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Originally published In Press as doi:10.1074/jbc.M200875200 on February 14, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16913-16919, May 10, 2002
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Peroxisome Proliferator-activated Receptor gamma  Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects*

Michael M. HayesDagger , Brian R. Lane§, Steven R. King, David M. Markovitz§||, and Michael J. CoffeyDagger **

From the Dagger  Divisions of Pulmonary and Critical Care Medicine,  Rheumatology, and || Infectious Diseases and the § Graduate Program in Cellular and Molecular Biology, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Received for publication, January 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPG) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. We investigated the role of PG in the replication of HIV-1 in primary macrophages. The cyPG, PGA1 and PGA2, inhibited HIV-1 replication in acutely infected human monocyte-derived macrophages (MDM). Because PGA1 and PGA2 have previously been shown to be peroxisome proliferator-activated receptor gamma  (PPARgamma ) agonists, we examined the effect of synthetic PPARgamma agonists on HIV replication. The PPARgamma agonist ciglitazone inhibited HIV-1 replication in a dose-dependent manner in acutely infected human MDM. In addition, cyPG and ciglitazone reduced HIV replication in latently infected and viral entry-independent U1 cells, suggesting an effect at the level of HIV gene expression. Ciglitazone also suppressed HIV-1 mRNA levels as measured by reverse transcriptase PCR, in parallel with the decrease in reverse transcriptase activity. Co-transfection of PPARgamma wild type vectors and treatment with PPARgamma agonists inhibited HIV-1 promoter activity in U937 cells. Activation of PPARgamma also decreased HIV-1 mRNA stability following actinomycin D treatment. In summary, our experimental findings implicate PPARgamma as an important factor in the suppression of HIV-1 gene expression in MDM by cyPG. Thus natural and synthetic PPARgamma agonists may play a role in controlling HIV-1 infection in macrophages.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

AIDS is characterized by numerous immunological abnormalities, alterations in lymphocyte populations, and immunosuppression with subsequent development of opportunistic infections (1). Many of these changes can be attributed to alterations in mediator production including cytokines (2) and eicosanoids (3, 4). Prostaglandins (PG)1 are one such group of mediators that are modulated in HIV infection (5, 6). In patients with AIDS, serum levels of PGE2 are significantly increased relative to serum levels of PGE2 in normal controls (6). Augmented levels of PG are also found in cerebral spinal fluid of AIDS patients (7, 8). Peripheral blood monocytes (PBM) and macrophages play multiple roles in primary HIV-1 infection as both producers of immune mediators and as reservoirs for the virus (9, 10). In vitro production of PGE2 and thromboxane B2 by PBM from AIDS patients is increased relative to normal controls (11, 12). Experimental infection of human PBM with HIV-1 also results in increased production of PGE2 relative to uninfected control cultures (13-16).

PG can be converted non-enzymatically to other compounds, including cyclopentenone PG (cyPG) (17). Primary PG, PGE1 and PGE2, can be converted to the cyPG PGA1 and PGA2, respectively, in aqueous solution or plasma over 12 h (18). It has been shown that cyPG have different biological activities from the primary PG. CyPG are actively incorporated into cells independent of PG receptors and are transferred to the nucleus. In the nucleus cyPG lead to cell cycle arrest at the G1 phase and inhibit the replication of a variety of viruses including both DNA and RNA viruses (19). There are several reports of cyPG inhibiting HIV-1 replication in vitro in T-cell lines (VB cells and CEM-SS cells), and in chronically infected macrophages (20, 21).

Mechanisms by which PGA1 and PGA2 act include activation of heat shock proteins (22) and peroxisome proliferator-activated receptors (PPAR) (23, 24). PPARs are ligand-inducible transcription factors belonging to the family of nuclear transcription factors that includes the classical steroid and thyroid hormone receptors (25). There are three PPAR isotypes, namely PPARalpha , PPARbeta , and PPARgamma . When activated by ligand binding, PPARs can bind to promoters in target genes and modulate gene expression (25, 26). PPARgamma is expressed upon activation of PBM and their subsequent differentiation into monocyte-derived macrophages (MDM). These nuclear receptors have been implicated in the regulation of glucose metabolism (27), cellular differentiation (28), tumor suppression (29), and inhibition of inflammation (30).

We have found that cyPG can inhibit HIV-1 replication following acute infection of human MDM. Furthermore, these natural PPARgamma agonists suppressed HIV-1 replication following phorbol myristate acetate (PMA) induction of latent HIV-1 infection in the monocytic U1 cell line. The synthetic PPARgamma agonist ciglitazone also inhibited the replication of HIV-1 following acute infection of human MDM with HIV-1 and in U1 cells. Mechanisms for the suppression of viral replication by cyPG and synthetic PPARgamma agonists include inhibition of HIV-1 transcription as well as a reduction in HIV-1 mRNA stability.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals

PGA1, PGA2, and ciglitazone (Cayman Chemical, Ann Arbor, MI) were dissolved in ethanol at 1 mg/ml and stored at -70 °C. WY-14643 (Cayman Chemical) was dissolved in Me2SO at 100 µM and stored at -20 °C. PMA (Sigma) was dissolved in Me2SO at 1 mM and stored at -20 °C.

MDM Cultures

Blood was drawn from healthy donors using heparin as an anticoagulant and layered on Ficoll-Hypaque (density 1.077 g/ml) (Amersham Biosciences). After centrifugation at 20 °C for 45 min at 800 × g, plasma was aspirated, and the interface layer was harvested and washed twice with cold phosphate-buffered saline (without Ca2+ or Mg2+, 0.1% bovine serum albumin). The cell pellet was resuspended to 2.5 × 106 cells/ml in cold DMEM (Invitrogen) with antibiotics/antimycotic and without FBS. Cells were plated at 5 × 105/well for 96-well plates or 3.125 × 106/well for 6-well plates. Plates were incubated at 37 °C with 5% CO2 for 2 h to allow PBM to adhere. Plates were rinsed three times with DMEM prewarmed to 37 °C. Plates were refed with DMEM (10% FBS) and incubated at 37 °C with 5% CO2. MDM culture viability was assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using a commercially available MTT assay kit (Roche Diagnostics Inc.) (31, 32).

HIV Infection of MDM and Culture of Latently Infected U1 Cells

PBM were differentiated to MDM, because of their greater susceptibility to HIV-1 infection in vitro, by incubation in culture for 5 days. The M-tropic strain, HIV-1BAL, was used to infect MDM. MDM cultures in 96-well plates were refed with 200 µl of the treatment compound diluted in DMEM (10% FBS) and incubated at 37 °C with 5% CO2 for 16-24 h. 20 µl of virus stock were added per well (estimated multiplicity of infection, 0.01), and incubated overnight at 37 °C with 5% CO2. Medium was aspirated, and the wells were refed. MDM cultures were refed (50% replacement) at intervals of 2-3 days. The monocytic cell line U1 is a clone of the cell line U937 and is latently infected with HIV-1 (33). U1 cells were plated in 96-well plates at 1 × 105 cells/well in 200 µl of RPMI 1640 (10% FBS). 20 µl of the treatment compound (10×) were added per well followed by incubation at 37 °C in 5% CO2 for 3 h. Induction of latent infection was accomplished by adding 20 µl of PMA) diluted in RPMI 1640 (10% FBS) to a final concentration of 0.1 µM (33, 34). The cultures were incubated for an additional 72 h at 37 °C in 5% CO2.

Measurement of HIV-1 Replication

Reverse Transcriptase (RT) Assay-- Samples (3 µl) were mixed with 12 µl of RT assay buffer containing 1 µCi/ml [32P]dTTP. The reaction mixture was incubated at 37 °C for 2.5 h. 3.5 µl of each sample was blotted on DEAE paper and air-dried. Blots were washed (with gentle agitation) three times in 2× SSC. Blots were then washed with 95% ethanol and air-dried (35). Incorporation of [32P]dTTP was measured using the PhosphorImager system (Molecular Dynamics) (36).

RT-PCR Protocol-- MDM, U937, and U1 cell lines were treated for 3 h with PGE2 prior to treatment with PMA (final concentration 107 M). At 40 h post-treatment cells were harvested and RNA extracted and purified (RNEasy 96 Kit, Qiagen, Valencia, CA). Quantitative RT-PCR was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). The platinum quantitative RT-PCR Thermoscript One-Step System reagents was used (Invitrogen). The primer sequences used were GCC TGG GCG GGA CCG and GTA CAG GCA GAA AGC AGC. The probe sequence is FAM-TGG CGA GCC CTC AGA TGC TGC-TAMRA. The ABI PRISM 7700 was cycled at 60 °C for 30 min, 95 °C for 5 min, 95 °C for 15 s (45 cycles), and 60 °C for 1 min (37, 38).

HIV mRNA Stability

MDM, U937, or U1 cells were plated in 96-well plates at a density of 1 × 105 cells/well (in 200 µl of RPMI 1640 with 10% FBS) and treated with PGE2 for 3 h (36 replicates for each treatment). U1 cells were subsequently stimulated with PMA at 0.1 µM. At 16 h post-PMA treatment, we added actinomycin D at a final concentration of 10 µg/ml to 18 wells of each treatment (and untreated control) to block further transcription. Three wells of each treatment (and untreated control) were harvested immediately and at intervals of 2 h (i.e. 2, 4, 6, 8, and 10 h). Next, we extracted and purified RNA (RNEasy 96 Kit). Quantitative RT-PCR was performed using the ABI PRISM 7700 sequence detection system (39-41).

Transfection

U937 and HeLa cell transfections were performed as described previously (42-45). Briefly, the U937 cell concentration was adjusted to 2 × 108 cells/ml in RPMI 1640 (with 10% FBS), and the plasmid DNA was mixed with 500 µl of U937 cell suspension in electroporation cuvette (Invitrogen). A typical transfection was with 5 µg of plasmid, e.g. LTR.luciferase with "carrier" DNA from salmon testes added to a total of 40 µg of DNA. Electroporation was performed at room temperature using a single-pulse Invitrogen Electroporator II set at 300 V and 1000 microfarad capacitance. The transfected cells were cultured overnight in 10 ml of RPMI 1640. The cells were washed and resuspended in RPMI 1640 to 2 × 105 viable cells/ml. Cells were plated in a 24-well tissue culture plate (1 ml/well) and treated with cyPG or ciglitazone for 3 h. Next they were stimulated with PMA at 0.1 µM to induce expression of luciferase from the LTR.luciferase plasmid. Cells were harvested, cell lysates prepared, and luciferase assay performed at 48 h post-treatment. The PPARgamma wild type (WT PPARgamma 1) and dominant negative (PPAR.af2) vectors (46) were transfected into U937 or HeLa cells.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CyPG Inhibit HIV-1BAL Replication in MDM-- PBM were incubated for 3-5 days in medium containing serum prior to infection with HIV-1BAL. Treatment of MDM cultures with PGA1 and PGA2 at a concentration of 10 -6 M at 16-24 h prior to infection with HIV-1 resulted in a significant decrease in viral replication as assessed by RT assay (Fig. 1). The inhibitory effect of PGA1 and PGA2 was dose-dependent. The addition of cyPG after HIV-1 infection was also effective in suppressing viral replication but to a lesser degree (data not shown). There was no significant decrease in cell viability between treatment groups (data not shown), as assessed by MTT assay.


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  		Fig. 1.   CyPG inhibit HIV-1BAL replication in MDM. Human PBM were plated at 1 × 10 5 cells/well in 200 µl of DMEM (10% FBS plus antibiotic/antimycotic) in a 96-well plate. At 3-5 days after plating, medium was aspirated and cultures refed with or without 10-8 M or 10-6 M PGA1 (A) or PGA2 (B) in triplicate. Cultures were incubated for 18-24 h in 5% CO2 at 37 °C. 20 µl of HIV-1BAL stock was added per well (estimated multiplicity of infection, 0.01) and incubated for 18-24 h. Medium was aspirated, and cultures were refed. At 2-3 day intervals cultures were refed (50% replacement). Post-infection samples were taken for RT assay at day 13. Results are shown as means ± S.E. expressed as a percentage of the value of the untreated control cultures. PGA1: 10-8 M, 82.6 ± 12.1% of untreated control, p = ns; 10-6 M, 36.5 ± 6.23% of untreated control, *, p = 0.0001, n = 5. PGA2: 10-8 M, 79.7 ± 10.7% of untreated control, p = ns; 10-6 M, 32.8 ± 4.7% of untreated control, **, p = 0.0001, n = 4.

CyPG Inhibit HIV-1 Replication in U1 Cells-- One mechanism by which cyPG could regulate HIV-1 replication would be at the level of viral entry and fusion with the cell membrane. Furthermore, other investigators have shown that other PG can down-regulate the expression of CCR5, the HIV-1 co-receptor (47). Therefore, we examined the effect of cyPG on CCR5 expression. We determined that cyPG had no significant effect on CCR5 expression with the mean fluorescence index of 48.8 ± 1.3 for untreated cells and 57.7 ± 10.5 for PGA2 treated cells (n = 3, p = ns). We next chose a cell type in which HIV-1 replication was fusion/entry-independent. In this system, we determined that treatment of the latently infected monocytic cell line, U1 cells, with PGA1 and PGA2, at a concentration of 10 -5 M 3 h prior to PMA induction of latent HIV-1 infection resulted in suppression of viral replication as assessed by RT assay (Fig. 2). There was no significant decrease in cell viability between treatment groups (data not shown) as assessed by MTT assay. These data with the U1 cells suggest that the effect of PGA1 and PGA2 on viral replication was not likely to be at the fusion/entry of HIV-1 but at a post-integration level, e.g. gene regulation.


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  		Fig. 2.   CyPG inhibit HIV-1 replication in U1 cells. U1 cells were suspended in RPMI 1640 (10% FBS) at 5 × 10 5 cells/ml. 200 µl of the cell suspension was aliquoted per well in a 96-well plate. 20 µl of the PGA1 or PGA2 at 10-4 M was added per well in triplicate. The treated cells were incubated for 3 h in 5% CO2 at 37 °C. 20 µl of PMA at 10-6 M was then added and the cultures incubated in 5% CO2 at 37 °C. Samples were then taken for RT assay at 72 h. Results are shown as means ± S.E. expressed as a percentage of the value of the untreated control cultures. PGA1: 10-4 M, *, p < 0.001, n = 5; **.PGA2: 10-4 M, p = 0.001, n = 5.

Synthetic PPARgamma Activators Inhibit HIV-1BAL Replication in MDM-- Because cyPG are natural agonists of PPARgamma , we next examined whether synthetic PPARgamma agonists would also suppress HIV-1 BAL replication in MDM. PBM were incubated for 3-5 days in medium containing serum prior to infection with HIV-1. Treatment of MDM cultures with the PPARgamma agonist ciglitazone resulted in a dose-dependent suppression of HIV replication, with the maximal effect occurring at 40 µM (Fig. 3A). The PPARalpha agonist WY-14643 (50 µM) added 16-24 h prior to infection with HIV-1 demonstrated no effect on HIV-1 replication (Fig. 3B). This was also true at higher concentrations (data not shown).


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  		Fig. 3.   Synthetic PPARgamma activator inhibits HIV-1 replication in MDM. Human PBM were plated at 1 × 10 5 cells/well in 200 µl DMEM as described in the legend for Fig. 1. A, the synthetic PPARgamma agonist, ciglitazone, was added in increasing concentrations (0-40 µM). B, the PPARgamma agonist (ciglitazone) or the PPARalpha agonist (WY-14643) were added at a concentration of 50 µM. Medium was aspirated and cultures refed. At 2-3-day intervals cultures were refed (50% replacement). Thirteen days post-infection samples were taken for an RT assay. Data are shown as means ± S.E. of triplicate wells, and results are expressed as RT activity (beta counts) (A) or as a percentage of the value of the untreated control cultures (B). A representative experiment of more than three performed is shown in A and B.

Synthetic PPARgamma Activators Inhibit HIV-1 Replication in U1 Cells-- Having demonstrated that the natural PPARgamma agonists cyPG suppressed HIV-1 replication in U1 cells, we next examined the ability of the synthetic PPARgamma agonist to suppress HIV-1 replication in the same cells. The PPARgamma agonist ciglitazone inhibited HIV-1 replication in PMA-stimulated U1 cells, whereas even higher doses of the PPARalpha agonist WY-14643 had no effect (Fig. 4). There was no significant decrease in cell viability between treatment groups (data not shown) as assessed by MTT assay.


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  		Fig. 4.   Synthetic PPARgamma activator but not PPARalpha activator inhibits HIV-1 replication in U1 cells. U1 cells were suspended in RPMI 1640 (10% FBS) at 5 × 10 5 cells/ml. 200 µl of the cell suspension was aliquoted per well in a 96-well plate. WY-14643 or ciglitazone was added per well in triplicate to a final concentration as indicated. The treated cells were incubated for 3 h in 5% CO2 at 37 °C. 20 µl of PMA at 10-6 M was added, and the cultures were incubated in 5% CO2 at 37 °C. Samples were then taken for an RT assay at 72 h. Data are shown as the means ± S.E. of triplicate wells, and results are expressed as RT activity (beta counts). A representative experiment of more than three performed is shown.

Natural and Synthetic PPARgamma Agonists Decrease HIV-1 mRNA Levels-- Because cyPG and PPARgamma agonists are able to block induction of HIV-1 from latency in the U1 model, we next wanted to determine whether the effect of PGA1 and PGA2 treatment was at the level of gene expression. Treatment of U1 cells with PGA1 and PGA2 decreased levels of HIV mRNA as measured by RT-PCR, in parallel with the decrease in RT activity (Fig. 5, A and B). Next we examined the effect of synthetic PPAR agonists on the levels of HIV mRNA in U1 cells. Ciglitazone, the PPARgamma agonist, but not the PPARalpha agonist WY-14643, suppressed HIV mRNA levels (Fig. 5B). These data strongly suggest that the effects of the natural and synthetic PPARgamma agonists occur at the level of HIV gene expression.


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  		Fig. 5.   Natural and synthetic PPARgamma agonists suppresses HIV-1 mRNA levels in U1 cells. U1 cells were suspended in RPMI 1640 (10% FBS) at 5 × 10 5 cells/ml. 200 µl of the cell suspension was aliquoted per well in a 96-well plate. PGA1 and PGA2 were added at a concentration of 10-4 M/well in triplicate. Ciglitazone and WY-46143 were added at a concentration of 40 and 50 µM, respectively. The treated cells were incubated for 3 h in 5% CO2 at 37 °C. 20 µl of PMA at 10-6 M was added, and the cultures were incubated for 72 h in 5% CO2 at 37 °C. Data are shown as means ± S.E. and expressed as a percentage of the value of the untreated control cultures. HIV-1 replication was determined by: A, RT assay: *, p = 0.002, **, p = 0.002, dagger , p = 0.001 (n = 3); and B, RT-PCR assay: *, p = 0.001, **, p = 0.002, dagger , p = 0.01 (n = 3).

PPARgamma Agonists Inhibit HIV-1 Promoter Activity-- We next wanted to determine whether PPARgamma agonists regulated HIV-1 promoter activity. U937 cells were transfected with LTR.luciferase, which was used as a measure of HIV-1 promoter activity. Ciglitazone treatment of U937 cells suppressed LTR.luciferase expression (Fig. 6A). The PPARgamma WT vector, which promotes PPARgamma activity, was co-transfected with the LTR.luciferase vector (Fig. 6B). Transfection of the PPARgamma WT vector alone into U937 cells in the absence of another stimulus suppressed HIV-1 promoter activity. When U937 cells co-transfected with LTR.luciferase and PPARgamma WT were subsequently treated with ciglitazone, PGA2, or 15dPGJ2, there was further suppression of HIV-1 promoter activity. These observations confirm that there is base-line PPARgamma expression in U937 cells, as indicated by the ability of ciglitazone to suppress LTR.luciferase expression in the absence of transfection with PPARgamma WT vector. These findings also support the hypothesis that increased expression of PPARgamma inhibits HIV gene expression, probably through regulation of HIV-1 promoter activity. To further confirm that PPARgamma expression is responsible for the regulation of HIV-1 promoter activity, we utilized the PPARgamma dominant negative vector, PPAR.af2, to block endogenous PPARgamma activity in U937 cells. Co-transfection of U937 cells with PPARgamma dominant negative vector and LTR.luciferase augmented HIV-1 promoter activity (Fig. 7A). In addition, the PPARgamma dominant negative vector boosted HIV-1 promoter activity in U937 cells stimulated with PMA (Fig. 7B).


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  		Fig. 6.   Synthetic PPARgamma agonists suppress HIV-1 promoter activity. U937 cells were transfected by electroporation with the LTR.luciferase vector (100 ng) with (B) or without (A) the PPARgamma WT vector, as described under "Experimental Procedures." A, the cells were treated with or without the PPARgamma agonist ciglitazone (50 µM). After overnight incubation the cells were treated with or without PMA for 2 h. Luciferase expression was determined by measuring relative light units/µg of protein. The data are normalized for total protein and beta -galactosidase expression. The data shown are the means ± S.E. and are expressed as a percentage of PMA-treated cultures. *, p < 0.001, n = 5. B, the U937 cells transfected with or without the PPARgamma WT vector were treated with the PPARgamma agonist ciglitazone, PGA2, or 15dPGJ2 for 3 h. The data are expressed as luciferase expression measured in relative light units (RLU)/µg of protein. A representative experiment of three performed is shown.


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  		Fig. 7.   HIV-1 promoter activity is reversed by inhibition of PPARgamma activity. U937 cells were transfected by electroporation with the LTR.luciferase vector (100 ng) with the PPARgamma dominant negative vector, PPAR.af2, and stimulated with (B) or without (A) PMA as described in the legend for Fig. 6. Luciferase expression was determined by measuring relative light units (RLU)/µg of protein. The data are normalized for total protein and beta -galactosidase expression. A, the data shown are the means ± S.E. luciferase expression expressed as a percentage of LTR.luciferase alone. *, p = 0.05, n = 3. B, mean data from an experiment performed in triplicate are shown.

Because cyPG have PPARgamma -independent effects and U937 cells have endogenous expression of PPARgamma , we next studied HeLa cells, which do not have any PPARgamma expression (48). In the absence of PPARgamma transfection, both cyPG and ciglitazone did not demonstrate suppression of HIV-1 promoter activity as measured by levels of LTR.luciferase expression (Fig. 8). Upon co-transfection of HeLa cells with PPARgamma WT and LTR.luciferase, in the absence of another stimulus, there was suppression of HIV-1 promoter activity. Following treatment with either cyPG or ciglitazone there was further inhibition of HIV-1 promoter activity. These observations suggest that the suppression of HIV-1 promoter activity by cyPG is PPARgamma -dependent. Furthermore, it confirms that the suppression of HIV-1 promoter activity by ciglitazone is through PPARgamma activation.


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  		Fig. 8.   Role of PPARgamma in the suppression of HIV-1 promoter activity by cyPG. HeLa cells were co-transfected by electroporation with the LTR.luciferase vector (100 ng) with or without the PPARgamma WT vector as described under "Experimental Procedures." The cells were treated with or without PGA2 or the PPARgamma agonist ciglitazone (Ciglit) for 3 h. After overnight incubation the cells were treated with or without PMA for 2 h. Luciferase expression was determined by measuring relative light units (RLU)/µg of protein. A representative experiment of three performed is shown.

Natural and Synthetic PPARgamma Agonists Decrease HIV mRNA Stability-- Having demonstrated that natural and synthetic PPARgamma agonist treatment suppresses HIV mRNA levels, we next wanted to determine whether there was also suppression at a post-transcriptional effect. If PPARgamma agonist pretreatment resulted in reduced mRNA levels (RT-PCR) upon treatment with actinomycin D, which blocks de novo mRNA synthesis, this would suggest that it was having an effect on HIV mRNA stability. The kinetics of the effect of ciglitazone pretreatment on HIV mRNA levels in U1 cells are demonstrated in Fig. 9A. From the early time points, ciglitazone suppressed HIV mRNA levels compared with untreated cells. Furthermore, ciglitazone pretreatment suppressed HIV-1 mRNA levels in U1 cells following the addition of actinomycin D, suggesting that it did reduce mRNA stability (Fig. 9, A and B). This observation suggests that the effect of ciglitazone on HIV-1 gene expression occurs both at the level of transcription and at the level of mRNA stability.


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  		Fig. 9.   Synthetic PPARgamma agonists suppress HIV-1 mRNA stability. A, U1 cells were preincubated with and without ciglitazone (Cig) (50 µM) for 3 h and then stimulated with PMA. At 16 h they were treated with or without actinomycin D (Act D) (10 µg/ml) (arrow). HIV-1 mRNA was determined by RT-PCR at 3, 16, 20, 24, and 28 h after treatment with or without ciglitazone. A representative experiment of three is shown as the mean and S.E. of triplicate wells. B, mean data ± S.E. from three individual experiments are shown. The left column demonstrates HIV-1 mRNA expressed as a percent of U1 cells treated and PMA at 12 h after treatment with actinomycin D. *, p = 0.05, n = 3. The right column demonstrates HIV-1 mRNA expressed as a percent of U1 cells treated with PMA and actinomycin D at 12 h after ciglitazone treatment. **, p = 0.008, n = 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In view of the antiviral effects of PG and the known increase in PG levels in macrophages in HIV-1 disease, we examined the effect of cyPG on HIV-1 replication. Our findings demonstrate the following. 1) Treatment with cyPG (i.e. PGA1 and PGA2) results in a significant decrease in HIV-1 replication in MDM and in latently HIV-1 infected U1 cells. 2) The mechanism of HIV-1 suppression replication did not appear to be at the fusion/entry level because cyPG also suppressed HIV-1 replication in the latently infected U1 cell line and reduced HIV-1 mRNA levels. 3) CyPG likely suppressed HIV-1 replication by activation of PPARgamma . 4) Synthetic PPARgamma agonists also suppress HIV-1 replication. 5) Finally, the mechanism of suppression of HIV-1 replication is at both the HIV-1 promoter level, as well as at the level of HIV-1 mRNA stability.

PPARs are ligand-inducible transcription factors that belong to the family of nuclear transcription factors. PPARalpha is expressed in freshly isolated PBM and is found in many tissues including the liver, heart, kidney, skeletal muscle, lung, and adipose tissue. PPARgamma is expressed abundantly in adipose tissue but also in skeletal muscle, liver, heart, and bone marrow stromal cells. PPARgamma exists as three isomers, PPARgamma 1 being expressed predominantly in macrophages. PPARgamma is also expressed upon activation of PBM and their differentiation into macrophages. The expression of PPARgamma occurs at 48-72 h after the culture of PBM in vitro (30, 49) and is also expressed in the monocytic cell line U1 (50). When activated by ligand binding, PPARs can activate promoters and modulate gene expression (25, 26).

PGA1 and PGA2 are natural ligands for PPARalpha and PPARgamma (23, 24, 51, 52). PGD2 can undergo dehydration to PGJ2 and ultimately Delta 12-PGJ2, which is produced in significant quantities in the human body (53). CyPG, which includes PGA1 and PGA2, can down-regulate gene transcription by a variety of effects that can be PPARgamma -dependent or -independent. PGA1 is reported to inhibit NF-kappa B activation in Jurkat, CEM-SS, and HeLa cells (54) and has been demonstrated to increase I-kappa Balpha expression. Increased expression of I-kappa Balpha would result in an increased association of I-kappa Balpha and NF-kappa B, which would prevent the translocation of NF-kappa B to the nucleus (55). PGA1 and PGJ2 have been shown to inhibit I-kappa B kinase, which increases I-kappa Balpha levels and prevents the translocation of NF-kappa B to the nucleus (56). In our model the effect of PGA1 and PGA2 on the suppression of HIV-1 infection appears to be PPARgamma -dependent.

The synthetic PPAR agonist WY-14643 is a specific PPARalpha activator (23, 57). A thiazolidinedione compound, ciglitazone, has been shown to be a specific PPARgamma activator (58). Both specific PPARalpha agonists and PPARgamma agonists up-regulate and down-regulate gene expression (25, 26). Our observation that both the natural PPARgamma agonists PGA1 and PGA2 and the synthetic specific PPARgamma agonist ciglitazone cause a decrease in HIV-1 replication in MDM and the U1 cell line suggests that PPARgamma is an important factor in the regulation of HIV-1 replication. One mechanism appears to be the inhibition of HIV-1 promoter activity. This could come about through a number of scenarios. Activation of PPARgamma is known to inhibit gene expression by antagonizing the transcription factors AP-1, STAT, and NF-kappa B (30, 59). In addition, PPARgamma may suppress HIV-1 replication by inactivation of NF-kappa B (60). Another potential mechanism of inhibition of HIV-1 promoter activity by PPARgamma is through down-regulation of macrophage cytokine production, e.g. tumor necrosis factor-alpha and interleukin-6, which are known to augment transcription of HIV-1 (61).

There is a dearth of information on the effect of PPARgamma on mRNA stability. The predominant effect of PPARgamma is on transcriptional regulation. In the studies that investigated the role of PPARgamma on post-transcriptional regulation, there was no effect on mRNA stability (62, 63). Our findings suggest that PPARgamma activation reduces HIV-1 mRNA stability. Furthermore there is little information available on the regulation of HIV-1 replication by affecting viral mRNA stability. Expression of the Rev protein affects stability and transport of viral (HIV-1) mRNA (64, 65). Regulation of such proteins by PPARgamma activation may be the mechanism by which ciglitazone reduces HIV-1 mRNA stability.

Both the cyPG (and their derivatives) and thiazolidinedione compounds have been investigated as possible therapeutic agents for the treatment of a number of clinical disorders, e.g. diabetes mellitus and neoplasms (50, 66). Thiazolidinediones, which include troglitazone, ciglitazone, and rosiglitazone (BRL49653) are in clinical use for the treatment of insulin-resistant diabetes (67, 68). In addition, there are data in the literature suggesting that natural PPARgamma agonists suppress replication of viruses other than HIV. PGA is reported to inhibit Sendai virus replication in African green monkey kidney cells (69). PGE1 and PGE2, which can be converted to PGA1 and PGA2, respectively, have also been demonstrated to inhibit measles virus replication in Vero cells (70). Herpes simplex virus replication is reported to be inhibited by PGD2, Delta 7-PGA1, and Delta 12-PGJ2 (71, 72).

In summary, PG are produced by macrophages infected with HIV-1 and are known to be elevated in patients with AIDS (6). Our findings demonstrate for the first time that cyPG, which are natural PPARgamma agonists, decrease replication of HIV-1 in MDM. These PG, generated by HIV-1 infection of macrophages, may in turn act to decrease viral replication in these same cells by transcriptional and post-transcriptional mechanisms. Another exciting possibility is that synthetic PPARgamma agonists, already on the market for the treatment of diabetes mellitus, may be utilized as agents in the fight against HIV-1.

    ACKNOWLEDGEMENT

We thank Dr. V. Chatterjee, Cambridge, UK, for providing the PPARgamma expression vectors.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AI36685 (to D. M. M.) and HL57885 (to M. J. C.), the General Clinical Research Center at the University of Michigan (Grant MOI-RR00042), the Medical Scientist Training Program of the University of Michigan (National Institutes of Health Grant NIGMS T32GM07863 to B. R. L.), and funds from the Harvey fellows programs (to B. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: University of Michigan Medical Center, 6301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0642. Tel.: 734-764-4554; Fax: 734-764-4556; E-mail: coffeym@umich.edu.

Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M200875200

    ABBREVIATIONS

The abbreviations used are: PG, prostaglandin(s); HIV, human immunodeficiency virus; PBM, peripheral blood monocyte; cyPG, cyclopentenone prostaglandin; PPAR, peroxisome proliferator-activated receptor; MDM, monocyte-derived macrophage; PMA, phorbol myristate acetate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT, reverse transcriptase; WT, wild type; STAT, signal transducers and activators of transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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