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The Journal of Immunology, 2002, 168: 274-282.
Copyright © 2002 by The American Association of Immunologists

Prostaglandin E2-Mediated Activation of HIV-1 Long Terminal Repeat Transcription in Human T Cells Necessitates CCAAT/Enhancer Binding Protein (C/EBP) Binding Sites in Addition to Cooperative Interactions Between C/EBP{beta} and Cyclic Adenosine 5'-Monophosphate Response Element Binding Protein1

Nancy Dumais, Salim Bounou, Martin Olivier2 and Michel J. Tremblay2

Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous work indicates that treatment of human T cells with PGE2 results in an increase of HIV-1 long terminal repeat (LTR) transcriptional activity. The noticed PGE2-mediated activation of virus gene activity required the participation of specific intracellular second messengers such as calcium and two transcription factors, i.e., NF-{kappa}B and CREB. We report in this work that the nuclear transcription factor CCAAT/enhancer binding protein (C/EBP) is also important for PGE2-dependent up-regulation of HIV-1 LTR-driven gene activity. The implication of C/EBP was shown by using a trans-dominant negative inhibitor of C/EBP (i.e., liver-enriched transcriptional inhibitory protein) and several molecular constructs carrying site-directed mutations in the C/EBP binding sites located within the HIV-1 LTR. Mutated HIV-1 LTR constructs also revealed the involvement of the two most proximal C/EBP binding sites. Data from cotransfection experiments with vectors coding for dominant negative mutants and gel mobility shift assays indicated that PGE2-mediated induction of HIV-1 LTR activity results from a cooperative interaction between C/EBP{beta} and CREB, two members of the basic leucine zipper family of transcription factors. Altogether these findings indicate that treatment of human T cells with PGE2 induces HIV-1 LTR activity through a complex interplay between C/EBP{beta} and CREB. Such a combinatorial regulation may represent a mechanism that permits a fine regulation of HIV-1 expression by PGE2 in human T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several signal transduction pathways have been shown to regulate the expression of target genes by inducing the phosphorylation of specific transcription factors (1). The second messenger cAMP mediates the transcriptional induction of numerous genes through protein kinase A (PKA)3-dependent phosphorylation of the CREB at Ser133 (2). CREB is a stimulus-induced 43-kDa basic leucine zipper (b-ZIP) transcription factor that binds to an octanucleotide cAMP-responsive element (CRE) (i.e., TGANNTCA) both as a homodimer and as a heterodimer in conjunction with other members of the activation transcription factor (ATF)/CREB superfamily of transcription factors (3, 4, 5). It is now believed that the transcriptional regulation of genes containing either CCAAT/enhancer binding protein (C/EBP) or ATF/CREB recognition sites may involve the heterodimerization between different members of the b-ZIP family. This is clearly illustrated by the recent demonstration that transcription of HIV-1 in monocytic cells is regulated by a synergistic interaction between ATF/CREB and C/EBP protein families (6). The C/EBP-related family of nuclear transcription factors constitutes a class of proteins characterized by their ability to bind the CCAAT consensus sequence, inducing either transcriptional activation or repression of target genes (7, 8, 9, 10). Members of this family include C/EBP{alpha}, C/EBP{beta} (also termed LAP, NF-IL6{alpha}, IL-6DBP, AGP/EBP), C/EBP{gamma} (previously defined as Ig/EBP-1), C/EBP{delta} (NF-IL6{beta}), and C/EBP{epsilon} (11). Interestingly, the regulatory sequences of HIV-1, which are located within the long terminal repeat (LTR), harbor three C/EBP sites that bind C/EBP{beta} (12), and these sites are essential to initiate virus replication in cells of the monocyte/macrophage lineage (13) and in endothelial cells as recently described (14). PKA and transcription factors of the ATF/CREB family may be critical for HIV-1 expression and regulation. In this regard, HIV-1 infection has been associated with sustained elevation of cAMP in T cell lines and in normal peripheral blood mononuclear lymphocytes (15). Moreover, HIV-1 replication has been shown to be modulated by intracellular levels of cAMP (16, 17). For example, activation of the cAMP/PKA pathway by cholera toxin enhances HIV-1 transcription in latently infected monocytoid U1 cells (18). It is still unknown whether the HIV-1 genome, especially the LTR, possesses CRE sequences. However, the downstream sequence elements located in the U5 domain of HIV-1 LTR has been proposed to act as 12-O-tetradecanoylphorbol-13-acetate/phorbol ester responsive element (TRE)-like CRE that bind both AP-1 and CREB/ATF, allowing the induction of HIV-1 LTR activity through both protein kinase C and PKA activation signals (19).

The clinical progression of diseases resulting from HIV-1 infection is controlled to a large extent by cellular processes that govern the transcriptional modulation of virus gene expression. A vast array of host- and virus-derived factors interacts with the HIV-1 LTR to influence transcriptional activity through a complex interplay between both positive and negative elements (20, 21). It has been postulated that such cofactors may be important in disease progression by enhancing cell-to-cell transmission or through up-regulation of HIV-1 expression in latently infected cell (22). In persons dually infected with HIV-1 and other pathogens, the inability of the host to develop an effective immune response may involve the participation of the immunosuppressive molecule PGE2, an oxygenated polyunsaturated fatty acid that contains a cyclopentane ring structure. PGE2 has been shown to modulate the immune response both in vitro and in vivo (23, 24). This hormone-like molecule has been implicated in decreasing T cell proliferation, IL-2 production, and IL-2R expression (24, 25, 26, 27, 28, 29). PGE2 shifts the balance of the cellular immune response away from Th1, favoring a Th2 response which drives humoral responses toward the production of IgE (30). However, more recent findings have depicted PGE2 as a pleiotropic molecule that can act either negatively or positively on the immune system (29). Depending on the cell type, binding of PGE2 to one of its six described receptors (EP1, EP2, EP3I, EP3II, EP3III, and EP4) can lead to phospholipase C activation, phosphatidylinositol turnover increase, activation of adenylate cyclase via cholera toxin-sensitive G{alpha}S proteins and mobilization of intracellular Ca2+ concentration (31). PGE2 overproduction has been reported in HIV-1-infected individuals (32, 33, 34, 35), and it may contribute to the immunosuppressive state observed in such individuals (36). We have previously shown that interaction of PGE2 with EP4 receptor subtype in human T cells can up-regulate HIV-1 replication via both NF-{kappa}B-dependent and -independent signal transduction pathways (16). More recently, we observed that the PGE2-mediated induction of HIV-1 LTR activity was resulting from synergistic interactions between two specific intracellular second messengers, i.e., calcium and CREB (our unpublished observations).

In this study, we have addressed the functional role played by C/EBP family proteins and C/EBP binding sites in regulating HIV-1 LTR transcription in human T cells following PGE2-dependent cAMP stimulation. Through transient transfection experiments with wild-type and mutated versions of HIV-1 LTR-driven luciferase constructs, we demonstrate the importance of the two most proximal C/EBP binding sites in the PGE2-mediated induction of virus transcription. Results from EMSAs revealed that C/EBP proteins were translocated through the nucleus following treatment of human T cells with physiological concentrations of PGE2. Our results also suggest that PGE2-induced increase in cAMP level results in the formation of heterodimeric complexes composed of C/EBP{beta} and CREB, which interact with the two most proximal C/EBP binding sites located within the HIV-1 LTR region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PGE2, PMA, and PHA were purchased from Sigma-Aldrich (St. Louis, MO), while TNF-{alpha} was from R&D Systems (Minneapolis, MN). Forskolin was obtained from BioMol (Plymouth Meeting, PA). A stock solution of PGE2 (1 mM) was prepared by dissolving the lyophilized product into absolute ethanol and was stored at -20°C until needed.

Cells and culture conditions

The parental lymphoid T cell line, Jurkat E6.1, was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.22% NaHCO3, and were maintained at 37°C in a 5% CO2 humidified atmosphere.

Plasmids

In our studies, we have used the constructs pLTR-LUC, pm{kappa}BLTR-LUC, p-205{Delta}LTR-LUC, pmC2LTR-LUC, pmC3LTR-LUC, and pmC2,3LTR-LUC, which have been kindly provided by Dr. K. Calame (Columbia University, New York, NY). pLTR-LUC and pm{kappa}BLTR-LUC contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF-{kappa}B-mutated (CTCACTTTCC) HIV-1HXB2 LTR (-453 to +80) (13). p-205{Delta}LTR-LUC is a truncated version of pLTR-LUC lacking the negative regulatory element (NRE) of HIV-1. In pmC2LTR-LUC, pmC3LTR-LUC, and pmC2,3LTR-LUC vectors, the C/EBP binding sites of HIV-1 LTR (C2 (-178 to -159 bp) and C3 (-120 to -109)) contain site-directed mutations generated by PCR to either each or both sites (13). pCMV-I{kappa}B{alpha}S32A/36A expresses a dominant negative form of NF-{kappa}B under the control of the CMV promoter (37). We have also used the commercially available pCRE-LUC vector, which contains five tandem repeats of the CRE sequence and a TATA box placed upstream of the luciferase reporter gene (Stratagene, La Jolla, CA). The KCREB construct consists of a CREB cDNA with a single amino acid mutation in the DNA-binding domain, cloned into a mammalian expression vector driven by the Rous sarcoma virus promoter. This plasmid was kindly provided by Dr. R. H. Goodman (Vollum Institute for Advanced Biochemical Research, Portland, OR). The pCMV-LIP (liver-enriched transcriptional inhibitory protein), a generous gift from Dr. K. Calame, encodes for a truncated C/EBP protein that has only the DNA-binding and leucin zipper domains and possess a dominant negative role in transcriptional regulation of gene expression (38).

Transient transfection by DEAE-dextran

Jurkat E6.1 cells (5 x 106) were first washed once in a TS buffer (25 mM Tris-HCl (pH 7.4), 5 mM KCl, 0.6 mM NaHPO4, 0.5 mM MgCl2, and 0.7 mM CaCl2) and resuspended in 0.5 ml of TS containing 5–40 µg of the indicated plasmids and 500 µg/ml DEAE-dextran (final concentration). The cells/TS/plasmid/DEAE-dextran mix was incubated for 25 min at room temperature. Cells were then diluted at a concentration of 1 x 106/ml using complete culture medium supplemented with 100 µM chloroquine (Sigma-Aldrich). After 45 min of incubation at 37°C, cells were centrifuged, washed once, resuspended in complete culture medium, and incubated at 37°C for 24 h. To minimize variations in plasmid transfection efficiencies, transfected cells were pooled 24 h after transfection and were next separated into various treatment groups, as follows. Transiently transfected cells were seeded at a density of 105 cells per well (100 µl) in 96-well flat-bottom plates. Cells were either left untreated or were treated with PHA/PMA (3 µg/ml and 20 ng/ml, respectively), TNF-{alpha} (2 ng/ml), forskolin (100 µM), and PGE2 (100 nM) in a final volume of 200 µl for a period of 8 h at 37°C. All experiments were performed three times and luciferase activity was evaluated within each for four different similarly treated samples by a modified version of a previously published procedure (39). Briefly, following the incubation period, 100 µl of cell-free supernatant were withdrawn from each well and 25 µl of 5x cell culture lysis buffer (25 mM Tris phosphate (pH 7.8), 2 mM DTT, 1% Triton X-100, and 10% glycerol) were added before incubation at room temperature for 30 min. An aliquot of cell extract (20 µl) was analyzed in 96-well plates using a microtiter plate luminometer (MLX Dynex Technologies, Chantilly, VA). Each well was injected with 100 µl of luciferase assay buffer (20 mM tricine, 1.07 mM (MgCO3)4 · Mg(OH)2 · 5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP, and 33.3 mM DTT). Light output was measured for 20 s with a 2-s delay. Values are expressed as relative light units as measured by the apparatus.

EMSAs

Nuclear extracts were prepared according to the microscale preparation protocol as described previously (16). In brief, Jurkat cells (106) were either left untreated or were treated with PGE2 (100 nM) or forskolin (100 µM) for 30 min at 37°C and the protein content was determined by the commercial BCA Protein Assay Reagent (Pierce, Rockford, IL). Nuclear extracts were incubated for 30 min at room temperature in the binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM DTT, 5 mM EDTA, 250 mM NaCl, 2 µg of poly(dI-dC), 10 µg of nuclease-free BSA fraction V) containing 1 ng of 32P-5' end-labeled dsDNA oligonucleotide. dsDNA (100 ng) was labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase in a kinase buffer (New England Biolabs, Beverly, MA). This mixture was incubated for 30 min at 37°C and the reaction was stopped with 5 µl of 0.2 M EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed trough a G-50 spin column. The dsDNA oligonucleotide, which was used as a probe, contained the consensus C/EBP-binding site corresponding to the sequence 5'-TGCAGATTGCGCAAT-3' and was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The sequences of the synthetic C/EBP of HIV-1 LTR oligonucleotides (C/EBP-110 and C/EBP-170) are respectively 5'-CTAGCATTTCATCACGTGGCC-3' and 5'-CATCGAGCTTGCTACAAGGG-3'. For cold competition experiments, we used commercial consensus sequences for CREB (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'), and NFAT (5'-TCGAGCCCAAAGAGGAAAATTTGTTTCATG-3') (Santa Cruz Biotechnology). In supershift experiments, nuclear extracts were preincubated for 30 min on ice with 1 µg of either specific anti-C/EBP{beta} or anti-CREB polyclonal Abs purchased from Santa Cruz Biotechnology. DNA-C/EBP complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gel containing 0.5x Tris-borate-EDTA. The gel was subsequently dried and autoradiographed.

Preparation of protein extracts and immunoblotting

Nuclear extracts were prepared by hypotonic detergent lysis procedures as previously described (40). Whole cell lysates were generated by extraction with radioimmune precipitation assay buffer. Cells were washed once with PBS, scraped, pelleted, and resuspended in radioimmune precipitation assay buffer (40). Protein extracts were then incubated for 20 min on ice with periodic shaking. Cellular debris were pelleted down by centrifugation at 14,000 rpm for 5 min, and the supernatant was collected and stored at -80°C. Nuclear or whole cell extracts were mixed with an equal volume of sample buffer, heated at 100°C for 5 min, and loaded on 12% sodium dodecyl sulfate polyacrylamide gels. Proteins were transferred to Immobilon membranes (Millipore, Bedford, MA) and probed with Abs against C/EBP{beta} (Santa Cruz Biotechnology). The blots were developed using the ECL detection system (Pierce). Blots were then evaluated using an alpha-imager spot density calculator.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment of human T cells with PGE2 leads to activation and nuclear translocation of C/EBP family of proteins

To define the capacity of PGE2 to mediate a possible activation of C/EBP family members in human T lymphoid cells, EMSAs were performed with a C/EBP probe that bears a consensus CCAAT sequence. When this probe was incubated with nuclear extracts from Jurkat cells stimulated with PGE2 (100 nM) for 15, 30, and 60 min, a sustained signal was detected at every time point (Fig. 1Go; compare lane 1 with lanes 2-4). These data demonstrate a rapid accumulation of C/EBP in the nucleus of Jurkat cells as early as 15 min after PGE2 treatment. The specificity of the signal was shown by competition experiments using a 50-fold excess of the unlabeled oligonucleotide (Fig. 1Go, lane 5). A similar complex was formed when cells were treated for 15 min with forskolin (100 µM), a powerful cAMP-inducing agent, and such complex was also competed away by an excess of the unlabeled C/EBP probe (Fig. 1Go; compare lanes 6 and 7). Data from this series of investigations indicate that a rise in cAMP levels mediated either by PGE2 or forskolin treatment can lead to nuclear translocation and activation of C/EBP transcription factors in human T cells.



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  		FIGURE 1. Treatment of human T cells with PGE2 results in nuclear translocation and activation of C/EBP. Jurkat cells were either left untreated or were treated with 100 nM PGE2 for various periods of time (0, 15, 30, and 60 min) and 100 µM forskolin for 15 min. The nuclear extracts (4 µg) were next incubated with a 32P-end-labeled synthetic double-stranded C/EBP probe (consensus sequence). Competition experiments were achieved by using 50-fold excess of cold C/EBP probe. An arrow on the left indicates the position of the specific complex bound by the C/EBP site probe.

 
Role of C/EBP binding sites in PGE2-mediated increase in HIV-1 LTR activity

Next, we assess the importance of C/EBP binding sites located within the HIV-1 LTR in the previously reported PGE2-dependent enhancement of virus transcription by using HIV-1 LTR reporter constructs carrying appropriate site-directed mutations. Three putative binding sites for C/EBP proteins have been described so far in the HIV-1 LTR domain (Fig. 2GoA), one located in the NRE (C1, located between -340 and -185 relative to the transcriptional start site) and the two others placed upstream from the two NF-{kappa}B binding sites (C2, from -178 to -159; C3, from -120 to -109). As depicted in Fig. 2GoB, deletion of the most distal C/EBP site did not diminish transcription relative to the wild-type construct in cells treated with PGE2. On the contrary, the p-205{Delta}LTR-LUC vector, which still bears the two most proximal C/EBP binding sites, is even slightly more active than the full-length LTR in response to PGE2 stimulation (compare 12.5- and 8.9-fold increases). Interestingly, the constructs containing mutations at either of the two most proximal C/EBP sites (i.e., pC2LTR-LUC and pC3LTR-LUC) had activities that were significantly decreased and were only minimally different from those of the construct lacking both proximal sites (i.e., pC2, 3LTR-LUC). These data suggest that the two most proximal C/EBP binding sites are functionally equivalent in the activation of HIV-1 LTR activity that is seen upon PGE2 treatment of human T lymphoid cells. It should be mentioned that deletion of each or both of the two most proximal C/EBP binding sites had no effect on LTR activity in Jurkat cells stimulated by the proinflammatory cytokine TNF-{alpha} (2 ng/ml) or the combination of PHA and PMA (3 µg/ml and 20 ng/ml, respectively) (data not shown).



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  		FIGURE 2. C/EBP binding sites are required for PGE2-induced activation of HIV-1 LTR transcription. A, Viral transcription is controlled by several cis-acting promoter elements that are located within the HIV-1 LTR. Three C/EBP binding sites that lie upstream of the tandem NF-{kappa}B sites are found in the HIV-1 LTR. B, Jurkat cells were transfected with 10 µg of various truncated and mutated HIV-1 LTR constructs depicted in A. C, In some experiments, cells were also cotransfected with pCMV-I{kappa}B{alpha}S32A/36A, a vector coding for a specific inhibitor of NF-{kappa}B. Such transiently transfected cells were either left untreated (control) or were treated with 100 nM of PGE2 for 8 h. Finally, cell lysates were evaluated for luciferase activity with a microplate luminometer. Results shown are the means ± SD of four determinations and are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1). These results are representative of three independent experiments.

 
We have previously shown that PGE2 mediates up-regulation of HIV-1 LTR activity partly via the ubiquitous mammalian transcription factor NF-{kappa}B (16). In an attempt to define the exact contribution of C/EBP binding site(s) in the noticed effect of PGE2 on HIV-1 LTR and to eliminate the potential interference made by NF-{kappa}B, cotransfection studies were conducted with a vector coding for a dominant negative repressor of NF-{kappa}B (i.e., pI{kappa}B{alpha}S32A/36A). As expected, the ability of PGE2 to activate the full-length HIV-1 LTR (i.e., pLTR-LUC) was reduced by cotransfecting Jurkat cells with pI{kappa}B{alpha}S32A/36A (compare Fig. 2Go, B and C). This observation is in agreement with our previous findings that indicate that PGE2 posi-tively affect the regulatory sequences of HIV-1 through both NF-{kappa}B-dependent and -independent signal transduction pathways (16). Cotransfection of Jurkat cells with the construct lacking the two most proximal C/EBP binding sites and the dominant negative repressor of NF-{kappa}B resulted in complete inhibition of PGE2-induced activation, indicating that the positive PGE2-dependent modulatory effect on HIV-1 LTR activity involves NF-{kappa}B and transcription factor(s) that can bind to C/EBP sites.

Functional role of C/EBP proteins in the induction of HIV-1 LTR transcription by PGE2

To further incriminate the involvement of C/EBP in PGE2-mediated effect on HIV-1 transcription, transient transfection experiments were conducted using the expression vector pCMV-LIP. This vector codes for a truncated form of C/EBP{beta}, LIP that acts as a trans-dominant negative regulator of C/EBP family activators. LIP contains the conserved basic leucine zipper domain which permits DNA binding and dimerization but lacks the strong transcriptional activation domain. Thus, inactive heterodimers are preferentially formed between LIP and C/EBP activators rather than self-heterodimers (13, 38). Cotransfection of Jurkat cells with the full-length HIV-1 LTR along with increasing amounts of pCMV-LIP resulted in a dose-dependent decrease of PGE2-mediated reporter gene activity (Fig. 3GoA). This inhibitory effect is cAMP dependent because the C/EBP dominant negative LIP also impairs HIV-1 LTR activity following forskolin stimulation, whereas it has no noticeable effect on PHA/PMA-induced activation.



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  		FIGURE 3. Involvement of the transcription factor C/EBP{beta} in PGE2-mediated up-regulation of HIV-1 LTR activity. Jurkat cells were transiently cotransfected with 10 µg of either pLTR-LUC (A) or pm{kappa}BLTR-LUC (B) along with increasing concentrations of pCMV-LIP, a trans-dominant negative form of C/EBP{beta} (filled bars, 0 µg; shaded bars, 10 µg; open bars, 20 µg; and checkered bars, 30 µg). Next, such transiently transfected Jurkat cells were either left untreated or were treated for 8 h with PHA/PMA (3 µg/ml and 20 ng/ml, respectively), forskolin (100 µM), and PGE2 (100 nM). Finally, cell lysates were evaluated for luciferase activity with a microplate luminometer. Results shown are the means ± SD of four determinations and are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1). These data are representative of three independent experiments.

 
The pm{kappa}BLTR-LUC construct contains the complete HIV-1 LTR region but lacks the two {kappa}B binding sites. This vector is thus ideal to identify the region(s) responsible for NF-{kappa}B-independent activation of HIV-1 transcription in human T cells treated with PGE2. Cotransfection of pm{kappa}BLTR-LUC and increasing concentrations of pCMV-LIP led to a marked decrease in both PGE2- and forskolin-mediated induction of LTR activity (Fig. 3GoB). These results are consistent with those obtained with the full-length HIV-1 LTR (Fig. 3GoA). Again, the introduction of LIP did not alter luciferase activity in cells stimulated with the combination of PHA and PMA.

PGE2 treatment leads to the formation of heterodimeric C/EBP{beta}-CREB complexes

EMSAs were next performed to examine proteins in Jurkat nuclear extracts which could bind to the two most proximal C/EBP binding sites in the HIV-1 LTR domain (called C/EBP-110 and C/EBP-170 for the purpose of these experiments). First, using an oligonucleotide probe representing the C/EBP-110 site, a more intense retarded complex was observed following treatment of Jurkat cells with either PGE2 or forskolin (Fig. 4GoA; compare lane 2 with lanes 3 (forskolin) and 4 (PGE2)). The formation of this complex was competed for by an excess of either the homologous (C/EBP-110) or heterologous (C/EBP-170) competitor (Fig. 4Go; compare lane 4 with lanes 5 (C/EBP-110) and 6 (C/EBP-170)). Interestingly, this complex was also competed away by an excess of both unlabeled consensus C/EBP (Fig. 4Go; compare lanes 4 and 7) and CRE (Fig. 4Go; compare lanes 4 and 8) but not a NFAT probe (Fig. 4Go; compare lanes 4 and 9). NFAT was chosen as a negative control because previous unpublished observations suggest that NFAT is not activated upon treatment of Jurkat cells with PGE2. The induced complex was almost totally abrogated when a C/EBP{beta} or CREB polyclonal antiserum was included in the EMSA reaction mixture (Fig. 4Go; compare lane 4 with lanes 10 (anti-C/EBP{beta}) and 11 (anti-CREB)), demonstrating that both C/EBP{beta} and CREB are major components of the C/EBP-110 binding site complex in Jurkat cells exposed to PGE2. Of interest to note was the formation of two distinct complexes (CI and CII) when using an oligonucleotide probe representing the C/EBP site which spans the -170-bp site in the HIV-1 LTR (Fig. 4GoB). Moreover, the second complex (i.e., CII) is primarily affected by an excess of unlabeled probes for either C/EBP-110, C/EBP-170, consensus C/EBP, or CRE. Ab against C/EBP{beta} or CREB altered the formation of the second complex without affecting the upper one, suggesting that neither CREB nor C/EBP{beta} is implicated in the formation of CI. We also used the consensus sequence for C/EBP as a probe for gel shift assays, and results were consistent with those obtained with the C/EBP-110 probe, except that competition with C/EBP and CRE caused a more complete disappearance of the complex induced by PGE2 treatment (Fig. 4GoC).



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  		FIGURE 4. PGE2 mediates binding of C/EBP{beta} and CREB to the two most proximal C/EBP binding sequences located within the HIV-1 LTR. EMSAs were performed with nuclear extracts from Jurkat cells either left untreated or treated with 100 nM PGE2 or 100 µM forskolin. Next, nuclear extracts were incubated with double-stranded radiolabeled oligonucleotide probes spanning either the C/EBP-110 (A), the C/EBP-170 (B), or consensus C/EBP binding site (C). Competition experiments were achieved by using 50-fold excess of the indicated cold probes. These reactions were also conducted in the absence or the presence of Ab directed against C/EBP{beta} or CREB. Arrows on the left indicate the positions of the specific DNA-protein complexes. The unreacted free probe is visible at the bottom of the gel. Lane 1 consists of the probe only.

 
To scrutinize the possible functional synergistic interaction between C/EBP and CREB within the context of the HIV-1 LTR, Jurkat cells were cotransfected with HIV-1 LTR constructs containing mutations in each of the two proximal C/EBP binding sites along with a vector coding for KCREB, a trans-dominant negative repressor of CREB. KCREB mutant contains a single base pair substitution in the DNA-binding domain (changing Arg287 to Leu) that destroys its DNA-binding ability and allow the quenching of factor(s) capable of associating with CREB by heterodimerization (41). Data obtained from this set of experiments demonstrate that addition of KCREB severely reduced PGE2-induced up-regulation of HIV-1 LTR activity (Fig. 5Go). Indeed, levels of diminution of 62.2 and 58.9% were observed with mC2LTR-LUC and mC3LTR-LUC constructs, respectively. A comparable reduction in HIV-1 LTR-driven gene expression was obtained with the other intracellular cAMP-increasing agent, forskolin. These experiments were repeated several times and gave consistent results. Altogether our data show that activation of the HIV-1 promoter in human T lymphoid cells with the cAMP inducer agent PGE2 involved the participation of both C/EBP and CREB transcription factors.



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  		FIGURE 5. Implication of both C/EBP and CREB in PGE2-mediated induction of HIV-1 LTR activity. Jurkat cells were transiently transfected with 10 µg of HIV-1 LTR molecular constructs bearing mutations in the C/EBP binding sites (A, pmC2LTR-LUC; B, pmC3LTR-LUC) either in the absence (open bars) or the presence of 20 µg of a construct coding for a dominant negative form of CREB (i.e., pKCREB) (filled bars). Cells were then stimulated with PHA/PMA (3 µg/ml and 20 ng/ml, respectively), TNF-{alpha} (2 ng/ml), forskolin (100 µM) or PGE2 (100 nM) for 8 h. Finally, cell lysates were evaluated for luciferase activity with a microplate luminometer. Results shown are the means ± SD of four determinations and are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1). These results are representative of three independent experiments.

 
Treatment of Jurkat cells with PGE2 results in nuclear translocation of C/EBP. In an attempt to demonstrate that PGE2 treatment of Jurkat cells modifies the amount of C/EBP{beta} in the nucleus, Western blot experiments were performed with whole cell lysates and nuclear extracts from treated cells. As shown in Fig. 6Go, a transient accumulation of C/EBP{beta} in the nucleus is seen over time following treatment of Jurkat cells with PGE2 (i.e., 2- to 3.5-fold increase). This noticed change in the nuclear concentration of C/EBP{beta} is not due to an increased synthesis of this protein because its concentration remained constant in whole cell extracts.



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  		FIGURE 6. PGE2 induces nuclear accumulation of C/EBP{beta} proteins in Jurkat cells. Jurkat T cells were treated with PGE2 (100 nM) and proteins were purified from cells at different time periods after treatment (0, 5, 10, 20, 30, and 60 min). The nuclear extracts (A) and whole cell proteins (B) (60 µg/lane) were directly loaded on a 12% SDS-polyacrylamide gel, transferred to a membrane, and revealed by an anti-C/EBP{beta} Ab. The relative protein levels were quantified by laser densitometry scanning (C).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The formation and production of elevated levels of inflammatory mediators such as PGE2 are a hallmark of HIV-1 infection (33, 34, 35). Prostaglandins play an important role in disease exacerbation by directly altering T cell functions or macrophage activation. Although PGE2 was initially envisaged as an immunosuppressive molecule that acts as a down-regulator of many aspects of B and T cell function and proliferation, recent findings support a role for PGE2 as a potentiator of Ig class switching and of cytokine and cytokine receptor synthesis (29). Given that PGE2 is a strong inducer of cAMP and that a 4-fold increase in intracellular levels of cAMP is seen in asymptomatic HIV-1-seropositive subjects as compared with uninfected controls (42), we previously assessed whether PGE2 could affect HIV-1 transcriptional activity in human T cells. Our findings demonstrate that PGE2 can act as a potent activator of HIV-1 LTR-driven transcription through an effect on both NF-{kappa}B-dependent and -independent signaling events (16). More recently, calcium and the CREB transcription factor were also found to be essential second messengers in the PGE2-mediated up-regulation of LTR activity in T cells (our unpublished observation). Although the binding of a member of the CREB family to the HIV-1 LTR via the CRE consensus sequence has not yet been described, it has been postulated that CREB can act indirectly on the regulatory elements of this retrovirus. For example, it has been shown that CREB interacts with HIV-1 LTR through an association with transcription factors such as TFIID and TFIIB (43, 44, 45) or with the adapter CBP; the latter is known to interact with the general transcription machinery (46). Recently, a recognition sequence for members of the ATF/CREB family was identified within the untranslated leader region of HIV-1 as a novel TRE-like CRE capable of binding both AP-1 and ATF/CREB (47). However, the U5 region of the HIV-1 LTR is absent from our molecular constructs, rejecting the possible implication of TRE-like CRE in the noticed PGE2-induced viral activation. A recent report has shown that dopamine treatment of HIV-1-infected T cells leads to the binding of CREB to the COUP-TF sequence that is located at the 5' end of the HIV-1 LTR in a region called the NRE (43). The various LTR constructs used in the present study do not bear the NRE, suggesting that the COUP-TF binding domain is not participating in PGE2-mediated effect. In the present study, we have analyzed in more details the precise binding site(s) or binding mechanism of the transcription factor CREB on HIV-1 LTR following treatment with PGE2.

The recent observation that complex interactions appear to occur between ATF/CREB and C/EBP in the context of the HIV-1 LTR (6) has prompted us to assess the ability of PGE2 to mediate C/EBP activation in human T lymphoid cells. The C/EBP family of nuclear proteins is a member of a larger superfamily of transcription factors characterized by the b-ZIP motif that also includes the ATF/CREB family (48). In a number of cell types, C/EBP{beta} has been shown to function as a cAMP-activated transcription factor (49, 50, 51). We found that treatment of Jurkat cells with PGE2 resulted in a noticeable induction of nuclear translocation and activation of C/EBP. Indeed, DNA mobility shift assays provided clear evidence that PGE2 and forskolin treatment of human T cells increases the level of specific protein-DNA complexes when the consensus C/EBP binding site is used as a molecular probe (Fig. 1Go). Although treatment of Jurkat cells with PGE2 did not alter the protein level of C/EBP{beta} in whole cell extracts, there was a redistribution of this protein from the cytoplasm to the nucleus upon exposure to PGE2 (Fig. 6Go).

A previous study has reported that C/EBP proteins and their binding sites are required for HIV-1 proviral induction in both promonocytic U937 cells and primary macrophages. In their experimental system, Henderson and colleagues (52, 53) have demonstrated that Jurkat and H9 T lymphoid cell lines express several C/EBP family transcriptional activators, including C/EBP{alpha}, C/EBP{beta}, and C/EBP{delta}, which are present primarily in the nuclear fraction. However, none of these proteins was found to be induced upon activation with the diacylglycerol analog PMA or the mitogenic agent Con A (53). We noticed that PGE2 and forskolin, both of which are known to induce an elevation of the intracellular cAMP level, were able to potently activate C/EBP in human T cells. The functional role played by C/EBP in PGE2-induced activation of virus transcription was demonstrated by transient transfection experiments using vectors carrying mutations in C/EBP binding sites that are present in the HIV-1 LTR. PGE2-mediated increase in HIV-1 LTR-driven luciferase activity was considerably reduced when Jurkat cells were transfected with molecular constructs bearing site-directed mutations within the two most proximal C/EBP binding site(s) (Fig. 2Go). C/EBP sites were not involved in the induction of LTR activity in response to PHA/PMA or TNF-{alpha} (data not shown). These data were expected, considering that such stimuli are unable to lead to an increase in cAMP level. The biological relevance of the HIV-1 C/EBP binding sites is provided by the observation that this sequence appears to be well conserved in primary HIV-1 isolates from AIDS patients over several years (54).

It has been previously demonstrated that individual C/EBP proteins can homodimerize or heterodimerize with other members of the C/EBP family of b-ZIP domain proteins to elicit specific cAMP-mediated transcriptional stimulation or repression (9, 49, 55). Moreover, it is now believed that transcriptional regulation of genes containing the recognition sites of either C/EBP or ATF/CREB may result from heterodimeric formation between different members in each of the C/EBP and ATF/CREB families (56). This mechanism may be used to respond to complex signals and transcriptional cues through single sequence elements including a response to cAMP, despite the absence of active CRE, AP1, and AP2 consensus nucleotide sequences (57, 58, 59). The best example is provided by the CFTR gene promoter that is controlled by interactions between C/EBP and ATF/CREB family members with CREB1 and ATF1 binding to the inverted CCAAT element of this gene to finely regulate its transcription (59). Although the absence of CRE certainly may not preclude ATF or CREB protein from targeting promoters devoid of such cis-acting elements, it is interesting to note that regulation of the somatostatin gene requires protein-protein interaction between C/EBP and ATF/CREB transcription factors to elicit a cAMP-dependent response through the CRE element (60). Inversely, C/EBP proteins have been shown to bind specifically to the phosphoenolpyruvate carboxykinase gene CRE with high affinity to promote cAMP-mediated transcriptional activation (61). In addition, previous studies identified C/EBP as an effector of cAMP-mediated transcription of the phosphoenolpyruvate carboxykinase gene through combined interactions with liver-specific transcription factors (62, 63). Experiments conducted with a vector coding for LIP suggested that C/EBP was playing a crucial role in activation of HIV-1 LTR-driven gene expression that is seen following treatment of human T cells with PGE2 (Fig. 3Go). It should be noted that the {beta}-isoform of C/EBP has been intimately linked with the cAMP signaling system, as exemplified by the reported capacity of cAMP to stimulate C/EBP{beta} gene expression (61) and translocation of C/EBP{beta} from the cytosol to the nucleus (49). Thus, we propose that the PGE2-dependent increase in HIV-1 LTR transcriptional activity is mediated in part by C/EBP{beta}. Our data indicate that the dominant negative form of C/EBP, i.e., LIP, has less impact on PGE2-mediated induction of HIV-1 LTR-driven activity than mutating the C/EBP binding sites, suggesting that factors in addition to C/EBP may be binding to C/EBP binding sites. Experiments are presently underway to address this issue.

Results gathered from mobility shift assays suggested that PGE2 treatment of human T cells results in nuclear translocation of C/EBP{beta}-CREB complexes that can bind to the two most proximal C/EBP sites located within the HIV-1 LTR (Fig. 4Go). The formation of heterodimeric complexes constituted of C/EBP{beta} and CREB transcription factors in response to PGE2 in T cells was clearly shown by cross-competition and supershift assays. The use of probes specific for the two most proximal C/EBP binding sites within the HIV-1 LTR (C/EBP-110 and -170) have confirmed the importance of both sites. Finally, the functional role played by CREB in PGE2-mediated activation of HIV-1 LTR was shown by using a dominant negative version of CREB (Fig. 5Go). Our findings that part of the PGE2-dependent positive modulatory effect on HIV-1 LTR activity is due to heterodimeric formation between C/EBP{beta} and CREB is perfectly in line with a recent study by Ross and coworkers (6). These investigators have indeed reported that HIV-1 LTR transcription is enhanced following stimulation of U937 monocytoid cells with IL-6 due to synergistic interactions between C/EBP and CREB transcription factors.

In summary, we have more deeply investigated the regulation of HIV-1 gene transcription following PGE2-mediated increase in intracellular cAMP levels in human T cells. We have identified C/EBP{beta} as a PGE2-activated transcriptional regulator of HIV-1 LTR in Jurkat cells and demonstrated that C/EBP binding sites are functionally important for virus transcription. We also suggest that functional and physical association between members of two important transcription factor families, i.e., C/EBP{beta} and CREB, are required for activation of HIV-1 transcription by PGE2. Our findings represent a further indication of the high complexity of the molecular mechanisms that regulate HIV-1 gene expression following treatment of human T cells with PGE2.


   Acknowledgments
 
We thank K. L. Calame for pLTRLUC, pm{kappa}BLTR-LUC, p-205{Delta}LTR-LUC, pmC2LTR-LUC, pmC3LTR-LUC, pmC2,3LTR-LUC, and pCMV-LIP; W.C. Greene for pCMV-I{kappa}B{alpha}S32A/36A; and R.H. Goodman for KCREB. We thank Dr. Corinne Barat for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a Canadian Institutes of Health Research Group grant to M.J.T. and M.O. (Grant MGC-14500). N.D is the recipient of a Ph.D. Fellowship from the Fonds de la Recherche en Santé du Québec/Fonds pour la Formation de Chercheurs et l’Aide à la Recherche-Programme Santé, and S.B. is the recipient of a Ph.D. Fellowship from the Canadian Institutes of Health Research. M.J.T. holds a Tier 1 Canada Research Chair in Human Immuno-Retrovirology. M.O. is the recipient of an Investigator Award from the Canadian Institutes of Health Research and is a Burroughs Wellcome Fund Awardee in Molecular Parasitology. Back

2 Address correspondence and reprint requests to Dr. Martin Olivier and Dr. Michel J. Tremblay, Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, RC-709, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail addresses: michel.j.tremblay@crchul.ulaval.ca and martin.olivier{at}crchul.ulaval.ca Back

3 Abbreviations used in this paper: PKA, protein kinase A; LTR, long terminal repeat; CRE, cAMP-responsive element; b-ZIP, basic leucine zipper; ATF, activation transcription factor; C/EBP, CCAAT/enhancer binding protein; NRE, negative regulatory element; LIP, liver-enriched transcriptional inhibitory protein; TRE, 12-O-tetradecanolyphorbol-13-acetate/phorbol ester responsive element. Back

Received for publication March 20, 2001. Accepted for publication November 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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