CREB, p-CREM, AP-1, and Ets transcription factor-1. The
binding of nuclear proteins from stimulated T cells to the -180
oligonucleotide diminished by 90% in the presence of anti-p-CREB
Ab and only by 10% in the presence of anti-p-CREM Ab (Figs. 2D, 3B, and 3D). The presence of
anti-jun or anti-fos Ab in the shift
assay reaction failed to change significantly the intensity of the
band. Specifically, the mean intensity (OD) of the band using nuclear
extracts from cells stimulated with PMA or PMA and ionomycin was 169
and 184, respectively. The mean intensity of the band in the presence
of anti-jun Ab was 136, p = 0.03,
whereas the mean intensity of the band in the presence of
anti-fos Ab was 145, p = 0.185 (Fig. 3, B and D). Similarly, the presence of anti-Elf
Ab (control) did not have any effect on this binding (Fig. 3B). These data indicate that the -180 site of the IL-2
promoter/enhancer represents a p-CREB binding site. The specificity of
this binding was also examined using excess unlabeled GATA, M1, and M2
oligonucleotides. The presence of excess unlabeled GATA, M1, or M2
oligonucleotide, unlike the excess -180 oligonucleotide, failed to
change significantly the intensity of the binding (Fig. 3A,
compare to Fig. 2C). Moreover, when we labeled the M2 probe
and used it in shift assay experiments using nuclear extracts from T
cells stimulated with PMA, the detected binding was comparable with the
binding of nuclear extracts from unstimulated T lymphocytes to the
-180 oligonucleotide (Fig. 3C). These results suggest that
the -180 site of the IL-2 promoter represents a p-CREB binding
site.
PKC- is involved in the phosphorylation of CREB and its
subsequent binding to the -180 site of the IL-2 promoter
To examine whether PKC- participates in CREB activation and
leads to IL-2 promoter/enhancer binding, normal T cells were incubated
with specific PKC and PKC- inhibitors before stimulation with PMA,
or PMA and ionomycin, as described in Materials and Methods,
and nuclear proteins were examined in immunoblotting and shift assay
experiments. It has been proposed that MEK and MAPKAP-K2 lie downstream
of PKC in CREB activation (34). To examine also the role
of these kinases in CREB activation in normal T cells, we used nuclear
extracts from T cells that were treated with SB203580 or PD98059,
specific MAPKAP-K2 and MEK inhibitors, respectively, prior to
stimulation with PMA or PMA and ionomycin. KT5720 was used as a
specific PKA inhibitor and W7 as a calcium/calmodulin inhibitor.
First, we performed immunoblot analysis of nuclear extracts from
unstimulated and PMA- and ionomycin-stimulated T cells treated with or
without inhibitors prior to stimulation. p-CREB protein levels were
abundant in nuclear extracts from stimulated normal T cells, but not in
nuclear extracts from unstimulated T cells (Fig. 4). The presence of rottlerin or
calphostin C decreased the p-CREB protein levels by 60% (Fig. 4A) and 90% (Fig. 4B), respectively. The
presence of PD98059, W7, KT5720, or SB203580 did not affect
significantly the p-CREB protein levels (Fig. 4B). Total
CREB protein levels were not affected in the presence of these
inhibitors, indicating that they only inhibited CREB activation. This
experiment suggested that CREB activation involves PKC-, which does
not require Ca2+ to be activated (9, 10). Subsequently, we determined whether p-CREB protein levels
are affected when normal T cells are stimulated only with PMA in the
presence of the same inhibitors. As expected, total CREB protein levels
were not affected, but the p-CREB protein levels were diminished by
95% in the presence of calphostin C and by 90% in the presence of
rottlerin (Fig. 5). The presence of
SB203580, PD98059, KT5720, or W7 did not affect the CREB protein levels
and did not decrease significantly the p-CREB protein levels (Fig. 5).
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A). Similar results were
obtained when normal T cells were stimulated with anti-CD3 and
anti-CD28 Abs (data not shown). In nuclear extracts from T cells
that were treated with PD98059 or SB203580 before stimulation, p-CREB
binding decreased by 25% (Fig. 6A). p-CREB binding of
nuclear extracts from normal T cells treated with forskolin in the
presence of KT5720, a specific PKA inhibitor, decreased by 50% (data
not shown). Similar results were obtained from cells treated with
KT5720 before stimulation with PMA and ionomycin (Fig. 6A).
It has been proposed that calcium/calmodulin kinase IV can
phosphorylate and therefore activate CREB. When normal T cells were
treated with W7, a calcium/calmodulin inhibitor, before stimulation
with PMA and ionomycin, p-CREB binding to the -180 oligonucleotide
decreased by 25% (Fig. 6A). We treated T cells with the
same inhibitors and then stimulated them with anti-CD3 and
anti-CD28 Abs. EMSA and subsequent densitometric analysis revealed
inhibitory patterns similar to those obtained when T cells were
stimulated with PMA and ionomycin (data not shown). Finally, we
examined p-CREB binding to the -180 oligonucleotide using nuclear
extracts from normal T cells that were treated with PMA alone and with
the same inhibitors. W7, SB203580, and PD98059 did not affect p-CREB
binding, whereas calphostin C prevented the binding to the -180
oligonucleotide and rottlerin decreased this binding by 70% (Fig. 6B). The results described above were generated with doses
that had been found in preliminary experiments to be optimal for each
inhibitor.
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isoform play a
critical role. Rottlerin prevents the formation of p300/p-CREB complexes
CBP and p300 are known to bind p-CREB, resulting in the formation
of heteromeric activator complexes that contribute to efficient
initiation of transcription. As shown above, PKC- has a central role
in CREB phosphorylation. To examine the role of PKC- in
p-CREB/CBP-p300 complex formation, we performed immunoprecipitation
experiments in stimulated T cells treated with or without rottlerin.
First, we immunoprecipitated nuclear extracts from unstimulated T cells
with anti-p300 and anti-CBP Abs, but we did not detect any
p-CREB/p300 complexes, as expected. In contrast, these complexes were
abundant in immunoprecipitates of nuclear extracts from T cells
stimulated with PMA and ionomycin (Fig. 7). The presence of rottlerin (Fig. 7, Table I) or calphostin C (data not shown)
inhibited completely the formation of p-CREB/p300 complexes. Similarly,
rottlerin and calphostin C inhibited the formation of p-CREB/p300
complexes in nuclear extracts from T cells stimulated with anti-CD3
and anti-D28 Abs (data not shown). When T cells were treated with
PD98059, SB203580, W7, or KT5720 before stimulation with PMA and
ionomycin, or with anti-CD3 and anti-CD28 Abs, the p-CREB/p300
complexes could be detected, but densitometric analysis revealed that
diminished by 
20% (data not shown). Thus, PKC- plays an
important role in CREB phosphorylation and activation and the
subsequent formation of p-CREB-CBP/p300 complexes that can affect
transcription.
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enhances the
-180 site-driven reporter activity
To prove that the -180 site of the IL-2 promoter is functional
and PKC- regulated in normal human T cells, we transiently
transfected fresh normal human T cells with a luciferase-reported
construct driven by two tandem -180 sites
(pGL2-(-180)x2) (32). As shown in
Fig. 8A, stimulation of
pGL2-(-180)x2-transfected normal T cells with
PMA and ionomycin for 6 h induced a mean 3-fold increase in
luciferase activity compared with stimulated cells transfected with
empty pGL2 vector. Normal T cells that were
cotransfected with pGL2-(-180)x2 construct and
a PKC--containing vector showed 2-fold increase in luciferase
activity compared with cells transfected with the
pGL2-(-180)x2 construct alone
(p < 0.001). When T cells were cotransfected
with pGL2-(-180)x2 and plasmids encoding
PKC- or PKC-, we did not observe a significant increase in
luciferase activity, compared with cells transfected with the
pGL2-(-180)x2 construct alone. Furthermore, T
cells were cotransfected with the pGL2-(-180)x2
and CREM construct in the presence or absence of the plasmid
encoding PKC-. The presence of CREM construct caused 70%
suppression of the -180-driven luciferase activity, which was limited
to 50% when a PKC- construct was also present (Fig. 8A).
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, PKC-, PKC-, CREM, or PKC-
and CREM. Stimulation of the cells that were transfected with the
intact IL-2 promoter with PMA and ionomycin caused a 23-fold (mean)
increase in luciferase activity compared with unstimulated cells.
Cotransfection with PKC- and PKC- constructs caused a 2.4- and
0.5-fold increase in luciferase activity compared with stimulated T
cells that were transfected with the whole IL-2 promoter alone.
Cotransfection with a PKC- construct revealed a 4-fold increase in
the reporter gene activity, indicating the important role of PKC- in
IL-2 production. To examine whether CREM can affect the activity of the
IL-2 promoter, we cotransfected T cells with a CREM construct in the
presence or absence of a plasmid encoding PKC-. The presence of the
CREM construct suppressed the IL-2 promoter-driven luciferase
activity by 65%, which was limited to 50% when PKC- was present
(Fig. 8B).
Finally, we transfected normal T cells with the -575/+57 IL-2 promoter
constructs in which the -180 site had been mutated (M1 and M2). Normal
T cells that were transfected with the M1 or the M2 construct displayed
60% and 85% decrease in luciferase activity, respectively, compared
with the cells that were transfected with the wild-type IL-2 promoter
construct, following stimulation with PMA and ionomycin for 6 h.
The presence of PKC- construct did not restore the IL-2
promoter-driven luciferase activity (Fig. 8c). The
transfection experiments described above were also examined using the
same constructs in Jurkat cells. As shown in Fig. 9, the pattern obtained after
transfecting Jurkat cells was similar to that observed in normal T
cells. Specifically, stimulation of
pGL2-(-180)x2-transfected Jurkat cells with PMA
and ionomycin for 6 h induced a 3.5-fold increase in luciferase
activity compared with stimulated cells transfected with empty
pGL2 vector. Jurkat cells that were cotransfected
with the pGL2-(-180)x2 construct and plasmids
encoding PKC- showed a 2-fold increase in luciferase activity
compared with cells transfected with the
pGL2-(-180)x2 construct alone. The presence of
PKC- or PKC- plasmids in the presence of
pGL2-(-180)x2 construct did not affect the
luciferase activity compared with Jurkat cells that were transfected
with the pGL2-(-180)x2 construct alone. The
presence of CREM construct caused a 75% decrease in luciferase
activity, which was limited by 50% when a PKC- construct was also
present (Fig. 9). Jurkat cells were also examined in transfection
experiments using a construct driven by the -575/+57 region of the
IL-2 promoter/enhancer alone or in the presence of plasmids expressing
PKC-, PKC-, PKC-, CREM, or PKC- and CREM. Stimulation
of the cells that were transfected with the intact IL-2 promoter with
PMA and ionomycin caused a 10-fold increase in luciferase activity
compared with unstimulated cells. Cotransfection with PKC- and
PKC- constructs caused a 1- and 0.5-fold increase in luciferase
activity compared with Jurkat cells that were transfected with the
whole IL-2 promoter alone (Fig. 9). Cotransfection with a PKC-
construct resulted in a 10-fold increase in the reporter gene activity,
confirming the results obtained from transfecting normal T cells with
the same constructs and further supporting the important role of
PKC- on IL-2 gene transcription (Fig. 9). The presence of the
CREM construct suppressed the IL-2 promoter-driven luciferase
activity by 50%. Cotransfection of the PKC- and the CREM
construct caused an 80% decrease in luciferase activity (Fig. 9).
Thus, the -180 site of the IL-2 promoter/enhancer is critical in the
transcriptional regulation of IL-2, and PKC- is involved in the
phosphorylation and subsequent binding of CREB to this site.
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) that no binding can be detected to the -180 site-defined
oligonucleotide if poly(dI · dC) is used as a nonspecific
competitor. Instead, we used poly(dG · dC) as a nonspecific
inhibitor in performing EMSA experiments and we observed that nuclear
extracts from fresh human T cells treated with anti-CD3 and
anti-CD28 Abs, or PMA alone, or PMA and ionomycin bind to the -180
site-defined oligonucleotide. In determining the specificity of this
binding, 1) we were able to eliminate it in the presence of sufficient
amounts of excess unlabeled oligonucleotide; 2) we were able to
eliminate the band in presence of anti-p-CREB Ab; and 3) the
presence of Abs directed against the components of AP-1 failed to
change the intensity of the band significantly. Therefore, we concluded
that the -180 site of the IL-2 promoter represents a p-CREB binding
site. Our experiments have also established that the -180 site is
functional because a luciferase reporter construct driven by two tandem
-180 sites increased its activity when transfected into normal T cells
that were stimulated with PMA and ionomycin. The importance of the
-180 site in the transcription of the IL-2 gene in human T cells is
further underscored by the fact that mutation of two bases within this
region limited the ability of the whole IL-2 promoter to drive the
expression of the reporter gene.
Different signals delivered by hormones, synaptic activity, growth
factors, stress, and inflammatory cytokines can enhance cascades that
lead to CREB phosphorylation (6, 7, 8). Recent reports have
shown that in T cells PKC- is involved in the activation of AP-1
(18, 33) and NF-B (19, 20), two
transcription factors that lie down- and upstream of the -180 site of
the IL-2 promoter/enhancer and influence the regulation of the IL-2
gene transcription (32, 33, 34). Our experiments, besides
establishing the binding of CREB to the -180 site of the IL-2
promoter, have demonstrated that PKC- is involved in the
phosphorylation and activation of CREB. This conclusion is supported by
several facts: first, p-CREB protein levels diminished in the presence
of calphostin C, a PKC inhibitor, and in the presence of rottlerin, a
specific PKC- inhibitor, but they did not change significantly in
the presence of inhibitors directed against other kinases important in
T cell signaling; second, the binding of p-CREB to the -180
oligonucleotide diminished in T cells previously treated with specific
PKC- inhibitors; third, transfection of T cells with a PKC-
construct increased the ability of the -180 site to drive the
transcription of the reporter gene, whereas transfection of T cells
with other PKC isoforms, including PKC- and PKC-, either failed
or only slightly affected the transcription of the -180 site reporter
construct. The behavior of the -180 site reporter construct was
mimicked by a whole (-575/+57 region) IL-2 promoter/enhancer-driven
reporter construct. Specifically, its activity increased when the
PKC- construct was cotransfected. In addition, mutation of two bases
in the -180 region limited its ability to drive transcription, and the
presence of a PKC- plasmid did not restore its activity. The central
importance of the -180 site in the IL-2 gene transcription should be
noted in view of the reports that have proposed that PKC-
participates in the activation of AP-1 and NF-B that bind to the
-150 and -200 sites of the IL-2 promoter, respectively
(18, 19, 20). Our experiments also showed that MAPKAP-K2 and
PKA inhibitors affected moderately the binding of p-CREB to the -180
oligonucleotide, suggesting that other kinases, besides PKC-, may be
involved in the phosphorylation of CREB. Whether phosphorylation by PKA
influences the DNA-binding function of CREB remains a controversial
point (6). PKA can activate CREB in normal human T
lymphocytes because forskolin, a PKA activator, and KT5720, a specific
PKA inhibitor, affected moderately p-CREB protein levels and p-CREB/DNA
binding to the IL-2 promoter/enhancer. Although our findings do not
allow us to pinpoint the cascade that leads to CREB phosphorylation
upon TCR engagement, we propose that PKC leads to the activation of
Ras-Raf-1-MEK-mitogen-activated protein kinase in a
Ca2+-independent manner (31). In
this pathway, PKC- seems to have an important role, although it is
possible that other isoforms may participate. In contrast, the observed
nuclear protein binding to the -180 site after stimulation with PMA
and ionomycin, and the decrease of this binding when T cells were
treated with W7, a calcium/calmodulin inhibitor, suggest that a
Ca2+-dependent pathway may also participate.
It has been established that CREB associates with the coactivators
CBP/p300, resulting in the formation of heteromeric activator
complexes, which interact with the general transcription apparatus. In
this communication, we have shown that this interaction occurs also in
fresh human T cells. CBP/p300 exhibits intrinsic histone
acetyltransferase activity, which facilitates transcription by directly
participating in chromatin remodeling at the level of inducible
promoters (39, 40). We have shown that rottlerin, a
specific PKC- inhibitor, prevented the formation of p-CREB-CBP/p300
complexes, whereas inhibitors of other pathways also known to activate
CREB only slightly diminished the formation of these complexes.
Therefore, PKC- displays a major role in CREB phosphorylation and
its subsequent activation and further engagement in the transcription
initiation complex.
The -180 site appears to have an important role in the regulation of IL-2 gene transcription as it relates to normal T cell function, anergy, and the expression of human disease. Our experiments show that in fresh human T cells and in Jurkat cells, p-CREB binds to this site and enhances the transcription of the IL-2 gene. Powell et al. (5) have shown that nuclear proteins from anergic T cell clones contain CREB/CREM heterodimers that bind to the -180 site of the IL-2 promoter and may suppress IL-2 transcription (5). Transgenic mice expressing a dominant-negative form of CREB display defective thymocyte proliferation and IL-2 production (22). Recently, we showed that T cells from patients with systemic lupus erythematosus, a systemic autoimmune disease characterized by decreased IL-2 production in vitro, express increased levels of p-CREM protein that binds to the -180 site of the IL-2 promoter. CREM was shown to suppress IL-2 gene transcription (32). Finally, Bodor et al. (41) have shown that repressor ICER can suppress the transcription of the IL-2 gene.
In conclusion, we have presented evidence that PKC- participates in
the phosphorylation of CREB in human T cells and promotes IL-2
transcription. Additional studies are needed to investigate how p-CREB
assumes a central role in the transcription of the IL-2 gene in view of
the fact that the IL-2 promoter defines additional sites for
well-established transcription factors such as AP-1, NF-AT, and NF-B
(42). Apparently, each factor contributes variable degrees
of transcriptional activity depending on the incoming signal and
probably through interaction with other transcription factors and
coactivators. Because IL-2 has a central role in the function of T
cells from normal individuals and patients with autoimmune diseases,
further studies should characterize the critical role of members of the
CREB family of proteins in the transcriptional regulation of IL-2. It
is probable that at any given time point, the rate of IL-2 gene
transcription is determined by the levels of positive and/or negative
isoforms of CREB/CREM/ICER present in the vicinity of the IL-2 promoter
and the availability of other transcription factors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George C. Tsokos, Walter Reed Army Institute of Research, Department of Cellular Injury, Building 503, Room 1A32, 503 Robert Grant Road, Silver Spring, MD 20910-7500.
3 Abbreviations used in this paper: CREB, cAMP-responsive element-binding protein; CREM, cAMP-responsive element modulator; CBP, CREB-binding protein; DAG, diacylglycerol; ICER, inducible cAMP early repressor; MAPKAP-K2, mitogen-activated protein kinase-activated protein kinase-2; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; PKA, protein kinase A; PKC, protein kinase C.
Received for publication December 1, 2000. Accepted for publication February 20, 2001.
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