Jul 12, 2007 - CCR5 signaling through phospholipase D involves p44/42 MAP-kinases and promotes HIV-1. LTR-directed gene expression. Sylvain Paruch,* ...
The FASEB Journal article fj.06-7325com. Published online July 12, 2007.
The FASEB Journal • Research Communication
CCR5 signaling through phospholipase D involves p44/42 MAP-kinases and promotes HIV-1 LTR-directed gene expression Sylvain Paruch,*,† Myle`ne Heinis,*,† Julie Lemay,*,† Guillaume Hoeffel,*,† Concepcio´n Maran˜o´n,*,† Anne Hosmalin,*,† and Axel Pe´rianin*,†,1 *Institut Cochin, Universite´ Paris Descartes, CNRS (UMR 8104), Paris, France; and †Inserm, U567, Paris, France ABSTRACT The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1 in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1 also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation,respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1BAL replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.—Paruch, S., Heinis, M., Lemay, J., Hoeffel, G., Maran˜o´n, C., Hosmalin, A., Pe´rianin, A. CCR5 signaling through phospholipase D involves p44/42 MAP-kinases and promotes HIV-1 LTR-directed gene expression. FASEB J. 21, 000-000 (2007)
Key Words: HIV-1 coreceptors 䡠 signal transducers 䡠 infection CCR5 is a chemoattractant receptor for several chemokines (MIP-1␣, MIP-1, and RANTES) that plays an important role in the early phases of infection by the human immunodeficiency virus 1 (HIV-1). CCR5 is a leukocyte coreceptor of HIV-1 (1) that interacts with the viral envelope glycoprotein gp120 after the latter binds to CD4, the main HIV-1 receptor. Only HIV-1 isolates that bind to CCR5 were shown to infect macro0892-6638/07/0021-0001 © FASEB
phages, which are major reservoirs of the virus (2). Structural protein rearrangements allow the viral transmembrane subunit gp41 to fuse the viral envelope with the membrane of target cells, thereby facilitating HIV-1 uptake. The provirus genome is subjected to reverse transcription (3, 4) and integration into the host genome, and can establish a latent infection (5, 6). HIV-1 replication is dependent on activation of a single promoter present in the long terminal repeat (LTR) region of HIV-1 genome that contains binding sites for the HIV-1 transactivator protein Tat (7, 8) and for various endogenous transcription factors (9, 10). Viral replication requires a state of activation of host cells since HIV-1 does not infect resting CD4⫹ T cells from peripheral blood (3, 11). Consistent with an important role for cellular signaling in HIV-1 infection, cell stimulation by mitogens such as phorbol esters up-regulates the activation of HIV-1 LTR and viral replication, supporting a role for protein kinase C (PKC) (12-14). Other signaling transducers have been shown to be involved, including G-proteins (15, 17), PI3-kinase (18), NF-B (19), and p44/42 MAP kinases (ERK1/2) (20), although relationships between these effectors remain unclear. Characterization of all of the signaling events mediated by CCR5 is needed to unravel HIV pathogenesis. Among the major intracellular signaling effectors activated by classical chemoattractants (formyl peptides, complement-derived C5a, platelet-activating factor), the phosphatidylcholine (PC)-specific phospholipase D (PLD) was lately identified in mammalian cells and proven to be an important source of several second messengers in most cell types (21, 22). So far, PLD activity in response to activation of the HIV-1 coreceptors CCR5 or CXCR4 remains uncharacterized. PLD cleaves PC into choline and a lipid second messenger, phosphatidic acid (PA). The latter is rapidly dephosphorylated into diglycerides (DG) (23) whereas choline 1 Correspondence: Institut Cochin, De´partement de Biologie Cellulaire, Pavillon G. Roussy 5e`me Etage, P502, 27, rue du Faubourg St. Jacques, 75014, Paris, France. E-mail: perianin@ cochin.inserm.fr doi: 10.1096/fj.06-7325com
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is converted into a second messenger, phosphorylcholine (24). Two distinct families of PLDs, PLD1 and PLD2, have been cloned in mammalian cells and share ⬃50% amino acid identity (25). However, both PLDs show a different intracellular distribution and are activated differently, suggesting they regulate different biological functions (23). We recently provided evidence that p44/42 MAP kinases use the PLD2 isoform to regulate the respiratory burst of HL-60 cells upon activation of fPR by formyl peptides (26). This receptor is also activated by HIV-1 gp41-derived peptides (27). We further showed that the PLD2 isoform is directly phosphorylated by ERK2 (26). This novel ERK/PLD2 pathway raises the possibility that some biological functions ascribed to ERK1/2 may actually be relayed by PLD. So far, the biological importance of PLD isoforms in CCR5 signaling and regulation of HIV-1 gene expression and replication remains unknown. In this study we characterized the PLD activity in response to activation of CCR5 in human HeLa cells stably expressing the HIV-1 coreceptor. Taking advantage of the expression of PLD variants and antisense oligonucleotides, we further investigated the contribution of both PLD isoforms, PLD1 and PLD2, to activation of HIV-1 LTR by the viral transactivator Tat and to virus replication. Our data indicate that CCR5 mediates PLD stimulation through activation of ERK1/2 and that the activity of both PLD isoforms strongly contributes to the transactivation of HIV-1 LTR and viral replication.
MATERIALS AND METHODS Materials Cell culture media, fetal calf serum (FCS), and oligofectamine were from Invitrogen (Carlsbad, CA, USA). FuGENE 6 reagent was from Roche Molecular Biochemicals (Basel, Switzerland). Amplex Red was obtained from Molecular Probes (Invitrogen). Antiphosphorylated ERK1/2 mouse monoclonal antibody was from New England Biolabs (Ipswitch, MA, USA). Rabbit polyclonal anti-ERK1, anti-MEK1, and peroxidase- or alkaline phosphatase-conjugated antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-HA (12CA5) and protease inhibitor cocktail tablets were from Roche. Mouse monoclonal anti PLD1 (clone SKB2) was from Upstate (Millipore, Billerica, MA, USA). Rabbit polyclonal anti PLD2 was purchased from Santa Cruz and was a kind gift from Dr. Blandine Ge´ny (Pasteur Institute, Paris, France). The soluble HIV-1 glycoprotein Gp120 was obtained through the NIH AIDS Research and Reference Reagent Program catalog of the National Institutes of Health (Bethesda, MD, USA). Thiolated oligodeoxynucleotides, antisense, and sense to PLDs were synthesized by Invitrogen and were used earlier with success in HL-60 cells (26). The sequence of oligodeoxynucleotides was derived from published coding sequences of PLD cDNA, i.e., PLD1 antisense (5⬘-CCGTGGCTCGTTTTTCAGTGACAT-3⬘), PLD2 antisense (5⬘-AGGGTCTTCTGGGTTACAGTCAT-3⬘), PLD1 (scrambled) sense (5⬘-TTCACGCCTGAATGTTCTCTGTGT-3⬘), and PLD2 (scrambled) sense (5⬘-TGTGCGGTTTATTCGGAGGCAAT-3⬘). SiRNA for PLD1 (hPLD1 target sequence (5⬘-AAGUUAAGAGGAAAUUCAAGC) and 2
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siRNA for PLD2 (hPLD2 target sequence 5⬘-GACACAAAGUCUUGAUGAG) were synthesized by Sigma-Proligo (SigmaAldrich, St. Louis, MO, USA) and designed according to Fang et al. (28) and Powner et al. (29), respectively. A siRNA without sequence similarity with any known mammalian gene (5⬘-CGUACGCGGAAUACUUCGA) was used as a negative control (30). All other reagents were purchased from Sigma. Plasmids Plasmids encoding wild-type HA-tagged PLD2 and PLD1 or their catalytically inactive variants, PLD2-DN (K758R) and PLD1-DN (K898R), were kind gifts from Dr . M. A. Frohman (31). Plasmid encoding the constitutively active MAPKK1 (MEK1) mutant in which the serine residues 218 and 222 were replaced by glutamic acid (MEK-SS/DD) in-frame with an HA-tag (32) was kindly donated by J. Pouyssegur (Institute of Signaling, CNRS, Nice, France). The plasmid pCTA allowing HIV-1 Tat expression was a kind gift from Dr. M. Marc Alizon (33). Cell lines and transfections HeLa P4.2 cells stably express CD4 and bear the Escherichia coli -galactosidase gene (LacZ) under transcriptional control of the HIV-1 LTR (33). MAGI-CCR5 cells constitutively expressing both CD4 and CCR5 (34) are derived from HeLa P4.2 cells and are routinely used for HIV-1 replication (35). Cells were cultured under standard conditions (DMEM, 10% FCS, 100 g/ml streptomycin, and penicillin) and transfected with 3 g of plasmid DNA of interest using FuGENE 6 according to the manufacturer’s instructions. The HeLa-Env-ADA and the HeLa P4.2 target cells used in the cell fusion assay were provided by Dr. M. Alizon (33). The HeLa-Env-ADA stably expresses the R5-HIV-1ADA strain and the HIV-1 transactivator Tat. The HeLa P4.2 stably expresses CD4 and bears the E. coli -galactosidase gene (LacZ) under transcriptional control of the HIV-1 LTR (33). The monocytic THP-1 cells (a kind gift from Dr. Philip G. Strange, University of Reading, Reading, UK) were grown in RPMI 1640 medium in standard conditions. Cell-cell fusion assays Target cells (⬃5⫻104 HeLa P4.2 cells) were dissociated with 5 mM EDTA and added to an equivalent number of adherent HeLa env-ADA in a 35-mm plate (33). After 24 h, cells were fixed with 0.4% glutaraldehyde and stained with the -galactosidase substrate X-Gal (5-bromo-4-chloro-3-indoyl--d-galactopyranoside). Blue-stained foci representing syncytia were counted under ⫻20 magnification. This model of cell fusion requires CCR5 expression since, in the absence of CCR5, basal values represented 1-4% of syncytia obtained with CCR5-transfected cells. HIV-1 infection assay MAGI cells were plated (106 cells/ml) in 24-well plates and transfected with an empty vector (pcDNA3, control) or with wild-type and inactive PLDs in conditions indicated in the figure legends. The monocytic THP-1 cells were transfected with 8 M sense or PLD1 or PLD2 antisense oligonucleotides in the presence of oligofectamine according to the manufacturer’s instructions. Twelve hours after transfection, the medium was removed and cells were infected with monocytotropic HIV-1BAL (10 ng Gag p24 protein/million cells) for 3 h at 37°C under 5% CO2 in DMEM supplemented with 1% FCS
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(36). Cells were washed extensively and cultured in the same medium. A fraction of the supernatant (100 l) was collected every 12 h for 4 days and stored at ⫺80°C to measure HIV-1 gene expression as determined by Gag p24 ELISA (Immunogenetics NV, Ghent, Belgium). In some experiments, THP-1 cells were treated with 100 and 300 ng/ml pertussis toxin for 18 h before infection and throughout the assay, as described (16). Cell viability was determined using the Trypan blue assay. PLD and immunoblot assays PLD activity was studied by measuring mass choline production (37) with some adaptations for fluorometric measurements, as recently reported (26). HeLa MAGI cells were starved with 0.05% BSA for 6-12 h, harvested with PBS containing 10 mM ETDA, and suspended in Hanks balanced salt solution (HBSS, pH 7.4). Cells were stimulated at a density of 2 ⫻ 106 cells/200 l, as described in the figure legends. Choline content was calculated from calibration curves obtained with exogenous choline chloride (5 to 50 pmol) and expressed as percent increase above basal control values. In some experiments, cells were transfected with either 3 g plasmid encoding PLD1-DN, PLD2-DN or with 30 nM siRNA using oligofectamine (Invitrogen) according to the manufacturer’s protocol. The cells were cultured for 36 h in 1% FCS, then starved in the presence of 0.05% BSA for 6 h before stimulation with MIP-1. Immunoblot experiments were performed under standard conditions using specific antibodies against phosphoERK1/2 (Thr202/Tyr204), ERK1/2, the HA epitope of PLD1 or PLD2), and endogeous PLDs, as described (26). Enhanced chemiluminescence was used to detect HPO-conjugated secondary antibody. In some experiments, membranes were stripped and reprobed with antibodies of interest. Quantification of phosphorylated proteins was performed with the NIH Image 1.62 software (National Institutes of Health, Bethesda, MD, USA). Statistical analysis Unless indicated otherwise, data represent means ⫾ se of at least 3 experiments. Differences between means were identified using a Student’s paired t test or the 1-way ANOVA test for differences between distinct cell populations. Statistically
significant differences between means were calculated with a threshold of P ⬍ 0.05, designated by an asterisk.
RESULTS CCR5-mediated PLD activity in MAGI cells is dependent on ERK1/2 activation PLD activity was studied in MAGI cells stably expressing CCR5 and CD4 by measuring the production of mass choline, as we previously reported (26). Treatment of cells in the presence of the specific CCR5 agonist, the macrophage inflammatory protein 1- (MIP-1), triggered a rapid and transient production of choline peaking at ⬃1-2 min (Fig. 1A). This PLD activity was proportional to an MIP-1 concentration up to 50 nM (Fig. 1B). We previously showed that PLD activity induced by the chemoattractant f-Met-Leu-Phe (fMLP) is dependent on activation of the p44/42 MAP kinases (ERK1/2) (26). To determine the contribution of ERK1/2 to CCR5-mediated PLD activity, MAGI cells were treated with a specific MEK1/2 antagonist, U0126, under conditions that blocked ERK activation (Fig. 1D). U0126 also abrogated MIP-1-induced choline production, suggesting a major contribution of ERK1/2 to PLD activity mediated after the interaction of CCR5 with MIP-1 (Fig. 1C). Since CCR5 also interacts with the HIV-1 envelope glycoprotein Gp120 as part of the virus entry process, we examined whether Gp120 stimulates PLD activity. Treatment of MAGI cells with soluble Gp120 (40, 80, and 160 nM) triggered a rapid and transient choline production proportional to the stimulus concentration (⬃75, 85, and 105% increase above basal choline values). This stimulation was inhibited by ⬃50% by U0126, indicating a significant contribution of ERK1/2. Next, the contribution of PLD isoforms PLD1 and PLD2 to choline production in MAGI cells was studied using the transient expression of a catalytically inactive
Figure 1. MIP-1- and Gp120-induced PLD activity is dependent on the activation of ERK1/2. HeLa P4.2 cells stably expressing CCR5 (MAGI-CCR5) were stimulated with 50 nM MIP-1 for varying times (A) or with various MIP-1 concentrations for 3 min (B). PLD activity is expressed as a percent increase (⌬ %) above basal values (393⫾27 pmol per 107 cells, n⫽5 experiments). C) Cells were treated without (control) or with 10 M U0126 for 10 min before stimulation with 50 nM MIP1- or 80 nM Gp120 for 2 min. PLD activity is expressed as a percent increase above basal control values (mean of 5 experiments, *P ⬍ 0.05). D) An immunoblot of phosphorylated ERK1/2 representative of 3 separate experiments. PLDS IN CCR5 SIGNALING TO HIV-1 INFECTION
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mutant of PLD2 (PLD2-DN) or PLD1 (PLD1-DN) (Fig. 2A). Both PLD mutants reduced basal PLD activity by ⬃30-35% (data not shown). The expression of PLD1-DN and PLD2-DN strongly inhibited MIP-1induced PLD activity (Fig. 2B). Because the expression of PLD variants could lead to side effects, the contribution of both PLDs was further examined using a siRNAbased approach. Treatment of MAGI cells with 30 nM siRNA for 48 h significantly reduced the expression of both PLDs by ⬃50-60% (Fig. 2C). Both siRNAs reduced basal PLD activity by ⬃40-50% whereas the MIP-1induced choline production was strongly inhibited (Fig. 2D), confirming a major contribution of both PLDs observed with the PLD mutants. Contribution of PLD isoforms to activation of the HIV-1 LTR To investigate the relevance of PLD activity in regulating HIV-1 infection parameters, we first used a cell-cell fusion model (syncytium) that mimics early steps of HIV-1 entry into CCR5/CD4⫹ cells and the activation of HIV-1 LTR (33). In this cell-cell fusion assay, the viral transactivator Tat is transferred from HeLa-Env-ADA cells to target HeLa P4.2 cells, which allows the LTRLacZ gene to be activated. Target cells that constitutively express CD4 were cotransfected with CCR5 and an empty vector (pcDNA3), with catalytically inactive PLD isoform PLD1-DN or PLD2-DN, or wild-type PLD1 or PLD2. Incubation of these cells with adherent HeLaenv-ADA resulted in the formation of CCR5-dependent syncytia, as revealed by -galactosidase-positive cells. Compared to control (pcDNA3), PLD1-DN and PLD2-DN both reduced the number of syncytium by ⬃50% (Fig. 3, P⬍0.05). Conversely, the expression of wild-type PLD2 (PLD2-wt) greatly boosted LTR activation, and the expression of wild-type PLD1 (PLD1-wt) provided a weak potentiation effect (P⬍0.05). To further determine whether PLDs selectively regulate the fusion process and/or LTR activation, we next used a cell fusion assay that does not rely on reporter gene activity, as described (33). Using FACS monitoring of fluores-
Figure 3. The contribution of PLD isoforms to the transactivation of HIV-1 LTR-LacZ. HeLa P4.2 cells were transiently cotransfected with CCR5 (0.8 g DNA) and either an empty plasmid (pcDNA3, 1.2 g DNA), a plasmid encoding catalytically inactive HA-PLD1 (PLD1-DN) or HA-PLD2 (PLD2-DN), or wild-type HA-PLD2 (PLD2-wt) or HA-PLD1 (PLD1-wt). After 12 h incubation at 37°C, cells were cocultured with HeLa-Env-ADA cells stably expressing the envelope of R5HIV-1 strain ADA and the transactivator Tat. Results show the number of syncytia expressed as a percentage of control values (n⫽3 experiments). *P ⬍ 0.05, significant difference between control (pcDNA3) and PLD variants.
cent dye transfer from fusing cells, we saw no significant differences in the presence of PLD variants in this assay (data not shown). Consequently, we investigated whether gene expression from the LTR could be upregulated by PLDs. The contribution of PLD isoforms to LTR activation was studied using HeLa P4.2 cells after cotransfection with Tat and the PLD variant-coding vectors or an empty vector. The activation of the HIV-1 LTR required Tat and was proportional to the amount of Tat DNA from 0.1 to 0.5 g (Fig. 4A). The expression of inactive
Figure 2. Contribution of PLD1 and PLD2 to MIP-1-induced choline production in MAGI cells. Cells were transfected with 3 g of an empty plasmid (pcDNA3, control) or with a plasmid encoding an inactive HA-PLD1 (PLD1-DN) or HA-PLD2 (PLD2-DN) and cultured for 48 h. A) A representative Western blot analysis of inactive PLD variants in cell particulate fractions as revealed by an anti-HA antibody. B) PLD activity induced by 25 and 50 nM MIP-1 for 3 min. C) The expression of PLD in MAGI cells was treated with PLD or control siRNA oligos (30 nM) for 48 h as revealed by immunoblot. D) PLD activity induced by 50 nM MIP-1 (n⫽3 experiments). The PLD activity in each group is expressed as percent increase above its control value. *P ⬍ 0.05, significant difference between control and treated groups. 4
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Figure 4. Effect of the expression of Tat and PLD variants on the activation of LTR-LacZ. A) HeLa P4.2 cells were transfected with varying amounts of a Tat-coding plasmid. Results represent the number of -galactosidase-positive cells (mean⫾sd of 4 experiments). HeLa P4.2 cells were cotransfected with 0.1 g Tat and with either an empty plasmid (control) or the inactive PLD1-DN, PLD2-DN (B), wild-type PLD1, or PLD2 (C). Data represent the number of -Gal-positive cells (mean⫾sd of 3 experiments). D) A representative immunoblot of PLD variants, Tat and ERK1/2, in cell lysates.
PLDs greatly inhibited the LTR response, reaching ⬃70% inhibition for both PLD variants (Fig. 4B). Conversely, the expression of wild-type PLD1 or PLD2 induced a great potentiation of the LTR response, and the degree of potentiation was similar for both PLDs (Fig. 4C). Since the activation of the LTR response was proportional to the amount of Tat (Fig. 4A), Tat expression was examined in cells transfected with the PLD variants. Immunoblot analysis of cellular extracts showed no significant alteration in the amount of Tat after expression of PLD variants (Fig. 4D). Since PLD activity appears to play an important role in regulating the LTR response, we wondered whether the activation of chemoattractant receptors such as CCR5 or the formyl peptide receptor fPR promotes the LTR response. To this end, HeLa P4.2 cells were cotransfected with Tat and either CCR5 or fPR. Stimulation of both receptors induced a significant potentiation of LTR activation compared to control (Fig. 5A). This effect was inhibited in the presence of the MEK antagonist U0126, suggesting a positive regulation by the ERK pathway. Consistent with this, the expression of a constitutively activated MEKK mutant (MEK-DD) markedly potentiated the transactivation of LTR (Fig. 5B).
variants progressively vanished, likely due in part to cell proliferation. To further examine the relevance of this observation in a more physiological cellular model, virus replication was studied in the monocytic cell line THP-1 (39). Our data confirm that this cellular model is appropriate to replication of HIV-1BAL (Fig. 6C). Since these cells are hardly transfectable with plasmid vectors encoding the PLD mutants under classical conditions, the contribution of PLD was studied after transfection of cells with the antisense oligonucleotides to PLD1 or PLD2 we used earlier in HL-60 cells (26). Treatment of THP-1 cells with 8 M antisense oligonucleotides inhibited the expression of both PLD by ⬃50-60% (Fig. 6D). Compared to control cells trans-
Contribution of PLDs to HIV-1 replication Activation of HIV-1 LTR by Tat is an essential step to virus replication (7, 8, 10). Since PLD activity strongly regulates the LTR response, we studied the contribution of PLD isoforms to replication of HIV-1 in MAGI cells constitutively expressing CCR5 and CD4. Incubation of MAGI cells with HIV-1-BAL led to a significant time-dependent production of HIV-1, as described (38). The transient expression of inactive PLD variants (Fig. 6B) partly but significantly inhibited the production of HIV-1 Gag p24 determined at 24 h of infection (Fig. 6A). Conversely, the expression of wild-type PLD1 or PLD2 boosted LTR activation. For longer periods of infection (48-72 h), the modulating effects of PLD PLDS IN CCR5 SIGNALING TO HIV-1 INFECTION
Figure 5. Priming of the activation of HIV-1 LTR upon stimulation of CCR5 and fPR and expression of MEK. A) HeLa P4.2 cells were cotransfected with Tat (0.1 g DNA) and CCR5 or fPR (0.8 g DNA), then treated in the absence (control) or presence of 10 M U0126 for 12 h before stimulation by MIP-1 or fMLP for 4 h. B) Cells were cotransfected with Tat (0.1 g DNA) and either an empty plasmid 0.8 g DNA) or a constitutively activated mutant of MEK (MEK-DD). Results represent the number of -galactosidase-positive cells expressed as a percentage increase above control values (n⫽4 experiments). *P ⬍ 0.05, significant differences between control and treated groups. 5
Figure 6. Contribution of PLD isoforms to HIV-1 replication. A) The production of HIV-1BAL in MAGI cells transfected with either an empty plasmid (pCDNA3) or the catalytically inactive HA-tagged PLD1 (PLD1-DN), PLD2 (PLD2-DN), wild-type PLD1 (PLD1-wt), or PLD2 (PLD-wt). The production of HIV-1 Gag p24 after 24 h of cell infection is expressed as a percentage of control values obtained with cells transfected with the empty plasmid (i.e., 78⫾5 pg/ml, mean of 4 experiments). B) A representative immunoblot of PLD variants in cell particulate fractions determined with an anti-HA antibody. C) THP-1 cells were treated with buffer or 8 M sense, antisense PLD1 (AS-D1), or PLD2 (AS-D2) for 12 h before infection with HIV-1BAL. Results are representative of 3 experiments and are expressed as pg Gag p24 /ml of supernatant. D) A representative immunoblot of endogenous PLD1 and PLD2 in the homogenates of THP-1 cells treated with sense, antisense oligonucleotides. *P ⬍ 0.05, significant difference between control and PLD variants.
fected or not with sense oligonucleotides, the antisense oligonucleotides to PLD1 or PLD2 strongly inhibited the production of Gag p24 after cell infection (Fig. 6C), which confirms the important role for both PLDs in HIV-1 replication parameters (Fig. 4). The efficiency of leukocyte infection with R5-HIV strains depends in part on the signaling initiated by trimeric G-proteins of Gi type (16), although this assumption has been a subject of controversy. We therefore examined the contribution of Gi in producing Gag p24 by HIV-1BAL-infected THP-1 cells. Treating cells with the G␣i inhibitor pertussis toxin (100 and 300 ng/ml) significantly reduced the production of Gag p24 after 3 days of infection (Fig. 7A) without altering cell viability (Fig. 7B). However, the PTX inhibitory effect was partial (40%), in agreement with the literature (16), and was not further increased with a higher toxin concentration (300 ng/ml).
DISCUSSION This study provides the first evidence that CCR5 activation by MIP-1 triggers substantial PLD activity (200300 p mol choline/107 MAGI cells) in a rapid, transient, and ERK1/2-dependent pattern similar to that of the formyl peptide receptor fPR in HEK and HL-60 cells (26). We also showed that HIV-1 enveloped glycoprotein Gp120 also stimulated strong PLD activity that was partially dependent on ERK. Both PLD1 and PLD2 contribute to CCR5-mediated PLD activity, as shown by the expression of inactive PLD mutants and the use of siRNA oligos. The mechanism by which ERK1/2 regulates the PLD activity was not studied in detail here since we previously showed that activated ERK phosphorylates the PLD2 isoform and stimulates PLD activity in membrane preparations in the presence of ATP (26). We also showed that the PLD2 isoform forms a complex with ERK1/2 in resting HEK cells and is phosphorylated on MAP kinases consensus (S/T-P) 6
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Figure 7. Effect of pertussis toxin (PTX) on HIV-1 replication. THP-1 cells were treated in the absence (control) or presence of 100 and 300 ng/ml pertussis toxin for 18 h before infection with HIV-1BAL and throughout the assay. Results are expressed as pg Gag p24 /ml of supernatant (A, a representative experiment of 3, *P⬍0.05). Cell viability was determined at day 4 of infection using the Trypan blue assay (B).
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upon stimulation of fPR. It remains unknown whether PLD1 can be activated by MAP kinases, although its sequence contains potential phosphorylation sites. The biochemical process by which activated CCR5 or fPR triggers the ERK/PLD2 pathway is not elucidated. However, PKC, a potent activator of ERK and PLD (26) that can be activated upon stimulation of CCR5 (40) or fPR (41), does not appear to play a major role, as we noted that the PKC antagonist GF109203X did not alter the PLD activity mediated by fPR (26) or CCR5 (data not shown). A similar observation was reported for the norepinephrine-stimulated PLD activity in vascular smooth muscle cells (42). An interesting hypothesis is that CCR5 or fPR-mediated PLD activity may occur through early recruitment of the Raf-MEK1/2-ERK1/2 module at the plasma membranes after the binding of -arrestins to activated CCR5 or fPR. This is suggested by the observation that -arrestin 2 is a scaffolding protein of the ERK1/2 module and stimulates its activation (43). Consistent with this, we reported that fPR stimulation in HEK cells increases the amount of ERK1/2 immunoprecipitated with PLD2 and the PLD2 phosphorylation by MAP kinases (26). The ERK/PLD pathway thus may be stimulated independent of PKC, presumably through the downstream effectors of heterotrimeric G-proteins such as Gi. Among these, the small G-protein Ras can be directly activated by the ␥ complex of Gi and by lipids generated by PI3-kinase-␥; the latter is also activated by the ␥ subunit of Gi (24). The importance of Gi in CCR5 signaling and in HIV-1 infection upon R5-virion binding to CCR5 has recently been reported in HOS cells using the Gi inhibitor PTX and a CCR5 mutant that does not activate Gi (16). We confirmed here that PTX significantly reduced the production of Gag p24 by THP-1 cells. The inhibition was observed 3 days after infection but not for shorter periods (24-48 h), consistent with the notion that CCR5 signaling through the Gi protein up-regulates HIV-1 postentry events (34, 15) but not HIV entry (44). However, the PTX inhibitory did not exceed 40% only, which indicates that Gi-independent signaling pathways may be important. Also, PTX-insensitive G-proteins like Gq should not be excluded since they interact with chemokine receptors (45), including CCR5 (46). This study shows that the catalytic activity of PLD1 and PLD2 plays a major role in regulating HIV-1 infection parameters. This is supported by the fact that the expression of inactive or wild-type PLDs respectively inhibited and boosted the LTR response (Fig. 4) together with the HIV-1 replication in MAGI cells (Fig. 6A). In addition, the inhibition of expression of both PLDs by antisense oligonucleotides drastically inhibited HIV-1 replication in THP-1 cells. PLD is a main source of lipid second messengers, such as phosphatidic acid (PA), a potent activator of PKC, and diglycerides, which activate other PKCs. Our observation that the PKC antagonist GF109203X strongly inhibited the LTR activation by Tat (⬃80%; data not shown) agrees with previous work (12-14) and is consistent with a main PLDS IN CCR5 SIGNALING TO HIV-1 INFECTION
contribution by the PLD pathway. A role for PA in regulating the LTR response has also been proposed (47). Our observation that the stimulation of CCR5 by MIP-1 or fPR by fMLP further increased the LTR activation through an ERK1/2-dependent process (Fig. 5) raises the possibility that HIV-1 postentry events can be regulated not only by CCR5 (34, 15), but also by other membrane receptors that stimulate ERK1/2 or PLD. The fPR is of particular interest since it can be activated by HIV-1-derived peptides (27) and by the HIV-1 transactivator Tat (48), suggesting a potential role for fPR in the amplification of viral infection. Pathways linking PLD activity to regulation of the transcriptional activation of LTR and viral replication are not elucidated. Our data suggest PLD preferentially regulates host-cell transcription factors that bind to the LTR sequence used here (⫺138 to ⫹83) rather than the viral protein Tat. Activation of LTR by Tat is essential for HIV-1 replication (7, 8, 10). Tat is expressed in the early steps of viral replication and interacts with the transactivation response element (TAR), allowing recruitment of cyclin T1 and CDK9, which phosphorylates the CTD of RNA pol II. Tat also induces nuclear translocation of ERK/12,which may lead to gene expression (49). In addition, Tat is secreted by infected cells and extracellular Tat stimulates various intracellular signaling events (48),which may promote gene expression. In this study, the expression of PLD variants did not alter the expression of Tat (Fig. 4C). This is consistent with an earlier observation that the PKC depletion in Jurkat and 293 cells did not alter the synthesis of Tat whereas LTR activation was strongly inhibited (13). However, regulation of the LTR response by PKC does not appear to involve phosphorylation of Tat or the phorbol esterresponsive enhancers of LTR that mediate basal transcription by mitogens (13). Among host cell transcription factors of the LTR (reviewed in ref. 50), NF-B was up-regulated by PLD (51). However, both PLDs repressed the level of p21 by different mechanisms: PLD1 by repressing p53 and PLD2 by down-regulating the activity of sp1 (52). The LTR response is also upregulated by reactive oxygen species (ROS) (14), activators of MAP kinases and NF-B (24). Note also that ROS production by stimulated phagocytes correlates with PLD activity (53, 26). Our data suggest that the PLD activity is important in HIV-1 replication as a potential target to lower the state of cellular activation, and consequently the HIV-1 LTR response and viral replication (Figs. 4, 6). In addition, our observation that PLD2 is finely regulated by ERK via phosphorylation of only a few sites (2 phosphopeptides vs. 12 for PKC␣) (26) makes it possible to identify structural motifs that could serve as templates to design PLD antagonists. One advantage of targeting the selective ERK/PLD coupling is that it would preserve basal PLD activation (Fig. 1C) and would selectively inhibit the priming properties of PLD activity mediated by various agonists at the origin of numerous cell dysfunctions. In conclusion, this study shows that activation of 7
CCR5 mediates a substantial PLD activity through the activation of ERK1/2. Both PLD1 and PLD2 contribute to this stimulation and make an essential contribution to the transactivation of HIV-1 LTR by Tat and to HIV replication. Since the PLD signaling pathway can be stimulated by various membrane receptors that promote HIV-1 LTR activation, PLD activity and ERK/PLD coupling open pharmacological perspectives to the design of potential PLD antagonists that could inhibit the state of cellular activation and the progression of HIV infection. This work is supported by l’Agence Nationale de la Recherche sur le Sida (ANRS). We thank Dr. Julie Overbaugh for providing MAGI-CCR5 cells obtained through the NIH-AIDS Research and Reference Reagent Program Division of AIDS (Bethesda, MD, USA). We thank Dr. P. G. Strange (University of Reading, UK) for providing the THP-1 cell line. We thank Drs. M. Alizon, A. Brelot, J. Toubas, and S. Marullo for their contributions. We thank Dr. B. Ge´ny (Institut Pasteur Paris, France) for providing the PLD2 antibody.
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Received for publication October 5, 2006. Accepted for publication May 31, 2007.
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