INTRODUCTION

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Infection with human immunodeficiency virus type-1 (HIV-1),¹ the etiologic agent of AIDS (1), leads to a progressive decline of CD4-expressing T cells resulting in impaired cellular immune functions. This infection is influenced by a complex interplay between viral and host factors, as well as by microbial agents termed cofactors. 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 cells (2). Mycoplasma (3), mycobacteria (4), viruses (5, 6), and the protozoan parasite Leishmania (7, 8) may act as cofactors for the pathogenesis of HIV-1 infection either by directly modulating virus replication or by inducing a more profound immunosuppressive state. During coinfections, 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 contain a cyclopentane ring structure.

PGE₂ are molecules that have been shown to modulate the immune response both in vitro and in vivo (9, 10). Macrophages, follicular dendritic cells, fibroblasts, and vascular endothelial cells synthesize PGE₂, while lymphocytes do not secrete this major product of arachidonic acid metabolism (11-14). A marked increase in PGE₂ production is generated in response to a variety of immunological stimuli including interleukin (IL)-1, tumor necrosis factor- (TNF-), antigen-antibody complexes, and lipopolysaccharide (15). Moreover, production of PGE₂ has been shown to be induced by infection with several pathogens such as Leishmania donovani (16), Leishmania major (17), Entamoeba histolytica (18, 19), Pseudomonas aeruginosa (20, 21), Staphylococcus epidermidis (22), Mycobacterium avium (23), herpes simplex virus type 1 (24), coxsackie virus (25), respiratory syncytial virus (26), and HIV-1 (27-30). PGE₂ has been implicated in decreasing T-cell proliferation, IL-2 production, and IL-2 receptor expression (15, 31-35). PGE₂ shifts the balance of the cellular immune response away from T-helper type 1 (Th1) favoring a Th2 response which drives humoral responses toward the production of IgE (36). However, more recent findings have depicted PGE₂ as a pleiotropic molecule that can act both negatively or positively on the immune system (15).

An overproduction of PGE₂ (as high as 10⁴ M) is seen in a number of disorders (e.g. allergy, hyper-IgE syndrome, Hodgkin lymphoma, trauma, sepsis, and transplantation), most of which are characterized by elevated Th2 and IgE responses (15, 36-38). As specified above, elevated levels of PGE₂ have also been reported in individuals infected with HIV-1 (27-30) and it has been postulated that this may contribute to the immunosuppressive state seen in such virally-infected patients (39). The mechanism(s) responsible for the enhanced prostaglandin formation is still undefined. The initial contact between the virus particle and its target cell might represent the crucial step leading to the production of PGE₂ by macrophages. This concept is supported by the finding that a significant production of endogenous PGE₂ is induced (20- to 40-fold increase) following incubation of primary human monocytes with the HIV-1 external envelope glycoprotein gp120 (40). However, in sharp contrast with this report, a previous study has demonstrated that interaction between gp120 and THP-1, a human monocytoid cell line, does not increase exogenous production of PGE₂ (39). It is important to specify that, unlike monocyte/macrophages, promonocytoid THP-1 cells are not at a terminal stage of differentiation. In addition, a monomer form of gp120 was used in this study which might not parallel physiological conditions where gp120 is under a multimeric form (41). Depending on the cell type, binding of PGE₂ to one of its six different described receptors (EP₁, EP₂, EP_3I, EP_3II, EP_3III, and EP₄) can lead to activation of phospholipase C, phosphatidylinositol turnover, activation of adenylate cyclase via cholera toxin-sensitive G_S proteins and mobilization of intracellular Ca²⁺ (42). Given that HIV-1 is highly dependent on intracellular signaling machinery for its life cycle, it is therefore possible that interaction of PGE₂ with its surface receptor(s) can modulate virus replication. A previous cellular study supports this postulate since PGE₂ was found to enhance HIV-1 replication in acutely infected T lymphoid cells (43).

The primary goal of the present work was thus to investigate the putative modulatory role of PGE₂ on the regulatory elements of HIV-1 (LTR) at both biochemical and molecular levels. For this purpose, we treated human T lymphoid cells stably and transiently transfected with different HIV-1 LTR-driven luciferase reporter gene vectors with concentrations of PGE₂ known to be found under physiological conditions. We then explored the intracellular second messengers participating in PGE₂-mediated signaling transduction pathway(s) and investigated DNA-binding transcriptional factor(s) and cell surface receptor(s) implicated in the PGE₂-dependent effect on HIV-1 transcription.

	EXPERIMENTAL PROCEDURES

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Reagents-- PGE₂, phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), indomethacin and dibutyryl-cAMP were purchased from Sigma. H7 was purchased from Seikagaku America Inc. (Tampa, FL). BAPTA/AM, IBMX, MDL-12,330A, and HA-1004 were purchased from BioMol (Plymouth Meeting, PA). The calcium inhibitor CAI was a generous gift from Dr. E. C. Kohn (National Institutes of Health, Bethesda, MD). AH 23848B was kindly provided by Dr. S. G. Lister (Glaxo Wellcome, United Kingdom). Trizol came from Life Technologies, Inc. (Grand Island, NY). Stock solutions of PGE₂ (1 mM) were prepared by dissolving the lyophilized product into absolute ethanol and were 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 (ATCC, Rockville, MD), while 1G5 was supplied by the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institute of Health (Rockville, MD). 1G5 is a clonal cell line derived from Jurkat E6.1 cells which has been stably transfected with a luciferase gene driven by the HIV-1 LTR (44). The cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.22% NaHCO₃, and were maintained at 37 °C in a 5% CO₂ humidified atmosphere.

Plasmids and Antibodies-- In our studies, we have used pLTR-LUC and pmBLTR-LUC that have been kindly provided by Dr. K. Calame (Columbia University, NY). These molecular constructs contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF-B-mutated (CTCACTTTCC) HIV-1_HXB2 LTR (453 to +80) (45). We have also used the pB-TATA-LUC vector which contains the minimal HIV-1 B region and a TATA box placed upstream of the luciferase reporter gene (46). This plasmid is a generous gift from Dr. W. C. Greene (The J. Gladstone Institutes, San Francisco, CA). The molecular construct pNF-B-LUC contains five (5) consensus sequences of NF-B-binding sites placed in front of the luciferase reporter gene (Stratagene, La Jolla, CA). NFAT-LUC contains the IL-2 minimal promoter with three tandem copies of NFAT-binding site placed upstream of the luciferase reporter gene (kindly provided by Dr. G. R. Crabtree, Howard Hughes Medical Institute, CA). Northern blot analyses were performed using human EP₁, EP₂, EP₃, and EP₄ subtypes cDNA fragments. The cDNAs for human prostanoid receptor EP₁ (1.3 kilobases), EP₃ (1.8 kilobases), and EP₄ (1.5 kilobases) were kind gifts from Dr. M. Abramovitz (Merck Frost, Qué, Canada). The cDNA for hEP₂ (1.1 kilobases) was generously provided by Dr. K. M. Kedzie (Allergan, Irvine, CA). The polyclonal antibody specific for EP₄ was obtained from Cayman Chemical (Ann Arbor, MI). This rabbit serum is directed against a synthetic peptide from the human EP₄ receptor.

Modulation of HIV-1 LTR Activity by PGE₂-- In order to assess whether PGE₂ could modulate HIV-1 LTR activity, 1G5 cells (5 × 10⁵) were either left untreated or treated with PHA (3 µg/ml), anti-CD3 antibody (clone OKT3 at 1 µg/ml), PMA (20 ng/ml), and TNF- (2 ng/ml: R&D systems, Minneapolis, MN) in a final volume of 200 µl for 1 h at 37 °C. Next, the cells were incubated in the absence or presence of 100 nM PGE₂ for 24 h at 37 °C. Dose-response experiments were done using a similar number of cells, washed once in phosphate-buffered saline (PBS, pH 7.4), and resuspended in 1 ml of fresh complete culture medium before incubation for 24 h at 37 °C with PGE₂ at final concentrations of 1, 10, 100, and 1000 nM. Kinetic experiments were done by incubating 1G5 cells with 100 nM PGE₂ for 2, 6, 8, and 24 h. In some experiments, 1G5 cells were pretreated for 1 h at 37 °C with second messenger inhibitors such as H7, HA-1004, BAPTA/AM, indomethacin, MDL-12,330A, and IBMX at subcytotoxic and subcytostatic concentrations prior to treatment with 100 nM PGE₂ for 8 h at 37 °C. The inhibitor CAI requires a pretreatment of at least 8 h for optimum inhibition and shows no acute interference with the growth properties of the cells (47). All experiments were performed three times and luciferase activity was evaluated for each quadruplicate samples by a modified version of a previously published procedure (48). Briefly, following the incubation period, 100 µl of cell-free supernatant were withdrawn from each well and 25 µl of cell culture lysis buffer (25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 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 mixed 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 dithiothreitol) and the sample was read in the counting chamber of a standard liquid scintillation counter equipped with a single-photon monitor software (Beckman Instruments, Fullerton, CA). Total photo-events were measured over a 30-s time lapse.

Transient Transfection by DEAE-dextran-- Jurkat E6.1 (5 to 10 × 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 0.5-1 ml of TS containing 10-20 µg of the indicated plasmids and 500 µg/ml DEAE-dextran (final concentration). The cells/TS/plasmid/DEAE-dextran mixture was incubated for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 × 10⁶ per ml using complete culture medium supplemented with 100 µM chloroquine (Sigma). 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 follow. Transiently transfected cells were seeded at a density of 10⁵ cells per well (100 µl) in 96-well flat-bottom plates. Cells were left untreated or were treated with TNF- (2 ng/ml), PHA/PMA (3 µg/ml and 20 ng/ml, respectively), and 100 nM PGE₂ in a final volume of 200 µl for a period of 8 h at 37 °C. Cells were then lysed and luciferase activity was assessed as described above.

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared as described previously (49). In brief, 1G5 cells (10⁶) were either left untreated or were treated with PGE₂ (100 nM) or TNF- (2 ng/ml) for 30 min at 37 °C. The incubation of cells with the stimulating agents was terminated by the addition of ice-cold PBS and nuclear extracts were prepared according to the microscale preparation protocol (50). Sedimented cells were resuspended in 400 µl of cold buffer A (10 nM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 nM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride). After 10 min on ice, the lysate was vortexed for 10 s and the samples were centrifuged for 10 s at 12,000 × g. The supernatant fraction was discarded and the pellet was resuspended in 100 µl of cold buffer B (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. Cellular debris were removed by centrifugation at 12,000 × g for 2 min at 4 °C and the supernatant fractions were stored at 70 °C until used. Ten micrograms of nuclear extracts were used to perform electrophoretic mobility shift assay. Protein content was determined by the commercial BCA Protein Assay Reagent (Pierce, Rockfold, IL). Nuclear extracts were incubated for 30 min at room temperature in 15 µl of buffer of a binding solution (100 mM HEPES, pH 7.9, 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol, 5 mM EDTA, 250 mM NaCl, 2 µg of poly(dI-dC), 10 µg of nuclease-free bovine serum albumin fraction V) containing 1 ng of ³²P-5'-end-labeled double-stranded (dsDNA) oligonucleotide. Double-stranded DNA (100 ng) was labeled with [-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 double-stranded DNA oligonucleotide, which was used as a probe, contained the consensus NF-B-binding site corresponding to the sequence 5'-ATGTGAGGGGACTTTCCCAGGC-3' and was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Ca). DNA·NF-B complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gel containing 50 mM Tris-HCl (pH 8.5), 200 nM glycine, and 1 mM EDTA. The gel was subsequently dried and autoradiographed.

Northern Blot Analysis-- Total RNA was extracted by the Trizol method (51, 52) from 1G5 and Jurkat E6.1 cells. Fifteen micrograms of total RNA were separated on formaldehyde-agarose gel (1% agarose, 1 × formaldehyde gel buffer MOPS, pH 7.0, 40 mM sodium acetate, 5 mM EDTA, 2.2 M formaldehyde). RNA was transferred to Hybond-N nylon membranes (Amersham) by capillary action using 10 × SSC (3 M NaCl, 0.3 M sodium citrate). RNA was fixed to the membrane by UV exposure and hybridized with radiolabeled probes for the EP receptors (EP₁, EP₂, EP₃, and EP₄) at 42 °C in 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 100 µg/ml denatured fragmented salmon sperm DNA. Blots were washed and autoradiographed at 70 °C.

Flow Cytometry-- Cell surface expression of hEP₄ receptor was evaluated by flow cytometry as follow. Cell lines 1G5 and Jurkat E6.1 (5 × 10⁵) were washed once in PBS containing 2% fetal bovine serum (PBS pH 7.4 + 2% fetal bovine serum (PBSA)). Cells were then resuspended in 100 µl of PBSA to which was added 0.5 µg of polyclonal rabbit anti-hEP₄ antibody, vortexed gently, and incubated for 30 min on ice. Cells were subsequently washed with PBSA and resuspended in 100 µl of PBSA containing fluorescein isothiocyanate-labeled chicken anti-rabbit IgG antibody (0.5 µg total) and further incubated for 30 min on ice. Cells were finally centrifuged and resuspended in 1% paraformaldehyde in PBS before being analyzed by flow cytometry (EPICS XL, Coulter Corp., Miami, FL).

Experiments with the EP₄ Receptor-specific Antagonist AH 23848B-- Assays with AH 23848B were performed by incubating 1G5 cells with 30 µM AH 23848B for 1 h at 37 °C (53). After this pretreatment, cells were incubated with 100 nM PGE₂ for 8 h at 37 °C. Experiments were performed three times and luciferase activity was monitored as described above.

	RESULTS

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Modulation of HIV-1 LTR-driven Gene Expression in T Cells by PGE₂-- In order to assess whether PGE₂ could affect the regulatory elements of HIV-1, we initially set out a dose-response experiment which was performed with PGE₂ in a 24-h incubation period. The results showed a steady increase of HIV-1 LTR activity in 1G5 cells starting at as little as 1 nM concentration of PGE₂ (Fig. 1A). In these experiments, PGE₂ was reconstituted in absolute ethanol to get a stock solution of 1 mM which was serially diluted in complete culture medium to get our working dilutions (1, 10, 100, and 1000 nM). Incubation of 1G5 cells with the concentration of ethanol corresponding to the one used with 1000 nM PGE₂ resulted in a 1.5-fold increase of HIV-1 LTR dependent activity, while no effect was seen with the equivalent ethanol concentration for 100 nM PGE₂ (data not shown). Therefore, subsequent experiments were carried out using 100 nM PGE₂ to avoid any putative influence of ethanol on PGE₂-mediated enhancement of HIV-1 LTR activity. Kinetics analyses were next performed to determine the optimal incubation time for this PGE₂-mediated HIV-1 LTR-driven activation. The maximal positive effect of PGE₂ was seen 8 h after the initiation of treatment (fold enhancement of 7.4) (Fig. 1B). Although this type of kinetic might be reminiscent of degradation of PGE₂, it is unlikely since similar kinetic of time-dependent HIV-1 LTR-driven luciferase activity were measured with activators such as TNF-, PHA, and PMA (data not shown). Moreover, preincubation of PGE₂ in complete culture medium for 24 h at 37 °C resulted in equal fold-induction of HIV-1 LTR-driven luciferase activity in 1G5 cells as compared with incubation with fresh PGE₂ (data not shown), which demonstrates that PGE₂ is fairly stable under these experimental conditions. Taken together, these data indicate that activation of HIV-1 regulatory elements by PGE₂ was rapid and transient, thereby suggesting that the effect was direct and was resulting from PGE₂-mediated signal transduction events.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Dose-dependent and kinetic analyses of PGE2-mediated positive effect on HIV-1 LTR activity. A, 1G5 cells were stimulated for 24 h with increasing doses of PGE2 (1, 10, 100, and 1000 nM). Cell lysates were evaluated for luciferase activity by scintillation count. B, 1G5 cells were either left untreated () or were stimulated with 100 nM PGE2 () for different time periods (2, 6, 8, and 24 h) prior to monitoring luciferase activity in cell lysates. Results shown are the means (±S.D.) of four determinations and are expressed in panel A as fold induction relative to basal luciferase activity in untreated control cells (considered as 1). These results are representative of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Activation of HIV-1 LTR-driven luciferase activity by several stimuli in the absence or presence of PGE2. 1G5 cells were either left untreated (control) or treated with PHA (3 µg/ml), OKT3 (1 µg/ml), PMA (20 ng/ml), or TNF- (2 ng/ml) in the absence () or presence () of 100 nM PGE2 for 8 h. Cell lysates were evaluated for luciferase activity by scintillation count. Results shown are the means (± S.D.) of four determinations and are representative of three independent experiments.

Signaling Events Involved in PGE₂-mediated Enhancement of HIV-1 LTR Activity-- Our results so far suggested that binding of PGE₂ to its cell surface receptors (EPs) triggers signal transduction that positively affect HIV-1 LTR expression. Several specific inhibitors were used to identify cellular element(s) participating to the signaling events involved in the PGE₂-induced effect on HIV-1 transcription. 1G5 cells were first pretreated with H7, a selective serine/threonine kinase inhibitor that can inhibit protein kinase A (PKA) (K_i = 3.0 µM), PKC (K_i = 6.0 µM), as well as PKG (K_i = 5.8 µM) (56). A dose-dependent inhibition of PGE₂-mediated HIV-1 LTR activation was seen when 1G5 cells were pretreated with H7 at concentrations sufficient to inhibit all of these enzymes (Fig. 3A). To more deeply scrutinize signaling molecule(s) implicated in PGE₂-induced positive effect on HIV-1 LTR activity, cells were next pretreated with HA-1004, a serine/threonine kinase inhibitor that preferentially inhibits PKA (K_i = 2.3 µM) and PKG (K_i = 1.3 µM) over PKC (K_i = 40.0 µM) (56). The PGE₂-mediated activation of HIV-1 transcription was almost completely abrogated by a pretreatment with HA-1004 at concentrations sufficient to inhibit both PKA and PKG, but not PKC (1 and 5 µM) (Fig. 3B). To clearly discriminate between PKA and PKG in the PGE₂-induced signaling cascade, 1G5 cells were pretreated with a specific inhibitor of adenylate cyclase (MDL-12, 330A) (57-59). Activation of HIV-1 LTR-mediated reporter gene expression by PGE₂ was totally abolished by concentrations of MDL-12,330A sufficient to completely inhibit cAMP activity (Fig. 3C). Results from these experiments hence demonstrated that PKA was an essential intracellular second messenger participating in the PGE₂-dependent up-regulating effect on HIV-1 LTR activity in T cells.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Identification of second messengers implicated in PGE2-mediated up-regulation of HIV-1 LTR activity. 1G5 cells were incubated for 1 h with H7 (1, 5, 10, and 20 µM) (panel A), HA-1004 (1, 5, 10, and 20 µM) (panel B), MDL-12,330A (50, 100, and 250 µM) (panel C), IBMX (1, 5, and 10 µM) (panel D), BAPTA/AM (1, 5, and 10 µM) (panel E), and for 16 h with CAI (0.1, 1.0, and 10 µM) (panel F) prior to treatment for 8 h with 100 nM PGE2. Cell lysates were evaluated for luciferase activity by scintillation count. Results shown are the means (± S.D.) 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.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Activation of HIV-1 LTR-driven luciferase activity by a combination of dibutyryl-cAMP, ionomycin, and PMA. 1G5 cells were either left untreated (control) or treated for 8 h with ionomycin (1 µM), PMA (20 ng/ml), and increasing concentrations of dibutyryl-cAMP (0, 25, 50, 100, and 200 µM). Cell lysates were evaluated for luciferase activity by scintillation count. Results shown are the means (± S.D.) of four determinations and are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

NF-B-dependent and -independent Signaling Pathways Are Involved in Activation of HIV-1 LTR by PGE₂-- To define the region(s) required for the activation of HIV-1 LTR transcription by PGE₂ at the molecular level, the parental Jurkat E6.1 cell line was transiently transfected with different HIV-1 LTR-driven luciferase constructs carrying either the full-length (pLTR-LUC) or modified versions of the HIV-1 LTR promoter (pmBLTR-LUC and pB-TATA-LUC). These latter constructs either contained the complete regulatory elements of HIV-1 mutated at the two NF-B-binding sites (pmBLTR-LUC) or the minimal HIV-1 B region and a TATA box (pB-TATA-LUC). Transiently transfected Jurkat E6.1 cells were then stimulated with TNF-, PHA/PMA, or PGE₂ for 8 h. In these experiments, stimulation with TNF- and PHA/PMA were used as positive controls since TNF- is known to activate HIV-1 transcription exclusively via NF-B (66), while the combination of PHA and PMA can enhance HIV-1 LTR activity via both NF-B-dependent and -independent signaling pathways (data not shown). As shown in Fig. 5A, we observed a 13.7-, 83.3-, and 12.1-fold increase in luciferase activity for the full-length HIV-1 LTR (pLTR-LUC) in the presence of TNF-, PHA/PMA, and PGE₂, respectively. With the molecular construct pmBLTR-LUC, as expected, no increase in HIV-1 LTR-driven gene activity was detected with TNF-, while PHA/PMA was still inducing HIV-1 LTR-dependent luciferase activity (8.5-fold increase). Interestingly, the luciferase-encoding vector mutated at both NF-B-binding sites of the LTR was still responding to PGE₂, although at a slightly lower level compared with the wild-type HIV-1 LTR construct (3.8- versus 12.1-fold). This experiment was repeated several times and gave consistent results. Therefore, data from experiments conducted with pLTR-LUC and pmBLTR-LUC were indicating that PGE₂-mediated positive effect on HIV-1 LTR-driven activity required both NF-B-dependent and -independent signal transduction pathways.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   NF-B-dependent and -independent activation of HIV-1 LTR by PGE2. A, Jurkat E6.1 cells were transiently transfected with pLTR-LUC, pmBLTR-LUC, or pB-TATA-LUC and were either left untreated or were treated for 8 h with TNF- (2 ng/ml), PHA/PMA (3 µg/ml and 20 ng/ml, respectively), or PGE2 (100 nM). B, Jurkat E6.1 cells were transiently transfected with pNF-B-LUC and were either left untreated or treated for 8 h with TNF- (2 ng/ml), PHA/PMA (3 µg/ml and 20 ng/ml, respectively), and PGE2 (100 nM). Cell lysates were evaluated for luciferase activity by scintillation count. Results shown are the means (± S.D.) 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.

View larger version (91K): [in this window] [in a new window]   Fig. 6.   Nuclear translocation and activation of NF-B by PGE2. 1G5 cells were either left untreated or were incubated for 30 min with either 100 nM PGE2 or 2 ng/ml TNF- (positive control). The nuclear extracts were next incubated with a 32P-end-labeled synthetic double-stranded NF-B probe. Lanes 1 and 2 are negative controls containing no extracts or extracts from untreated cells, respectively. Lane 3 represents a positive control containing cells stimulated with TNF- (2 ng/ml), while lane 4 are cells treated with PGE2. Lanes 5 and 6 represent a 100 X competition with the unlabeled probe for NF-B with TNF- and PGE2, respectively. The position of the specific complex bound by the B site probe is indicated by an arrow on the left side.

Surface Expression of the Human PGE₂ Receptor EP₄ Subtype in the Studied T Lymphoid Lineages-- PGE₂ is known to bind to specific protein receptors on a large array of target cells. Previous cDNA cloning and pharmacologic experiments have identified six different PGE₂ receptors (EPs) (68). Northern blot analyses were then performed to evaluate PGE₂ receptor(s) expression on the T lymphoid cell lines used in the present study. The cDNAs for hEP₁, hEP₂, hEP₃, and hEP₄ were hybridized with total RNA from 1G5 and Jurkat E6.1 cells. Results indicated that the hEP₄ gene was expressed on both 1G5 and Jurkat E6.1 cell lines, while EP₁, EP₂, and EP₃ subtype receptors were not expressed (data not shown). Flow cytometry analysis was also carried out with a polyclonal antibody specific for EP₄ receptor and confirmed its presence on the surface of Jurkat E6.1 (Fig. 7A) and 1G5 (Fig. 7B) cells. The identity of the prostaglandin receptor on T lymphoid 1G5 cells was directly addressed using a subtype selective pharmacologic antagonist. For this purpose, 1G5 cells were pretreated with AH 23848B (30 µM), a selective antagonist of human EP₄ receptor, prior to the addition of PGE₂. AH 23848B was found to abrogate PGE₂-mediated up-regulation of HIV-1 LTR activity in 1G5 cells (Fig. 8). Altogether these results indicate that the hEP₄ receptor is involved in the PGE₂-mediated activating effect on HIV-1 transcription in T lymphoid cells.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Cytometry analysis of PGE2 receptor subtype 4 on studied cell lines. Flow cytometric analyses were performed using a saturating concentration of polyclonal anti-human EP4 antibody in combination with fluorescein isothiocyanate-labeled chicken anti-rabbit IgG antibody (panel A, Jurkat E6.1 cells; and panel B, 1G5 cells). The solid lines represent background fluorescence.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Pharmacological study of the PGE2 receptor with the selective antagonist AH 23848B. 1G5 cells were either left untreated or were treated with increasing concentrations of the selective antagonist of human EP4 AH 23848B (0, 5, 15, and 30 µM) for 1 h prior to incubation for 8 h with PGE2 (100 nM). Next, luciferase activity was monitored as described under "Experimental Procedures." Results shown are the means (± S.D.) 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.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Immune and inflammatory responses are triggered by microorganisms such as bacteria, viruses, and protozoan, all known to be potential opportunistic pathogens in HIV-1-positive patients. The formation and production of elevated levels of inflammatory mediators such as PGE₂ is a hallmark of the HIV-1 infection (28-30). Prostaglandins play a role in disease exacerbation by directly altering T-cell functions or macrophage activation. Although it was thought that PGE₂ is primarily 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 immunoglobulin class switching and cytokine and cytokine receptor synthesis (15). This PGE₂-dependent positive effect on the immune response and the observation that higher levels of PGE₂ are detected in HIV-1-infected individuals (2-5-fold increase) have been the compelling force for our investigation. Knowing that PGE₂ is a good 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 (69), it was thus of prime importance to study the putative effect of PGE₂ on the regulatory elements of HIV-1.

We therefore asked whether the proinflammatory PGE₂ molecule had the ability to modulate HIV-1 transcription in T cells. This particular cell type was chosen since T cells are considered to be a major cellular reservoir for HIV-1 in the human peripheral blood (70). In this report, we present evidence indicating that PGE₂ up-regulates HIV-1 LTR-driven reporter gene expression in human T cells (Fig. 1). Our results are indicative of an optimal signal after 6 to 8 h of treatment. This is very similar to time kinetics of HIV-1 LTR activation by PMA, PHA, and TNF- agents which directly act on the HIV-1 promoter. Although we cannot refute a possible indirect mechanism for HIV-1 LTR activation by PGE₂ which would involve production of cytokines, time course experiments suggested that a more direct process might be at the basis of the effect of PGE₂ on HIV-1 LTR. Interestingly, a specific synergistic HIV-1 LTR activation was observed using both PGE₂ and TNF- (Fig. 2). This might be accounted by the fact that the effect exerted by TNF- is exclusively via NF-B, while PGE₂ acts also on region(s) other than NF-B in the HIV-1 LTR (see below). Previously described synergistic activation by NF-B and other factors have been reported (66, 67). The induction of HIV-1 LTR activity by PGE₂ thus support a previous cellular study showing a 2.5-fold increase in virus production following the addition of exogeneous PGE₂ to MT-4 cells acutely infected with HIV-1 (43).

In the present study, the involvement of specific intracellular second messengers in PGE₂-mediated up-regulation of HIV-1 LTR activity has been dissected using several signal transduction inhibitors. Experiments with indomethacin, a potent inhibitor of the cyclooxygenase pathway and thus of PGE₂ production, suggest that only exogeneous PGE₂ plays a role in the activation of HIV-1 LTR-driven gene expression. These results were expected based on studies that T cells had a limited capacity to metabolize arachidonic acid to prostaglandins (71-73). An earlier report indicated that interaction between PGE₂ and an adenylate cyclase-coupled stimulatory receptor leads to activation of adenylate cyclase, hydrolysis of ATP, enhanced turnover of intracellular cAMP, and binding to PKA (74). Our findings are clearly supportive of this signaling cascade since we found that PGE₂-induced enhancement of HIV-1 LTR dependent activity requires the participation of adenylate cyclase, cAMP, and protein kinase A (Fig. 3, A-D). Using MT-4 cells, another group has shown that elevation of cAMP levels resulted in HIV-1 replication (62). It is also well known that cAMP-dependent pathways regulate the immune effector functions of lymphocytes and macrophages. For example, during immune response, cAMP exhibits positive regulatory effects at low concentrations whereas inhibitory effects are seen at high concentrations (75). Many of the earlier studies have shown that PGE₂ interaction with T cells in vitro resulted in an elevation of the cAMP level (35) and that such elevated intracellular cAMP levels were responsible for the proliferative disturbances in T cells (76-78). Data from our experiment with the calcium chelator BAPTA/AM and the calcium inhibitor CAI are suggestive of the importance of Ca²⁺ in the PGE₂-induced activation of HIV-1 transcription (Fig. 3, E and F). However, given that there is no published report indicating Ca²⁺ influx through the EP₄ receptor, our results with BAPTA/AM and CAI, two inhibitors of intracellular calcium mobilization, lead us to postulate that PGE₂ could generate calcium release from intracellular storage organelles. All these results were supported by data shown in Fig. 4 indicating that up-regulation of HIV-1 LTR requires the implication of cAMP and calcium, as well as the participation of the NF-B transcription factor.

Several agents known as potent activators of HIV-1 transcription (e.g. PMA, PHA, TNF-, and anti-CD3 antibody) are all acting through a common mechanism, namely via the nuclear translocation of the transcription factor NF-B which binds to the enhancer region of the HIV-1 LTR (79). This transcription factor is sequestered in the cytoplasm due to its physical association with the inhibitor named IB. NF-B is a pleiotropic transcription factor that controls the expression of a wide variety of genes, including cytokines such as IL-1, IL-2, IL-6, IL-8, interferon-, and TNF-, as well as known genes for some cell adhesion molecules including ICAM-1 and VCAM-1. Its importance in the regulation of HIV-1 gene expression has been stated in numerous studies (80). Results from mobility shift assays suggest that the PGE₂-mediated effect on HIV-1 LTR activity is due to activation of the transcription factor NF-B by PGE₂. This is in agreement with the previous demonstration that PGE₂ activates NF-B in the macrophage-like cell line J774 (81). The fact that we have noticed that both NF-B and Ca²⁺ are key elements in the PGE₂ effect on HIV-1 transcription is of interest considering that calcineurin, a Ca²⁺/calmodulin-dependent serine/threonine protein phosphatase, has been reported to activate NF-B through the inactivation of IB (82). Moreover, researchers had earlier found that cAMP-mediated enhancement of PKA might be involved in the dissociation of IB from NF-B (79). Recent studies have revealed that NF-B is regulated through phosphorylation of the p65 subunit by PKA which is directly regulated by intracellular levels of cAMP (83). Our experiments hence support the notion that PGE₂ might be activating the transcription factor NF-B via cAMP/PKA and calcium signaling pathways in human T lymphoid cells. However, our experiments were performed with B-driven reporter gene constructs (pB-TATA-LUC and pNF-B-LUC) and HIV-1 LTR-based vectors (pLTR-LUC and pmBLTR-LUC), furthermore, suggest that NF-B-binding regions and another element(s) in the HIV-1 LTR are involved in the activation of HIV-1 LTR-dependent transcription induced by PGE₂.

NFAT is an immediate-early activation factor that plays a crucial role in T-cell activation and commitment processes through its control of IL-2 gene activation (84). Based on the demonstrated synergistic effect between NFAT and NF-B on the activation of HIV-1 transcription (67) and the proposed PGE₂-induced NF-B-independent pathway, we looked at the putative role of NFAT in the effect of PGE₂. We found that NFAT was not involved in PGE₂-dependent activation of HIV-1 LTR-driven luciferase activity. These data were expected considering that PGE₂ has been reported to inhibit NFAT activity (85). PGE₂ could have the capacity to modulate several signal transduction pathways through its effect on transcription factors regulated by cAMP such as the cAMP response-element binding factor, the activating protein-1 (38), and Sp1 (86). The involvement of these three transcription factors in the observed NF-B-independent activation of HIV-1 LTR mediated by PGE₂ is currently under investigation.

Finally, by Northern blot assays, flow cytometric analyses, and pharmacological studies, we demonstrated that studied T lymphoid cell lines (Jurkat E6.1 and 1G5) express the EP₄ receptor subtype on their surfaces and that EP₁, EP₂, and EP₃ receptors seem not to be expressed (Figs. 7 and 8 and data not shown). This finding is in accord with previous studies that have found by Northern blot analysis that the EP₄ gene is expressed on T lymphoid cells such as Molt-4, KM-3, and Jurkat E6.1 (87-89). It has been demonstrated that EP₄ receptors are coupled to adenylate cyclase via a stimulatory G protein (G_s) and that such activation results in an enhancement of intracellular cAMP levels (53, 68). Interestingly, PGE₂ has been shown to lead to an increase in intracellular cAMP levels partly via the EP₄ receptor (90), a finding which lend credence to the potential implication of the EP₄ receptor in the PGE₂-induced up-regulation of HIV-1 LTR activity.

Because of their intrinsic intracellular obligatory parasitic form of life, viruses depend heavily on cell metabolic machinery for their replication. Thus, changes in cellular metabolism might influence the viral life cycle. The experiments reported here highlight the positive action of PGE₂, a powerful cAMP-inducing agent, on the regulatory elements of HIV-1. The presented data suggest that elevated levels of PGE₂ detected in HIV-1-infected persons or induced by opportunistic pathogens might actively participate to immunological disturbances associated with AIDS and modify the pathogenesis of this retroviral disease by inducing a higher viral load. Finally, high concentrations of PGE₂ (up to 100 µM) found in seminal fluids of HIV-1-infected persons might directly enhance virus replication and facilitate viral transmission during sexual activities (40).