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(Journal of Leukocyte Biology. 2006;79:378-387.)
© 2006 by Society for Leukocyte Biology

Suppression of MIP-1ß transcription in human T cells is regulated by inducible cAMP early repressor (ICER)

Oxana Barabitskaja*, James S. Foulke, Jr.*, Shibani Pati*, Josef Bodor{dagger} and Marvin S. Reitz, Jr.*,{ddagger},1

* Institute of Human Virology, University of Maryland Biotechnology Institute, and
{ddagger} Department of Microbiology and Immunology, University of Maryland, Baltimore;
{dagger} Department of Pathology, Columbia University, New York, New York

1 Correspondence: Institute of Human Virology, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201. E-mail: reitz{at}umbi.umd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Local production of macrophage inflammatory protein-1ß (MIP-1ß), a ß-chemokine that blocks human immunodeficiency virus type 1 (HIV-1) entry into CD4+ CC chemokine receptor 5+ target cells, may be a significant factor in resistance to HIV-1 infection and control of local viral spread. The mechanisms governing MIP-1ß expression in T cells, however, are not well understood. Our results suggest that MIP-1ß RNA expression in T cells is dynamically regulated by transcriptional factors of the cyclic adenosine monophosphate (cAMP) responsive element (CRE)-binding (CREB)/modulator family. Transient transfection of primary human T cells with 5' deletion and site-specific mutants of the human MIP-1ß promoter identified an activated protein-1 (AP-1)/CRE-like motif at position –74 to –65 base pairs, relative to the TATA box as a vital cis-acting element and a binding site for inducible cAMP early repressor (ICER). Ectopic expression of ICER or induction of endogenous ICER with the cAMP agonists forskolin and prostaglandin E2 resulted in the formation of ICER-containing complexes, including an ICER:CREB heterodimer to the AP-1/CRE-like site and inhibition of MIP-1ß promoter activity. Our data characterize an important binding site for the dominant-negative regulator ICER in the MIP-1ß promoter and suggest that dynamic changes in the relative levels of ICER and CREB play a crucial role in cAMP-mediated attenuation of MIP-1ß transcription in human T cells.

Key Words: chemokine • promoter • CREB


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus type 1 (HIV-1) is suppressed by soluble factors secreted by activated CD8+ T cells [1 ], of which the C-C or ß chemokines, macrophage inflammatory protein-1ß (MIP-1ß), MIP-1{alpha}, and regulated on activation, normal T expressed and secreted (RANTES), are the most significant [2 ]. These chemokines inhibit viral entry into CD4+ CC chemokine receptor 5 (CCR5)+ target cells through blocking or down-modulating CCR5, which is required as a coreceptor by the majority of HIV-1 isolates [3 4 5 6 7 8 9 10 11 ]. Despite apparently equal potencies of inhibition in vitro, their antiviral activities in vivo may differ. Although there are conflicting data about correlations between RANTES and MIP-1{alpha} expression and progression to AIDS [12 , 13 ], higher levels of MIP-1ß expression by peripheral blood (PB) T cells, activated in vitro with lectin or specific antigen, have consistently correlated with lack of infection in HIV-exposed individuals and with a more favorable clinical course in HIV-infected people [14 15 16 17 18 19 20 21 22 23 ]. These data suggest that production of MIP-1ß at the site of infection may be a factor in resistance to HIV-1 and inhibition of local viral spread. The mechanisms governing MIP-1ß expression in PB T cells, however, are not clearly understood. MIP-1ß transcription is likely to be dynamically modulated by the interplay of positive and negative transcriptional factors. This kind of dual regulation of transcription has been demonstrated for other cytokine and chemokine genes, including interleukin (IL)-2, IL-4, granulocyte macrophage-colony stimulating factor, tumor necrosis factor {alpha}, and LD78{alpha} [24 , 25 ].

Activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A signal transduction pathway in PB T cells results in expression of inducible cAMP early repressor (ICER) and inhibits a number of T cell proliferative and effector functions [26 27 28 ]. ICER is a truncated product of the cAMP-responsive element (CRE)-modulator (CREM) gene, which is transcribed via an internal promoter, P2. ICER occurs as four isoforms generated by alternatively spliced transcripts (ICER I, ICER I{gamma}, ICER II, and ICER II{gamma}). Each isoform retains the CREM DNA binding and leucine zipper domains but lacks the CREM transactivation domain [29 , 30 ]. As a consequence, ICER binds to CRE and activated protein-1 (AP-1) DNA binding motifs and can interact via protein–protein interactions with some transcriptional factors, including nuclear factor of activated T cells (NF-AT), through its rel homology domain, which facilitates DNA binding and interactions of NF-AT with the AP-1 complex [26 ]. As a result of the lack of the transactivation domain, however, ICER fails to recruit CRE-binding protein (CREB)-binding protein and p300 (CBP/p300) and thus represses CREB- and Jun-mediated transcription. Uncoupling of CBP/p300 may abrogate early stages of transcriptional initiation as a result of the lack of CBP/p300-associated histone-acetyl-transferase activity and a failure to maintain the transcriptionally competent conformation of chromatin [31 32 33 ]. Here, we identify a proximal AP-1/CRE-like site upstream from the human MIP-1ß promoter at position –74 to –65 base pairs (bp), relative to the TATA box as an essential transcriptional element that confers positive and negative regulation of the MIP-1ß transcription by members of the CREB/activating transcription factor (ATF) and CREM protein families in primary T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and culture conditions
Heparinized PB was drawn from healthy donors at the National Institutes of Health (NIH) Blood Bank (Bethesda, MD). Peripheral blood mononuclear cells were resuspended in medium plus 10% fetal bovine serum, activated for 72 h with 2 µg/ml phytohemagglutinin (PHA)-L and 10 U/ml recombinant human (rh)IL-2, washed with Dulbecco’s phosphate-buffered saline, and cultured for an additional 12–14 days in the presence of rhIL-2. By flow cytometry, the cells were typically 95–98% CD3+ T lymphocytes with a 4:1 ratio of CD8+ cells to CD4+. The non-CD3+ cells were CD14+ (<1%), CD19+ (<1%), and CD56+ (variable between donors as a result of variable numbers of cells with a CD56+CD3+ phenotype, corresponding to natural killer-type T cells).

Flow cytometry
Cells were stained with mixtures of four differently colored fluorochrome-conjugated monoclonal antibodies (mAb; including CD3, CD4, CD8, CD14, CD19, and/or CD56) or matching isotypes (all from BD Biosciences, San Jose, CA) and analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

Human MIP-1ß promoter/reporter constructs
Human genomic DNA was isolated from H9 CD4+ T cells. The human MIP-1ß promoter region (–1065 to +43 bp, relative to the TATA box) was amplified by polymerase chain reaction (PCR) using sense (5'- ccctgtacccagctcaattc-3') and antisense (5'-gctgtgtcctgtgctgatac-3') primers. The PCR product was cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and used to generate a series of 5' nested deletion mutants of the MIP-1ß promoter by PCR using the antisense primer: 5'-tctcctcgag-tcctgtgctgatactgagtt-3' (an overhang with an XhoI cloning site is underlined) and six sense primers with common 5' extensions (5'-ttccggtacc) containing a KpnI cloning site: 5'-tgtacccagctcaattctgt-3', 5'-agcagcacagttcttgtcta-3', 5'-ttgtagcaggtgtgaa-3', 5'-ttggctgtaccacttc-3', 5'-agccatgacatcatct-3', 5'-ccatggaaattccactcact-3'. The PCR products were digested with KpnI and XhoI and cloned into pGL3-Basic vector (Promega, Madison, WI). Site-directed mutations were made using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: sense 5'-ctagcttcaggagccatgaattgtccctctaccatggaaattccac-3' and antisense 5'- gtggaatttccatggtagagggacaatcatggctcctgaagctag-3'.

His-tagged ICER
A construct containing human ICER II cDNA [27 ] was the template for PCR amplification with the following primers: sense 5'-ggcctggtacctatggctgtaactggagatgaaac-3' (a KpnI site is underlined) and antisense 5'-tggccctcgagctaatctgttttgggagagcaaatgtc-3' (an XhoI site is underlined). The PCR products were digested with KpnI and XhoI and cloned into plasmid cDNA3.1 (pcDNA3.1)/His B (Invitrogen) to give pICER II-His.

Electroporation and luciferase assays
PB T cells were electroporated with 3 µg MIP-1ß promoter constructs and 4–5 x 106 cells/0.1 ml human T cell nucleofector solution in AMAXA cuvettes with a Nucleofector I (AMAXA Biosystems, Koeln, Germany) using the T-20 program. After the rest period, cells were activated with 2 µg/ml PHA-L for 4 h. Luciferase was assayed using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). In some experiments, 6 µg pICER II-His or vector alone was coelectroporated with MIP-1ß promoter constructs (3 µg pDNA) into PB T cells. Transfection efficiencies were determined using a reporter plasmid expressing green fluorescent protein-C1 (BD Biosciences). Luciferase activity was expressed in arbitrary units and compared by normalizing relative light units to the protein content of lysates as measured by bicinchoninic acid (BCA) assays (Pierce Biochemicals, Rockford, IL). After the rest period, cells were treated with 100 nM prostaglandin E2 (PGE2) or 0.1 mM forskolin and harvested for nuclear extracts 4–5 h poststimulation. All values were correcting by subtracting background values obtained with a promoterless luciferase construct. When 30 µg DNAs (pcDNA3.1/His vector alone or with cloned ICER II) were used for electroporation of 50 x 106 PB T cells, the conditions were not altered.

Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared from 50 x 106 PB T cells by the method described by Masquilier and Sassone-Corsi [32 ] with minor modifications. Protein quantification was by BCA assay. MIP-1ß promoter-specific oligonucleotides were synthesized and purified by high-pressure liquid chromatography (AE grade; Midland Certified Reagents Co., TX). The overhangs were different for sense (5'-tagc) and antisense (5'-atgg) oligonucleotides, which were annealed, labeled by fill-in with Klenow enzyme and {alpha}-32P-deoxy-cytidine 5'-triphosphate at room temperature (20 min), and purified using Elutip-D columns (S&S, Keene, NH). EMSA binding reaction mixtures (20 µl) contained 0.2 µg each poly[dI-dC] and poly[dG]/poly[dC], 4 µg nuclear protein, and 20–100 pg-labeled oligonucleotide probe (20–100 pg) and were incubated 20 min at room temperature. In some experiments, 2–4 µg antibodies or immunoglobulin G (IgG) controls were preincubated with nuclear extracts on ice or at room temperature for 45 min, then probe was added, and the reaction was incubated an additional 20 min. Monoclonal anti-His G antibody was from Invitrogen; mouse anti-human CREB-1 X-12 (sc-240) mAb, rabbit polyclonal anti-human CREM clone X-12 (sc-440), anti-Fos clone K-25 (sc-253), anti-Jun clone D (sc-44), and clone H-79 (sc-1694) antibodies and oligonucleotides for gel shift [CREB consensus (sc-2504), CREB mutant (sc-2517) sequences] were from Santa Cruz Biotechnology (CA); and rabbit anti-human CREM CS4 sera were described previously [24 ]. Samples were electrophoresed on 5% polyacrylamide gel, which was dried and analyzed by the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Quantitative reverse transcriptase (RT)-PCR
Aliquots of PB T cells treated as described were collected in parallel with samples used for nuclear extract preparations. Synthesis of first-strand cDNA (RT reaction) used standard conditions including 2.5 µM random hexamer, 1 µg total cellular RNA, and 200 U SuperScript II RT for 1 h at 42°C, followed by 5 min at 99°C. Quantitative real-time RT-PCR was performed with a QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA) and a 5700 thermocycler (Applied Biosystems, Foster City, CA). The human MIP-1ß gene was amplified with the following primers: sense 5'-atgctagtagctgccttctg-3' and antisense 5'-ggctgctggtctcatagtaa-3'. The yield of MIP-1ß PCR product per each sample was normalized to the yield of PCR product generated using an rRNA 18S primer set (Ambion, Austin, TX). As a DNA contamination control, equal amounts of RNA were used without the RT step.

Western blots
Nuclear extracts used in EMSA assays were analyzed by Western blot. Proteins (15 µg/lane) were denatured (70°C, 10 min) in 1x lithium dodecyl sulfate loading buffer, resolved by 4–12% Bis-Tris (NuPAGE)-sodium dodecyl sulfate gel electrophoresis, and transferred to Optitran nitrocellulose membranes (S&S). After blocking with 5% nonfat dried milk, proteins were visualized with rabbit anti-human CREM (X-12) or mouse anti-human CREB-1 (X-12) at 1:200 dilutions followed by anti-rabbit heavy and light chain IgG or anti-mouse IgG conjugated to horseradish peroxidase at 1:2000 dilutions. The blots were developed using the enhanced chemiluminescence detection system (Amersham-Pharmacia Biotech, Piscataway, NJ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of an ICER binding site in the MIP-1ß promoter
The human MIP-1ß promoter region (–1065 to + 43 bp, relative to the TATA box) was amplified by PCR of genomic DNA from H9 CD4+ T cells and cloned. The DNA sequence was identical with GenBank entry accession number S56704, containing the region 5' to the MIP-1ß open-reading frame. As described in Materials and Methods, a series of nested 5' deletion mutants of the MIP-1ß promoter with a common 3' position (+37 bp, relative to the TATA box) was cloned into pGL3 Luc, a promoterless reporter vector, and transiently transfected into PB T cells, which were harvested and assayed for luciferase reporter gene activity 4 h post-PHA stimulation, a time at which induction of endogenous MIP-1ß mRNA reaches a maximum (not shown). The highest activity in PB T cells was obtained with the 485 bp (pGL3-485wt) MIP-1ß promoter (Fig. 1A ). The longest segment, pGL3-1062wt, had a slightly but consistently lower activity than pGL3-485wt. The MIP-1ß promoter segments pGL3-264wt to pGL3-78wt showed a progressive decline of reporter gene activity with decreasing length. Deletion to the –59-bp position eliminated most (98%) of the promoter activity, indicating the presence of critically important cis element(s) in the fragment from –78 to –59 bp.


Figure 1
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  		Figure 1. Involvement of a proximal AP-1/CRE-like site in MIP-1ß promoter activity in human T cells. (A) PB T cells were electroporated with the indicated promoter/reporter constructs and stimulated with PHA-L. PGL3-Luc empty reporter was used as a control. The 5' ends of the nested 5' deletion mutants pGL3-1062 to pGL3-59 are numbered relative to the TATA box. The wild-type and the AP-1/CRE-like site-specific mutants (mAP-1/CRE) were tested in parallel. Promoter activity is expressed as percent stimulated luciferase expression, expressed in percent relative luminescence units (RLU), relative to that obtained with pGL3-1062wt. Results (mean values±SD) of at least five independent experiments are shown. (B) Sequence of the MIP-1ß promoter region. The sequence between the 5' borders of the pGL3-79 and pGL3-59 nested deletion mutants, indicated by numbers in parentheses, contains an overlapping AP-1 site and a CRE-like motif. The indicated 6-bp mutation was introduced by site-directed mutagenesis into the two MIP-1ß promoter constructs, which were tested for reporter gene activity (see Fig. 2B ). The AP-1/CRE-like site is underlined, and the mutations are marked with asterisks.

 
Analysis of the promoter with the MatInspector program [34 ] identified overlapping consensus sites for AP-1 and CRE-like binding motifs at position –74 to –65 bp relative to the TATA box (Fig. 1B) . These AP-1/CRE-like sites were disrupted by site-directed mutagenesis of pGL1062wt (pGL3-1062 mAP-1/CRE) and pGL3-264wt (pGL3-264 mAP-1/CRE). DNA sequence analysis confirmed the 6-bp mutations (Fig. 1B) . Both constructs with the AP-1/CRE-specific mutations, similar to the shortest, wild-type 5' nested deletion mutant of the MIP-1ß promoter, also lacking the AP-1/CRE-site (pGL3-59wt), retained only ~2% of the activity of the pGL3-1062wt promoter.

To determine whether human ICER can attenuate MIP-1ß promoter activity, His-tagged ICER II was coelectroporated into PB T cells along with the different nested promoter constructs. As shown in Figure 2A , ICER II expression significantly reduced the activity of all but the shortest promoter. Similar experiments performed with the nested MIP-1ß promoters and expression constructs for ICER II, ICER I, and ICER I{gamma} gave virtually identical results (not shown), indicating that different ICER isoforms have similar effects on MIP-1ß promoter activity. Consequently, all subsequent experiments used the His-tagged ICER II plasmid. The activity of the shortest promoter (pGL3-59wt), although low relative to longer constructs, was virtually unaffected by ICER II (4.5–4.2 RLU, 7% reduction), suggesting that it lacks a functional binding site. The activities of the AP-1/CRE-mutated promoters, pGL3-1062mAP-1/CRE and pGL3-264mAP-1/CRE, were also only slightly reduced by ICER expression (Fig. 2B) compared with the activities of the cognate wild-type promoters.


Figure 2
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  		Figure 2. Sensitivity of MIP-1ß promoter mutants to ICER. Primary PB T cells were electroporated with the MIP-1ß promoter/pGL3 Luc reporter constructs and a vector encoding a His-tagged human ICER II isoform or vector control pcDNA 3.1/His and then stimulated with PHA-L. Shown are the effects of His-tagged ICER II on the activity of (A) 5' nested, deleted promoters containing the wild-type AP-1/CRE-like site or (B) pGL3-1062 and pGL3-264 promoter constructs containing a wild-type (wt) or the mutated AP-1/CRE-like site. Promoter activity is expressed as the percent luminescence units relative to that obtained with pGL3-1062wt. Although the relative values with the weakest promoters are quite low, the absolute values are 20- to 50-fold above the values obtained with a promotorless pGL3 plasmid, and the results are quite consistent. Results (mean values±SD) of at least five independent experiments are shown.

 
Recombinant ICER binds to the MIP-1ß promoter at the proximal AP-1/CRE-like site
Oligonucleotides, based on the MIP-1ß promoter, which contained the wild-type or a mutated AP-1/CRE-like site, were synthesized for EMSA analyses of binding in vitro to purified bacterial recombinant human ICER II. The 6-bp mutation in the oligonucleotide was identical to that introduced into the MIP-1ß promoter reporter constructs. Additional MIP-1ß probes, containing the NF-AT site (–177 to –166), the composite NF-AT and AP-1/fos-jun sites (–150 to –127), the AP-1 site (–98 to 90), and the overlapping NF-AT and NF-{kappa}B sites (–59 to –47), were also synthesized. The wild-type AP-1/CRE-like site showed preferential and specific binding to recombinant ICER II. All other probes bound ICER only at low levels, and binding was not competed with excess unlabeled, homologous probes (not shown).

To evaluate whether ectopically expressed, rhICER binds in vivo to the AP-1/CRE-like site alone or in a complex with endogenous protein(s), PB T cells were electroporated with ICER cDNA cloned into pcDNA3.1/His or with empty vector alone and treated with PGE2 for 4–5 h. Nuclear extracts prepared from PB T cells electroporated with His-tagged ICER bound MIP-1ß wild-type probe and formed complexes not observed with control nuclear extracts (Fig. 3A , compare lane 2 with lane 1). The presence of His-tagged ICER in the three novel complexes, designated as His-I, His-II, and His-III, was ascertained by supershift of these complexes by anti-His mAb but not by mouse IgG (Fig. 3A , compare lanes 5 and 6). In contrast, anti-His antibody did not shift any complexes formed by nuclear extracts from control PB T cells (Fig. 3A , lane 4). Moreover, the His-I, His-II, and His-III complexes were supershifted by anti-His and anti-CREM antibodies (Fig. 3B , lanes 2 and 5) but not by mouse and rabbit IgG (Fig. 3B , lanes 4 and 9). Anti-CREB antibody did not affect the intensity of complexes His-II and His-III but appeared to shift the complex His-I (Fig. 3B , lane 3). In contrast, the intensity of all three His-tagged ICER-containing complexes was unchanged by incubation with anti-Fos or two different anti-Jun antibodies (Fig. 3B , lanes 6–8).


Figure 3
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  		Figure 3. ICER ectopically expressed in primary PB T cells binds specifically to the MIP-1ß AP-1/CRE-like site and forms complexes with endogenous proteins. Nuclear extracts were prepared from PB T cells transfected with a pcDNA3.1 vector encoding a His-tagged human ICER II isoform or with empty vector and analyzed by supershift EMSA. (A) Autoradiogram of acrylamide gel containing the wild-type MIP-1ß oligonucleotide probe incubated with nuclear extracts from PB T cells transfected with control vector (lanes 1, 3, and 4) or the His-tagged ICER II vector (lanes 2 and 5–7) in the absence or presence of monoclonal anti-His antibody or mouse control IgG. Complexes containing ICER, labeled His-I, -II, and -III, which were formed by incubation with nuclear extracts from cells transfected with His-tagged ICER, were recognized and completely supershifted (S) by anti-His mAb (lane 6) but not by mouse IgG (lane 5). Anti-His antibody failed to supershift complexes A and B formed by nuclear extracts from PB T cells transfected with empty vector (lanes 1 and 4). (B) Nuclear extracts from PB T cells transfected with a vector encoding His-tagged ICER II were tested by supershift EMSA for binding of other transcriptional factors to the wild-type MIP-1ß probe as indicated. The antibodies used included anti-His (lane 2), anti-CREB (lane 3), mouse control IgG (lane 4), polyclonal antisera against CREM (lane 5), or Fos (lane 6), anti-Jun (mAb D and H-79; lanes 7 and 8), and rabbit IgG (lane 9).

 
Endogenous ICER binds to the proximal AP-1/CRE-like site of the human MIP-1ß promoter
To determine whether endogenous ICER also binds to the AP-1/CRE-like site, nuclear extracts from PB T cells treated with cAMP-elevating agonists (forskolin or PGE2) for 4–5 h were examined by EMSA. Nuclear extracts from treated cells (Fig. 4A , lanes 4 and 6) bound the MIP-1ß wild-type probe and formed complexes not observed with nuclear extracts from untreated cells (lanes 3 and 5). The mobilities of these novel complexes, designated as I and II, were similar to those of the His-tagged ICER-containing complexes His-I and His-III (lane 2). Two slower moving bands, which were evident in untreated cells, labeled A and B, disappeared when cells were electroporated with the ICER II expression plasmid but not when cells were treated with forskolin or PGE2. A shift of complexes I and II, along with the appearance of a high molecular weight complex near the origin of the gel in the lane treated with anti-CREM rabbit antibody (X-12; Fig. 4B , lane 3) but not by rabbit IgG (lane 4), indicates that these cAMP agonist-induced complexes contain proteins of the CREM family. As the X-12 antibody does not cross-react with CREB and as ICER is the major cAMP-inducible product of the CREM gene, complexes I and II likely contain endogenous ICER. Indeed, a Western immunoblot with anti-CREM X-12 antibody confirmed that endogenous ICER protein expression was induced in PB T cells in response to PGE2 and forskolin (Fig. 4C , lanes 3–6). It is not surprising that endogenous ICER protein levels in PB T cells were lower than the level of His-tagged ICER in electroporated PB T cells relative to the levels of CREB protein in each sample (compare lanes 2, 4, and 6). Perhaps because of the lower expression levels, endogenous ICER formed only two complexes with cellular proteins instead of the three formed with His-tagged ICER and did not compete the binding of complexes A and B with the AP-1/CRE-like probe (Fig. 4A , compare lanes 1 and 2 with lanes 3 and 4 and lanes 5 and 6).


Figure 4
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  		Figure 4. Complexes induced by cAMP agonists bind specifically to the AP-1/CRE-like site of the MIP-1ß promoter and contain endogenous ICER. (A) A 32P-radiolabeled wild-type MIP-1ß oligo probe was incubated with nuclear extracts prepared from human PB T cells untreated (lanes 3 and 5) or treated with forskolin (lane 4) or PGE2 (lane 6) and subjected to EMSA. Lanes 1 and 2 show migration of the probe incubated with extracts from PB T cells transfected with control vector or one encoding His-tagged ICER II, respectively. (B) Nuclear extracts from human PB T cells untreated (lane 1) or treated with PGE2 (lanes 2–4) were tested by supershift EMSA with the wild-type MIP-1ß probe using polyclonal anti-CREM antibody (lane 3) or rabbit IgG control (lane 4). (C) Western immunoblot assays of nuclear extracts from PB T cells transfected with control vector or one encoding His-tagged ICER II (lanes 1 and 2, respectively) and PB T cells nontransfected and untreated (lanes 3 and 5) or treated with PGE2 (lane 4) or forskolin (lane 6). Anti-CREM polyclonal antiserum was used to detect His-tagged and endogenous ICER and a monoclonal anti-CREB antibody to detect CREB-1.

 
The specificity of the cAMP agonist-induced complexes I and II with the AP-1/CRE-like site was further evaluated by competitive and supershift EMSAs. Complex I with the wild-type MIP-1ß probe (Fig. 5A , lane 1) was eliminated completely by competition with unlabeled MIP-1ß wild-type probe (lanes 2 and 3) and an unrelated probe containing a CREB consensus sequence (lane 6) but not by an excess of a MIP-1ß mutant probe, in which the AP-1/CRE-like site was disrupted (lanes 4 and 5) or by a CREB mutant probe (lane 7). Binding of complex I to the MIP-1ß wild-type probe was decreased by competition with an AP-1 consensus sequence (lane 8), but competition was nonspecific, as a mutated AP-1 sequence also competed (lane 9). Binding was unaffected by an excess of NF-AT wild-type oligo (lane 10). Complex I was supershifted by two different anti-CREM antibodies (lanes 12 and 13) and by an anti-CREB antibody that does not cross-react with ICER (lanes 16 and 20) but not by anti-Jun, anti-Fos, anti-ATF-1, and anti-ATF-2 antibodies or by rabbit and mouse IgG. An antibody that recognizes phosphorylated CREB did not affect the intensity of complex I (lane 22).


Figure 5
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  		Figure 5. Endogenous ICER-containing complexes differ by the absence/presence of CREB. Shown is the migration of complexes I and II (A and B, respectively) bound to 32P-labeled wild-type MIP-1ß oligo probe in the absence (lane 1) or presence of 100- and 1000-fold excess unlabeled self-competitor (lanes 2 and 3) or MIP-1ß mutant oligo probe with the disrupted AP-1/CRE-like site (lanes 4 and 5). Competitions by a 1000-fold excess of unlabeled oligo hetero-competitors containing a CREB consensus sequence (lane 6) or CREBm mutant (lane 7), an AP-1 consensus sequence (lane 8) or AP-1m mutant (lane 9), and a NF-AT consensus sequence (lane 10) are shown. Supershift assays for the proteins contained in complexes I and II were performed using anti-CREM CS4 and X-12 (lanes 12 and 13), anti-Jun D (lane 14), anti-Fos (lane 15), antiphospho-CREB (anti-pCREB; lane 22) polyclonal antibodies, or a corresponding rabbit IgG control (lanes 11 and 23) or anti-CREB (lanes 16 and 20), anti-ATF-1 (lane 19), anti-ATF-2 (lane 21) mAb, or a mouse IgG control (lanes 17 and 18).

 
Complex II with the MIP-1ß wild-type probe (Fig. 5B , lane 1) was also eliminated in the presence of homologous MIP-1ß wild-type oligo (lanes 2 and 3), as well as by the consensus CREB-containing oligo (lane 6) but was unaffected by an excess of the MIP-1ßm (lanes 4 and 5) or CREB mutant (lane 7) oligos. An excess of NF-AT wild-type oligo did not affect the intensity of complex II (lane 10), and (as with complex I) wild-type and mutant AP-1 oligos competed to a similar extent (lanes 8 and 9). In contrast with complex I, complex II was supershifted only by anti-CREM antibodies (lanes 12 and 13) but not by anti-CREB antibodies (lanes 16 and 20) or any of the other tested antibodies and IgG controls. Taking together, these data confirm that endogenous ICER-containing complexes I and II bind specifically to the AP-1/CRE-like site of the MIP-1ß promoter. Complex I appears to contain an ICER:CREB heterodimer. Complex II may consist of an ICER homodimer.

Complexes A and B are constitutively present and are not increased by treatment with forskolin or PGE2. In contrast to complexes I and II, complexes A and B were supershifted by antiphospho-CREB and anti-CREB antibodies (Fig. 6 , lanes 4 and 7). Anti-CREM, anti-ATF-1, anti-ATF-2, anti-C/EBP, anti-Fos, and two different anti-Jun antibodies or IgG controls failed to supershift these complexes. The finding that complexes A and B contained phosphorylated CREB (the active form) together with the data suggesting that ICER-containing complexes competed with these complexes for binding to the AP-1/CRE-like site of the MIP-1ß promoter raised an obvious question about a negative regulatory role for the ICER-containing complexes in MIP-1ß RNA expression. As shown in Figure 7 , an increase in relative levels of the ICER-containing complexes I and II compared with the phosphor-CREB-containing complexes A and B inversely correlated with levels of MIP-1ß mRNA.


Figure 6
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  		Figure 6. Two constitutive complexes bound to the AP-1/CRE-like site are specifically recognized by anti-CREB and antiphospho-CREB antibodies. Shown is an autoradiogram of an acrylamide gel containing the complexes A and B bound to the wild-type MIP-1ß probe incubated with mAb against ATF-1 (lane 2), ATF-2 (lane 3), or CREB (lane 7), polyclonal antisera against phosphor-CREB (lane 4), CCAAT/enhancer-binding protein (C/EBP; lane 6), CREM (lane 8), Fos (lane 9), or Jun (clones H-79 and D; lanes 10 and 11), or mouse and rabbit IgG controls (lanes 1 and 5, respectively).

 

Figure 7
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  		Figure 7. MIP-1ß RNA expression in primary T cells inversely correlates with the intensity of endogenous ICER-containing complexes bound to the AP-1/CRE-like site of the MIP-1ß promoter. Nuclear extracts from human PB T cells untreated or treated with cAMP agonists (PGE2 or forskolin) were incubated with the MIP-1ß oligo probe and analyzed by EMSA. Densities of endogenous ICER-containing complexes (I and II) and phosphor-CREB-containing complexes (A and B) were evaluated by PhosphoImager. Density of endogenous ICER-containing complexes (I and II) is expressed as a percent of total density of all four complexes (I, II, A, and B). Samples of RNA from PB T cells were collected in parallel with nuclear extracts, and MIP-1ß RNA was measured by quantitative real-time RT-PCR. Levels of MIP-1ß RNA were normalized for 18S rRNA and expressed as percent expression relative to an untreated sample. The samples were obtained from five different donors. The straight line was plotted with GraphPad Prism 4 (GraphPad Software, San Diego, CA) using the Deming (Model II) linear regression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our analyses of the MIP-1ß promoter identified positive and negative regulatory elements within the 485 bp upstream from the TATA box. Promoters, which included sequences upstream from –485 bp, were somewhat reduced in activity, suggesting the presence of additional negative regulatory element(s). Deletion of the region –78 to –59 bp relative to the TATA box eliminated 98% of the full-length activity. Similarly, reduced activities were observed with promoters containing a mutation of the AP-1/CRE-like site identified at –74 to –65 bp, suggesting that this site is critical for MIP-1ß transcription.

Bodor et al. [35 ] have shown that ICER expression correlates negatively with RNA expression of several cytokines and chemokines in human PB T cells and in thymocytes of ICER transgenic mice. ICER contains CREM DNA binding and leucine zipper domains but lacks other domains involved in transcriptional activation. Consequently, it binds to CREs, but its interactions with other proteins obviously differ from those of CREM, altering its transactivating activity [26 , 29 ]. We hypothesized that ICER, alone or in complexes with other nuclear proteins, could suppress MIP-1ß transcription in human T cells by binding to an AP-1/CRE-like site, which we identified in the promoter. We confirmed that ICER indeed attenuates MIP-1ß promoter activity. Ectopic overexpression of ICER II strongly blocked MIP-1ß promoter activity in PHA-stimulated PB T cells. Studies with 5'-deleted promoters suggested that the AP-1/CRE-like site at position –74 to –65 bp upstream from the TATA box is an important binding site for ICER. Deletion of sequences 5' to position –59 as well as mutations of the identified AP-1/CRE-like site eliminated most of the inhibitory ability of ICER, although a slight response persisted. Thus, the possibility that ICER also targets an upstream site(s) cannot be ruled out. However, any contribution appears to be relatively minor.

DNA binding studies provided clear evidence that the AP-1/CRE-like site of the MIP-1ß promoter binds ICER. Purified recombinant ICER II preferentially bound in vitro to the MIP-1ß probe containing the AP-1/CRE-like site. Furthermore, ectopically expressed ICER bound to the MIP-1ß probe containing the AP-1/CRE-like site and formed multiple complexes in PB T cell nuclei. Several lines of evidence indicate that endogenous ICER also binds to this site. Treatment of PB T cells with the cAMP-elevating agonists, PGE2 and forskolin, resulted in induction of multiple complexes similar in mobility to His-tagged ICER-containing complexes. Cold competition experiments showed that these cAMP-induced complexes were specifically bound to the AP-1/CRE-like site and were supershifted by anti-CREM antibody. The intensity of the cAMP-induced complexes correlated directly with levels of endogenous ICER protein in nuclear extracts and inversely with levels of MIP-1ß mRNA. Taken together, our results indicate that cAMP-sensitive complexes, bound to the AP-1/CRE-like site of the MIP-1ß promoter, contain endogenous ICER and suggest that these complexes attenuate transcription from the natural MIP-1ß promoter.

To identify the proteins that heterodimerize with ICER in PB T cells, ICER-containing complexes were examined by supershift experiments. The slowest migrating complex was recognized by anti-CREM and anti-CREB (but not antiphospho-CREB) and by anti-His when recombinant His-ICER was present. These complexes likely contain ICER:CREB heterodimers in which CREB is not phosphorylated. Lower molecular weight ICER-containing complexes were supershifted only by anti-CREM and likely contain ICER homodimers and/or heterodimers with other transcription factors. The presence of AP-1/Jun-Fos protein family members was excluded by nonspecific competition of binding to an AP-1 consensus sequence and more importantly, the failure of several anti-Jun and anti-Fos antibodies to supershift any ICER-containing complexes. These data suggest that ICER does not form complexes with Jun or Fos and the AP-1/CRE site in the MIP-1ß promoter but binds instead as a homodimer or a heterodimer with CREM-related proteins and with CREB, in agreement with previous reports [30 , 36 37 38 39 ].

The steady presence of the ICER:CREB complex (complex I), bound to the AP-1/CRE-like site following activation with cAMP agonists, is consistent with an important role for this complex in suppression of the MIP-1ß promoter activity in primary T cells. We found that 75–90% inhibition of MIP-1ß transcription with PGE2 or forskolin treatment correlated with a predominant formation of the ICER:CREB complex (complex I), a slight reduction of two CREB-containing, cAMP-insensitive complexes, and little difference in CREB protein levels in nuclear extracts. In contrast, overexpression of His-tagged ICER resulted in total depletion of the cAMP-insensitive complexes containing CREB, a significant drop in levels of CREB protein in nuclear extracts, and a shift toward formation of ICER:ICER or ICER:CREM-related complexes (complex II). Based on these observations, we suggest that relatively low concentrations of ICER are sufficient for formation of an ICER:CREB complex that inhibits the MIP-1ß promoter. Higher ICER expression leads to the formation of ICER-containing complexes with lower molecular weights than that of the ICER:CREB heterodimer. We speculate that by such a mechanism, ICER-mediated attenuation of MIP-1ß transcription in human T lymphocytes can occur following its rapid induction without necessarily depleting other CREB-containing complexes and perhaps without having a similar effect on expression of other CREB-dependent genes. Under this scenario, reduction of the MIP-1ß promoter activity could occur at relatively low concentrations of ICER by binding of ICER:CREB heterodimers to the AP-1/CRE-like site at position –74 to –65 bp. Whether ICER binding to nonphosphorylated CREB prevents its phosphorylation or facilitates its dephosphorylation is not clear.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH Grant R01 HL63647. The authors are grateful to Drs. George Lewis and Sayed Abdel-Wahab for help with flow cytometry analyses.

Received May 9, 2005; revised September 26, 2005; accepted September 28, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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