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Originally published In Press as doi:10.1074/jbc.M608307200 on December 11, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4288-4300, February 16, 2007
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Activation of JNK-dependent Pathway Is Required for HIV Viral Protein R-induced Apoptosis in Human Monocytic Cells

INVOLVEMENT OF ANTIAPOPTOTIC BCL2 AND c-IAP1 GENES*

Sasmita Mishra{ddagger}1, Jyoti P. Mishra{ddagger}1, and Ashok Kumar{ddagger}§2

From the Departments of §Pathology and Laboratory Medicine and {ddagger}Biochemistry, Microbiology, and Immunology, University of Ottawa K1H 8M5 and Infectious Disease and Vaccine Research Centre, Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada

Received for publication, August 30, 2006 , and in revised form, November 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus (HIV) accessory protein viral protein R (Vpr) plays a key role in virus replication and induces cell cycle arrest and apoptosis in various cell types including T cells and neuronal and tumor cells following infection with Vpr-expressing HIV isolates or exposure to the extracellular Vpr protein. The C-terminal Vpr peptide encompassing amino acids 52–96 (Vpr-(52–96)) is required for exerting the apoptotic effects, whereas the N-terminal Vpr-(1–45) peptide is responsible for virus transcription. We demonstrate that Vpr-(52–96) induced apoptosis in human promonocytic THP-1 cells and primary monocytes through the mitochondrial pathway in a caspase-dependent manner. To understand the regulation of Vpr-induced apoptosis, we investigated the signaling pathways, particularly the MAPKs, and the transcription factors involved. Although both Vpr-(52–96) and Vpr-(1–45) peptides induced phosphorylation of all the three members of the MAPKs, Vpr-(52–96)-activated JNK selectively induced apoptosis in monocytic cells through the mitochondrial pathway as determined by using JNK inhibitors SP60025, dexamethasone, curcumin, and JNK-specific small interfering RNAs. Furthermore Vpr-(52–96)-induced apoptosis was mediated by inhibition of downstream antiapoptotic Bcl2 and c-IAP1 genes whose expression could be restored following pretreatment with JNK-specific inhibitors. Overall the results suggest that Vpr-(52–96)-activated JNK plays a key role in inducing apoptosis through the down-regulation of antiapoptotic Bcl2 and c-IAP1 genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is an active process mediated by programmed signaling pathways that can be initiated by a variety of intracellular and extracellular stimuli. Apoptosis is essential to many biological processes including functional self-organizational processes in the immune and central nervous systems, morphogenetic changes in embryonic development, tissue homeostasis, and normal cell turnover (1). It also plays a critical role in the pathogenesis of a variety of diseases including cancer and infectious diseases (1, 2). Modulation of apoptosis may be closely associated with the outcome of a disease process. For example, resistance to apoptosis is one of the important mechanisms by which cancer cells evade destruction by the immune system (1). Therefore, effective therapies against such diseases should ideally induce efficient cytotoxicity to override apoptotic resistance. Recently the viral protein R (Vpr)3 of the human immunodeficiency virus (HIV) has been proposed as a novel antiproliferative agent in vivo because of its ability to induce apoptosis in normal and tumor cell lines, including leukemialymphoma, colon, and cervical carcinoma cell lines (3, 4), and its ability to transduce cells effectively without carriers or receptor targeting strategies (5, 6).

Vpr, a 14-kDa 96-amino acid protein, is the most conserved and multifunctional regulatory protein. It plays a key role in virus replication by causing nuclear translocation of the HIV-1 preintegration complex and transcriptional regulation of the HIV long terminal repeat (7–10). It has also been shown to induce cell cycle arrest at the G2/M phase and apoptosis in a variety of cell types including T cells, neutrophils, and neuronal cells following infection with Vpr-expressing HIV isolates or exposure to the extracellular Vpr protein (7, 11). Recently Vpr was detected in the sera and cerebrospinal fluid of HIV-infected subjects at levels similar to those of p24 antigen (5, 12). Moreover circulating Vpr was found to be biologically active in inducing virion production from latently infected cells, inducing apoptosis and causing depletion of bystander cells in lymphoid tissues during HIV infection (5, 12).

Multiple functions of Vpr have been attributed to its different domains (13). The Vpr protein is characterized by three well defined {alpha}-helices: 17–33, 40–48, and 55–83 surrounded by flexible negatively charged N-terminal and basic C-terminal domains (14). Furthermore the amphipathic {alpha}-helix 55–83 overlaps with a leucine-rich domain that contains a short leucine zipper-like motif (13–16). Mapping studies performed on lymphocytes or isolated mitochondria revealed that the N-terminal amino acids 1–51 Vpr protein (Vpr-(1–51)) is required for virion incorporation and nuclear localization, whereas the C-terminal amino acids 52–96 domain (Vpr-(52–96)) induced cell cycle arrest and apoptosis (5, 6, 17, 18). This apoptotic effect was mimicked by the Vpr-(52–96) but not by the Vpr-(1–51) moiety (19). There is evidence to suggest that Vpr induces apoptosis by directly targeting the mitochondrial permeability transition pore complex, a polyprotein complex organized around the two most abundant proteins of the inner and outer mitochondrial membranes, causing their permeabilization (9, 19). The NMR structure of the Vpr domain critical for its mitochondrial effects is comprised of an 11-amino acid helical 71HFRIGCRHSRI82 peptide with three positively charged Arg residues clustered on one side of the helix. Substitution of these Arg residues was shown to abolish Vpr-mediated apoptotic potential (9, 15, 20).

Monocytic cells in general have a slow turnover rate, are resistant to apoptotic pathways, and hence survive over a prolonged period of time. It is believed that resistance to apoptotic pathways in mononuclear phagocytes may facilitate establishment of persistent infection with intracellular organisms such as HIV (21). During HIV infection, persistently infected monocytic cells survive HIV replication and serve as a major reservoir of HIV in lymphoid tissues at all stages of disease, and their eradication presents a key challenge to eliminate virus reservoirs (21). Because the Vpr protein has been shown to induce apoptosis in normal and tumor cells (3), it has been suggested that Vpr or a Vpr derivative could be useful as an antiproliferative or cytotoxic agent for therapeutic purposes (3, 4). Therefore, in this study, we first investigated whether HIV Vpr can induce apoptosis in human monocytic cells by using Vpr-(1–45) and Vpr-(52–96) peptides as a model. Our results suggest that Vpr-(52–96) peptide selectively induced apoptosis in primary monocytes and leukemic promonocytic THP-1 cells through the mitochondrial pathway and in a caspase-dependent manner. Subsequently we investigated the signaling pathways, in particular the MAPKs, and the transcription factors involved in the regulation of genes mediating Vpr-induced apoptosis. Although both Vpr-(1–45) and Vpr-(52–96) peptides induced phosphorylation of all three p38, ERK, and JNK MAPKs, Vpr-(52–96)-induced apoptosis was regulated selectively through JNK activation. Furthermore Vpr-induced apoptosis was mediated by the down-regulation of antiapoptotic genes Bcl2 and cellular inhibitor of apoptotic protein 1 (c-IAP1) through the activation of upstream JNK MAPK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Monocytes from Peripheral Blood Mononuclear Cells, Cell Lines, and Reagents—THP-1, a promonocytic cell line derived from a human acute lymphocytic leukemia patient, was obtained from the American Type Culture Collection (Manassas, VA). Monocytes were isolated from peripheral blood mononuclear cells by autoMACS positive selection. Blood was obtained from healthy volunteers after approval of the protocol by the ethics review committee of the Children’s Hospital of Eastern Ontario, Ottawa, Canada. Peripheral blood mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque (GE Healthcare) as described previously (22). The cell layer consisting mainly of mononuclear cells was collected and washed twice in phosphate-buffered saline containing 2% EDTA followed by incubation with autoMACS CD14 microbeads (Miltenyi Biotech Inc., Auburn, CA) for 15 min at 4 °C. Cells obtained were washed and subjected to autoMACS positive selection separation according to the manufacturer’s instructions. Cells thus obtained contained >95% CD14+ monocytes. Cells were cultured in IMDM (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 µg/ml gentamicin, 10 mM HEPES, and 2 mM glutamine. PD98059, an inhibitor of mitogen-activated protein/ERK kinase-1, which selectively blocks the activity of ERK MAPK and has no effect on the activity of other serine/threonine protein kinases including Raf1, p38, and JNK MAPK, was purchased from Calbiochem (23, 24). The pyridinyl imidazole SB202190 (Calbiochem), a potent inhibitor of p38 MAPK, has no effect on the activity of ERK or JNK MAPKs (24, 25). SP600125, a JNK inhibitor (Biomol, Plymouth Meeting, PA), is a reversible ATP competitive inhibitor with >300-fold selectivity versus related MAPKs including ERK1 and p38 (26). Dexamethasone (DXM), 9{alpha}-fluoro-16{alpha}-methylprednisolone, and curcumin from Calbiochem were used as JNK inhibitors as well. Z-VAD-fmk, the broad spectrum caspase inhibitor, was obtained from Calbiochem. All other chemicals used for electrophoresis and immunoblot analysis were obtained from Sigma.

Cell Stimulation with Vpr Peptides—Vpr peptides were synthesized by Genemed Synthesis Inc., San Francisco, CA, by automated solid-phase synthesis using 9-fluorenylmethoxycarbonyl and were purified by reverse-phase high pressure liquid chromatography (>95%) followed by analysis with electrospray mass spectrometry. The amino acid sequence of these peptides is as follows: Vpr-(52–96), DTWAGVEAIIRILQQLLFIHFRIGCRHSR IGVTRQRRARNGASRS; Vpr-(1–45), MEQAPEDQGPQREPYNEWTLELLEELKSEAVRHFPRIWLHNLGQH. Because of the high propensity of Vpr peptides to bind to proteins, cells (1 x 106/ml) were treated with peptides in an isotonic buffer (13 mM HEPES, 2.4% glucose, 68 mM NaCl, 1.3 mM KCL, 4 mM Na2HPO4, 0.7 mM KH2PO4, pH 7.2) for 30 min followed by addition of culture medium as described previously (27).

Western Blot Analysis—Cells were treated with the indicated concentration of inhibitors for 2 h followed by stimulation with Vpr peptide(s) for various times for analysis of MAPK activation and Bcl2/c-IAP1 expression by Western immunoblotting as described earlier (22, 28). Briefly total proteins were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membranes were probed with either anti-phospho-MAPKs, anti-c-IAP1 (29), anti-Bcl2, or anti-phospho-Bcl2 antibodies (Cell Signaling Technology, Danvers, MA) followed by donkey anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase (Amersham Biosciences). To control for total proteins, the membranes were stripped of the primary antibodies and reprobed with rabbit polyclonal antibodies specific for the total p38, ERK, or JNK MAPKs (Santa Cruz Biotechnology) or with mouse anti-beta-actin monoclonal antibodies (Sigma). All immunoblots were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology).

Propidium Iodide (PI) Staining—Briefly cells (1 x 106/ml) were washed with phosphate-buffered saline containing 1% fetal bovine serum, fixed with methanol for 15 min at 4 °C, and treated with 1 µg/ml RNase A followed by staining with 50 µg/ml PI (Sigma) at 4 °C for 1 h. The DNA content was analyzed by using a FACScan flow cytometer (BD Biosciences) as described earlier (28).

Annexin-V and PI Staining—Cells were collected, washed with phosphate-buffered saline, and then stained with fluorescein isothiocyanate-labeled annexin-V and PI (Molecular probes, Eugene, OR) for 15 min at room temperature in the dark and analyzed by flow cytometry (30). Annexin-positive cells were plotted as histograms.

JC-1 Staining for Determination of Mitochondrial Membrane Potential (MMP)—JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) is a lipophilic carbocyanine that exists in a monomeric form and is able to accumulate in mitochondria. In the presence of high {Delta}{psi}, JC-1 can reversibly form aggregates that after excitation at 488 nm emit in the orange/red channel (FL-2). The collapse in {Delta}{psi} provokes the decrease in the number of JC-1 aggregates and a consequent increase in JC-1 monomers (FL-1) (31). Cells were stained with 10 µg/ml {Delta}{psi}-sensitive probe JC-1 (Molecular probes) in IMDM containing 10% fetal calf serum for 15 min at room temperature in the dark and analyzed by flow cytometry as described previously (31). JC-1 monomers and aggregates are presented as dot blots. Apoptotic cells with depolarized mitochondria are represented in the lower right quadrant of the graph. All histograms and dot blots were generated using WinMDI version 2.8 software.

Transient Transfection with Stealth JNK siRNA—THP-1 cells were transiently transfected with stealth siRNA (Invitrogen) specific for either JNK1 or JNK2 using FuGENE 6 (Roche Diagnostics) as described previously (22). Briefly a 50 pM concentration of either stealth JNK or control RNA was incubated for 45 min at room temperature with 3 µl of FuGENE 6 in 100 µl of serum-free IMDM to allow formation of RNA-liposome complexes. These complexes were then added to the cell suspension (106/ml) for 24 h followed by treatment with Vpr peptide(s) for either 2 or 24 h after which cells were analyzed for JNK phosphorylation by Western blot analysis or for apoptosis, cell cycle arrest, and mitochondrial membrane potential, respectively.

Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays were performed as described earlier (22, 28). Briefly cells were treated with Vpr peptide(s) for 3 h in the presence or the absence of indicated inhibitors. Subsequently nuclear proteins were harvested from cell pellets (5 x 106) following lysis with lysis buffers. The nuclear proteins (5 µg) were mixed with 32P-labeled specific oligonucleotide probes for 20 min, and the resulting complexes were separated on a 5% non-denaturing gel. The oligonucleotide probes containing sequences corresponding to the CREB (32), NF{kappa}B, and Sp1 binding sites (32) in the Bcl2 promoter were as follows: CREB: sense, 5'-GAA CCG TGT GAC GTT ACG CA-3'; antisense, 5'-TGC GTA ACG TCA CAC GGT TC-3'; NF{kappa}B: sense, 5'-TGC CAA GAG GGA AAC ACC AGA ATC AA-3'; antisense, 5'-TTG ATT CTG GTG TTT CCC TCT TGG CA-3'; and Sp1: sense, 5'-CAG AGG AGG GCT CTT TCT TTC-3'; antisense, 5'-GAA AGA AAG AGC CCT CCT CTG-3'. The oligonucleotide probes containing sequences corresponding to the CREB and NF{kappa}B binding sites in the c-IAP1 promoter (GenBankTM accession number AF070674 [GenBank] ) were as follows: CREB: sense, 5'-GGG CGC GCT GAC GTC ATC GTG CGT-3'; antisense, 5'-ACG CAC GAT GAC GTC AGC GCG CCC-3'; and NF{kappa}B: sense, 5'-GAG AAA GGC TAG TCC CTT TTC-3'; antisense, 5'-GAA AAG GGA CTA GCC TTT CTC-3'. To determine specificity of transcription factor binding, parallel electrophoretic mobility shift assay reactions were set up with 100–200-fold excess of unlabeled specific and nonspecific oligonucleotides for 20 min prior to the addition of corresponding labeled probe. Supershift experiments were also performed by using mouse anti-NF{kappa}B p50 and p65, anti-CREB, and anti-Sp1 antibodies (Santa Cruz Biotechnology). The gel was dried and exposed to x-ray film (Eastman Kodak Co.).

RNA Isolation and Semiquantitative RT-PCR Analysis—Total RNA was isolated from cells using RNeasy minispin columns combined with DNase I treatment (Qiagen, Mississauga, Canada). Total RNA (1 µg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences) as described previously (22, 28). Equal aliquots (5 µl) of cDNA were subsequently amplified for Bcl2, c-IAP1, and beta-actin. The oligonucleotide primer sequences used for Bcl2 (Biomol), c-IAP1 (33), and beta-actin (Stratagene, La Jolla, CA) were as follows: Bcl2: sense, 5'-TTC TTT GAG TTC GGT GGG GTC-3'; antisense, 5'-TGC ATA TTT GTT TGG GGC AGG-3'; c-IAP1: sense, 5'-AGC TGT TGT CAA CTT CAG ATA CCA CT-3'; antisense, 5'-TGT TTC ACC AGG TCT CTA TTA AAG CC-3'; and beta-actin: sense, 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3'; antisense, 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3'. The amplification conditions for Bcl2, c-IAP1, and beta-actin were as follows: denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min. After 30 cycles, the amplified Bcl2, c-IAP1, and beta-actin fragments were resolved by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.

RNase Protection Assay (RPA)—Cells were treated with various concentrations of Vpr peptide(s) for 0–6 h followed by isolation of total RNA. RPA was performed by using the BD Riboquant package kit (BD Biosciences) with a multiprobe template set of anti- and proapoptotic genes of the Bcl2 family along with internal controls (glyceraldehyde-3-phosphate dehydrogenase/beta-actin) according to the manufacturer’s protocol (34). Briefly 32P-labeled riboprobes of defined length were generated using T7 RNA polymerase and 50 ng of DNA template in the presence of 150 µCi of [{alpha}-32P]UTP (Amersham Biosciences). Template DNA was digested with RNase-free DNase followed by precipitation of labeled RNA. Total RNA (5 µg) was mixed with 6.2 x 105 cpm 32P-labeled riboprobe in hybridization buffer (40 mM PIPES, 1 mM EDTA, 0.4 M NaCl, and 80% formamide) and incubated for 5 min at 90 °C followed by 12 h at 56 °C. The hybridized RNA duplexes were digested with RNase and proteinase K followed by extraction with phenol and precipitation by the addition of equal volumes of 4 M ammonium acetate and 2 volumes of ethanol. Labeled RNA samples were resolved on 6% urea denaturing gels, dried, and visualized by autoradiography.


Figure 1
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  		FIGURE 1.
Vpr-(52–96)-induced apoptosis is mediated by the mitochondrial pathway. Cells (0.5 x 106/ml) were stimulated with various concentrations of Vpr-(52–96) or Vpr-(1–45) peptide for 24 h followed by staining with intracellular PI (A and B, left panel) and annexin-V/PI for measurement of apoptosis (B, middle panel) and JC-1 for mitochondrial membrane potential (B, right panel). The graphs shown are a mean ± S.D. of three experiments performed in triplicate. con., concentration.

 
Statistical Analysis—Means were compared by the two-tailed Student’s t test. The results are expressed as mean ± S.D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Vpr-(52–96) Peptide Induces Apoptosis through the Mitochondrial Pathway in THP-1 Cells—The C-terminal region of Vpr was shown to cause cell cycle arrest and apoptosis in T cells (9, 27). To determine whether monocytic cells exposed to Vpr peptides undergo apoptosis, THP-1 cells were treated with Vpr-(52–96) or Vpr-(1–45) peptides for 24 h followed by measurement of apoptosis. Vpr-(52–96), in contrast to the Vpr-(1–45) peptide, induced apoptosis in a dose-dependent manner as determined by PI and annexin-V/PI staining (Fig. 1, A and B, left and middle panels). In the apoptotic pathway, mitochondria play a key role by releasing caspase-activating apoptotic factors such as cytochrome c during MMP loss (35). To elucidate the role of MMP in Vpr-induced apoptosis, Vpr-(52–96) or Vpr-(1–45) peptide-treated cells were stained with JC-1 and analyzed by flow cytometry. Vpr-(52–96), in contrast to the Vpr-(1–45) peptide, induced loss of MMP in a dose-dependent manner (Fig. 1B, right panel) suggesting a distinct involvement of mitochondria in Vpr-(52–96)-induced apoptosis.

Cytochrome c release from mitochondria results in sequential procaspase activation and ultimately apoptosis (35). To determine the involvement of caspases in Vpr-(52–96)-induced apoptosis, the effect of the broad spectrum caspase inhibitor Z-VAD-fmk was examined. Pretreatment of cells with Z-VAD-fmk resulted in a significant reduction of apoptosis in a dose-dependent manner as determined by PI and annexin-V/PI staining (Fig. 2, A and B). However, Z-VAD did not affect MMP as determined by JC-1 staining (Fig. 2C). Because Z-VAD-fmk blocks DNA fragmentation and not the mitochondrial damage (36), the results suggest that loss of MMP is regulated independently of caspases in monocytic cells.

Vpr-(52–96)-activated JNK Selectively Induces Apoptosis—Because MAPKs play a key role in cell proliferation and apoptosis (24), we investigated the role of the MAPK pathway in Vpr-induced apoptosis. Treatment of cells with either Vpr-(52–96) or Vpr-(1–45) peptides resulted in strong activation of p38, ERK, and JNK MAPKs. Phosphorylation of ERK and JNK MAPKs became apparent at about 10–30 min following treatment and was sustained up to 4 h (Fig. 3). By using specific inhibitors of the MAPK family members such as SB202190 for p38, PD98059 for the ERK, and SP600125 and DXM for JNK MAPKs, we demonstrated that prior treatment of cells with PD98059, SB202190, and SP600125 inhibited Vpr-(52–96)- and Vpr-(1–45)-induced phosphorylation of the respective MAPKs in a dose-dependent manner (Fig. 3, A, B, and C). To determine the involvement of MAPKs in Vpr-induced apoptosis, cells were treated with inhibitors specific for p38, ERK, or JNK MAPKs for 2 h followed by stimulation with Vpr-(52–96) or Vpr-(1–45) peptides for 24 h. As observed earlier, Vpr-(52–96) treatment significantly induced apoptosis as determined by PI staining. Interestingly pretreatment of cells with JNK inhibitors SP600125 and DXM (Fig. 4A) and curcumin (data not shown) significantly reversed the Vpr-(52–96)-induced apoptosis. In contrast, neither SB202190 nor PD98059 affected Vpr-(52–96)-induced apoptosis (Fig. 4A). Similar observations were made when cells were analyzed by staining with either annexin-V/PI or JC-1 (data not shown).


Figure 2
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  		FIGURE 2.
Vpr-(52–96)-induced apoptosis is caspase-dependent. THP-1 cells (0.5 x 106/ml) were treated with 1.5 µM Vpr-(52–96) peptide in the presence or the absence of various concentrations of Z-VAD-fmk for 24 h followed by staining with intracellular PI (A), annexin/PI (B), or JC-1 (C). The values shown in the lower right quadrant in C indicate percentage of apoptotic cells. The results shown are a representative of three different experiments.

 
Poly(ADP-ribose) polymerase (PARP) recognizes DNA strand breaks and is implicated in DNA repair and apoptosis (37). On activation, PARP gets cleaved into two fragments of 89 and 24 kDa; this occurs early in the apoptotic response as a result of the activity of caspase 3. Because PARP cleavage has been used as a marker of apoptosis (37), we determined whether Vpr-(52–96)-induced JNK phosphorylation and associated apoptosis involves PARP activation and cleavage. Vpr-(52–96) induced cleavage of PARP (Fig. 4B) was inhibited following pretreatment of cells with SP600125 and DXM indicative of reversion of the apoptotic process (Fig. 4B).

To confirm that Vpr-(52–96)-induced apoptosis involved JNK activation, we used stealth siRNA specific for JNK1 and JNK2 to knock down endogenous JNK expression. THP-1 cells were transfected with siRNA for either JNK1, JNK2, or control siRNA for 24 h followed by treatment with Vpr peptides for another 2 h. Transfection of cells with either JNK1 or JNK2 siRNAs significantly inhibited endogenous expression of JNK1 and JNK2 (Fig. 5A, left panel). Vpr-(52–96) stimulation of cells transfected with either siRNAs specific for JNK1 or JNK2 did not induce phosphorylation of JNK1 and JNK2 compared with the cells transfected with the control siRNA (Fig. 5A, right panel). Cell cycle analysis of Vpr-(52–96)-treated cells transfected with either JNK1 or JNK2 siRNA revealed a significant decrease in apoptosis from ~32 to ~17% (PI staining, Fig. 5, B and C, left panel), from ~40 to ~20% (annexin-V/PI staining, Fig. 5B, middle panel), and from ~50 to ~25% (JC-1 staining, Fig. 5C, right panel).

Vpr-(52–96)-induced Apoptosis Is Mediated via Down-regulation of Bcl2 and c-IAP1 Genes—The Vpr-(52–96) peptide may cause apoptosis either by up-regulating the expression of proapoptotic genes or by down-regulation of antiapoptotic genes. The effect of Vpr-(52–96) peptide on pro- and antiapoptotic gene expression was determined by RPA using a kit containing probes for the Bcl2 family of proapoptotic (BikB, Bik, Bad, Bax, and Bak) and antiapoptotic genes (BclW, BclX, Bcl2, and Mcl1) in THP-1 cells treated with either Vpr-(52–96) or Vpr-(1–45) peptides for 3 and 6 h. Vpr-(52–96) peptide selectively inhibited Bcl2 gene expression that was detected at 3 h, and significant down-regulation was observed by 6 h of treatment as compared with the cells treated with the Vpr-(1–45) peptide (Fig. 6, A and B). There was no significant change in the level of other apoptotic (BikB, Bik, Bad, Bax, and Bak) or antiapoptotic genes (BclW, BclX, and Mcl1). The effect of Vpr-(52–96) peptides on Bcl2 expression was confirmed by RT-PCR and Western immunoblotting (Fig. 6, C and E). We also investigated the effect of Vpr peptides on the expression of c-IAP1, a member of the IAP family of antiapoptotic genes, by RT-PCR analysis. Treatment of cells with the Vpr-(52–96) and not the Vpr-(1–45) peptide significantly reduced the expression of the c-IAP1 gene. Neither of these peptides affected c-IAP2 expression as determined by RT-PCR (data not shown) and Western blot analysis (Fig. 6, C and D).


Figure 3
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  		FIGURE 3.
Vpr-(52–96) and Vpr-(1–45) peptides activate p38, p42/44, and JNK mitogen-activated protein kinases. A, B, and C, left panel, THP-1 cells (1.0 x 106/ml) were treated with a 1.5 µM concentration of either Vpr-(52–96) or Vpr-(1–45) peptides for 0–240 min, and cell extracts were subjected to Western blot analysis using anti-phospho-p42/44 (indicated by arrows as pp42/44), anti-phospho-p38 (pp38), or anti-phospho-JNK (pp46/54) antibodies. To normalize protein loading, the membranes were stripped and reprobed either with anti-p42/44, anti-p38, or anti-JNK antibodies, respectively. A, B, and C, right panel, cells (1.0 x 106/ml) were pretreated with the indicated concentrations of either PD98059 (PD)(A), SB202190 (SB)(B), SP600125 (SP) or DXM (C) for 2 h prior to stimulation with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptides. Total proteins (30 µg) were subjected to Western blot analysis using either anti-phospho-p42/44 (pp42/44), anti-phospho-p38 (pp38), or anti-phospho-JNK antibodies (pp46/54) after which the membranes were reprobed either with anti-p42/44, anti-p38, or anti-JNK antibodies, respectively. The results shown are representative of three different experiments.

 
JNK Activation Mediates Vpr-(52–96)-induced Down-regulation of Bcl2 and c-IAP1 Genes—Because JNK inhibitors significantly inhibited Vpr-(52–96)-induced apoptosis, it was of interest to determine whether JNK activation mediates down-regulation of Bcl2 and c-IAP1 genes. Treatment of THP-1 cells with the Vpr-(52–96) peptide reduced the expression of Bcl2 and c-IAP1 as compared with Vpr-(1–45)-treated cells, and prior treatment with SP600125 or DXM restored their expression as determined by RT-PCR and Western blot analysis. In contrast, pretreatment with SB202190 did not affect Vpr-(52–96)-induced down-regulation of either Bcl2 or c-IAP1 genes (Fig. 6, C, D, and E). Down-regulation of Bcl2 expression alone may not be enough to exert its antiapoptotic effects. Interleukin-3 and erythropoietin were shown to prevent apoptosis through phosphorylation of Bcl2 at Ser70 residue (38). Therefore, we determined whether the Vpr-(52–96) peptide modulated Bcl2 phosphorylation at Ser70 and whether JNK inhibitors reversed this phosphorylation. THP-1 cells exhibited a high basal level of Bcl2 phosphorylation at the Ser70 residues that was abrogated following treatment with Vpr-(52–96) peptide. Furthermore pretreatment of cells with SP600125 and DXM in contrast to SB202190 restored Vpr-(52–96)-mediated inhibition of Ser70 phosphorylation (Fig. 6E).

Vpr-(52–96)-activated JNK Inhibits Bcl2 and c-IAP1 Transcription—The Bcl2 gene is regulated by multiple transcription factors, namely NF{kappa}B, CREB, and Sp1 (32). Therefore, Vpr-(52–96)-activated JNK may down-regulate Bcl2 transcription by inhibiting the activities of either NF{kappa}B, CREB, and/or Sp1. We first investigated whether Vpr-(52–96) peptide inhibited the binding of NF{kappa}B, CREB, and Sp1 to their binding sites present in the Bcl2 promoter by gel shift assay. The untreated cells exhibited basal binding of NF{kappa}B, CREB, and Sp1 to their respective probes (Fig. 7, A, B, and C). The specificity of transcription factor binding was demonstrated by competition with specific and nonspecific oligonucleotides and supershift analysis with respective mouse monoclonal antibodies (Fig. 7, A, B, and C, left and middle panels). Interestingly Vpr-(52–96) peptide inhibited the binding of NF{kappa}B, CREB, and Sp1 to their respective probes. To determine whether Vpr-(52–96) peptide-activated JNK caused the inhibition of NF{kappa}B, CREB, and Sp1 binding, cells were pretreated with SP600125, DXM, or SB202190 for 2 h followed by stimulation with Vpr-(52–96) peptides. In contrast to SB202190, SP600125 and DXM pretreatment restored the binding of NF{kappa}B, Sp1, and CREB to their respective probes (Fig. 7, A, B, and C, right panels).

The above results suggested that Vpr-(52–96)-induced apoptosis may also be due to the inhibition of c-IAP1 expression. Because NF{kappa}B and CREB have been shown to regulate c-IAP1 transcription (39, 40), we investigated whether Vpr-(52–96)-activated JNK may down-regulate c-IAP1 transcription by inhibiting the activities of either NF{kappa}B and/or CREB transcription factors. The results show that untreated THP-1 cells exhibited constitutive binding of NF{kappa}B and CREB to their oligonucleotide probes corresponding to their binding site in the c-IAP1 promoter. The specificity of NF{kappa}B and CREB binding was demonstrated by competition with specific and nonspecific oligonucleotides and supershift analysis with respective mouse monoclonal antibody (Fig. 7, D, left and middle panels, and E, left panel). Treatment of cells with the Vpr-(52–96) peptide inhibited the binding of NF{kappa}B and CREB. Treatment of cells with SP600125 or DXM prior to Vpr-(52–96) stimulation restored the binding of both NF{kappa}B and CREB transcription factors (Fig. 7, D and E, right panels). These results suggest that Vpr-(52–96) peptide-activated JNK selectively inhibited the transcription of Bcl2 and c-IAP1 by inhibiting the binding of transcription factors to their binding sites in the Bcl2 and c-IAP1 promoters, respectively.


Figure 4
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  		FIGURE 4.
Vpr-(52–96)-activated JNK selectively induced apoptosis in THP-1 cells. A, cells (0.5 x 106/ml) were pretreated with the indicated concentrations of either SP600125 (SP), DXM, PD98059 (PD), or SB202190 (SB) for 2 h prior to stimulation with 1.5 µM Vpr-(52–96) peptide for 24 h followed by intracellular PI staining. The results shown are mean ± S.D. of three different experiments. B, cells (1 x 106/ml) were pretreated with the indicated concentrations of SP600125 or DXM for 2 h prior to stimulation with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptide for 12 h. Cell lysates were subjected to Western blot analysis for PARP expression by using anti-PARP antibodies. The results shown are a representative of three different experiments.

 
Vpr-(52–96)-induced Apoptosis Is Mediated by Inhibition of Bcl2 and c-IAP1 Genes through JNK Activation in Primary Monocytes—We next investigated whether primary monocytes exposed to Vpr-(52–96) peptides undergo apoptosis and whether Vpr-(52–96)-induced apoptosis is regulated through the selective activation of JNK in a manner similar to that observed in THP-1 cells. Similar to the results obtained with THP-1 cells, the Vpr-(52–96) but not the Vpr-(1–45) peptide induced apoptosis in a dose-dependent manner as determined by PI (Fig. 8A) and annexin/PI staining (data not shown) in CD14+ purified monocytes. Furthermore Vpr-(52–96)-induced apoptosis was found to be caspase-dependent as prior treatment of monocytes with caspase inhibitor Z-VAD-fmk resulted in a significant reduction of apoptosis (data not shown). To determine that Vpr-(52–96)-induced apoptosis is regulated through the activation of JNK MAPKs, we first demonstrated that both Vpr-(1–45) and Vpr-(52–96) peptides induced the phosphorylation of JNK MAPKs (Fig. 8B). Subsequently we demonstrated that prior treatment of monocytes with SP600125 (Fig. 8C), DXM, or curcumin (data not shown) significantly reversed Vpr-(52–96)-induced apoptosis in a dose-dependent manner as determined by PI and annexin/PI staining (Fig. 8C).

To further determine whether the Vpr-(52–96)-induced apoptosis is regulated by Bcl2 and cIAP-1 genes through JNK activation, monocytes were treated with SP600125 for 2 h followed by treatment with the Vpr-(52–96) peptide for 12 h. Unstimulated monocytes expressed both Bcl2 and c-IAP1 genes constitutively. Treatment of cells with the Vpr-(52–96) peptide, unlike the Vpr-(1–45) peptide, abrogated the expression of both Bcl2 and c-IAP1 genes at both the RNA and protein levels (Fig. 8, D, E, and F). Similar to the results obtained with THP-1 cells, exposure of monocytes to SP600125 prior to treatment with the Vpr-(52–96) peptide restored the expression of Bcl2 and c-IAP1 genes to the levels observed in unstimulated cells. In contrast, exposure of monocytes to SB202190 prior to treatment with the Vpr-(52–96) peptide did not affect the expression of Bcl2 and c-IAP1 genes (Fig. 8, D, E, and F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The primary aim of this study was to investigate the signaling pathways, particularly the role of MAPK, and the transcription factors involved in the regulation of Vpr-induced apoptosis in human monocytic cells. Our results suggest that Vpr-(52–96) induced apoptosis in primary monocytes and the promonocytic THP-1 cells in a caspase-dependent manner. Although both Vpr-(1–45) and Vpr-(52–96) peptides induced phosphorylation of p38, ERK, and JNK MAPKs, Vpr-(52–96)-induced apoptosis was regulated selectively through JNK activation. Furthermore Vpr-(52–96)-induced apoptosis was attributed to the down-regulation of antiapoptotic genes Bcl2 and c-IAP1 through the activation of upstream JNK MAPKs.


Figure 5
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  		FIGURE 5.
JNK-specific siRNAs reverse Vpr-(52–96)-induced apoptosis in THP-1 cells. A, JNK-specific siRNA reduced endogenous and Vpr-(52–96)-stimulated phospho-JNK. Cells (1 x 106/ml) were transfected with either JNK1, JNK2, or control stealth siRNA for 24 h (left panel) followed by stimulation with 1.5 µM Vpr-(52–96) peptide for 2 h (right panel). Cell lysates were analyzed by Western blot analysis using anti-JNK (left panel) or anti-phospho-JNK antibodies (right panel). The membranes were reprobed with total anti-p38 antibodies. The results shown are a representative of three different experiments. B and C, JNK-specific siRNAs reversed Vpr-(52–96)-induced apoptosis. THP-1 cells (1 x 106/ml) were transfected with either JNK1, JNK2, or control stealth siRNA for 24 h followed by stimulation with 1.5 µM Vpr-(52–96) peptide for 24 h and staining with intracellular PI (B and left panel of C), annexin/PI (C, middle panel), or JC-1 (C, right panel). The results shown in C represent a mean ± S.D. of three independent experiments. Cont., control.

 
It is well established that cell cycle arrest occurs by interfering with the activity of the Cdc2-cyclin B1 complex, a central regulator of the G2 to mitosis transition (41). In general, this complex is inactivated by Wee1 kinase and activated by Cdc25c phosphatase (41). Vpr mediates cell cycle arrest by phosphorylation and inactivation of Cdc25c phosphatase via activating the ataxia-telangiectasia and Rad3-related (ATR) DNA damage response pathway (42, 43). The Rad17 and Hus1 proteins involved in the ATR pathway were shown to be required for cell cycle arrest by Vpr (44). Vpr was also shown to bind Cdc25c directly and inhibit Cdc25c phosphatase activity (43, 45), perhaps blocking cyclin B1-Cdc2 redundantly with the ATR pathway. Recently Jacotot et al. (19) demonstrated that Vpr-(52–96)-induced apoptosis may occur through a direct effect on the mitochondrial permeability transition pore complex and specifically on the mitochondrial adenine nucleotide translocator, a component of the permeability transition pore complex. This event leads to permeabilization of the outer mitochondrial membrane with consequent release of apoptosis-inducing factor and cytochrome c (9, 19). Cytochrome c interacts with Apaf-1 and procaspase 9 to create an apoptosome, the caspase activation complex that causes activation of other caspases resulting in apoptosis (8, 9, 19). Our results suggest that Vpr-(52–96) peptide induced apoptosis by causing a dramatic reduction of MMP in human monocytic cells.

Although several studies have implicated the JNK pathway in pro- as well as antiapoptotic cellular functions (46), there is little information on the role of MAPKs in Vpr-induced apoptosis. Recently Vpr-induced apoptosis was shown to be associated with down-regulation of ERK MAPK pathway in a 293 fibroblast cell line (47). Our results suggest that Vpr-induced apoptosis in human monocytic cells is regulated through the activation of JNK MAPKs. Pretreatment of cells with JNK inhibitors SP60025 and DXM or transfection with JNK-specific siRNAs significantly inhibited Vpr-induced apoptosis. Although MAPKs have been reported to regulate HIV replication in T cells and latently infected cells (10, 48), this is the first report describing a novel pathway involving JNK activation in Vpr-induced apoptosis.

The JNK group of MAPKs is encoded by three genes: jnk1 and jnk2 genes, which are ubiquitously expressed, and the jnk3 gene expressed primarily in the heart, testis, and brain (49). Targeted disruption of jnk1 or jnk2 genes results in mice that exhibit defects in apoptosis, development and functions of effector T cells, and cytokine production (46). However, disruption of both jnk1 and jnk2 genes resulted in embryonic death (24, 46). Although a distinct role for JNK1 and JNK2 in apoptosis and immunological and developmental processes is not clear at present, some studies have suggested a differential involvement for JNK1 and JNK2 (24, 46, 50). We used siRNAs specific for JNK1 and JNK2, both of which prevented Vpr-mediated cell death to equal levels suggesting redundancy between the two JNK isoforms at least with respect to Vpr-induced apoptosis in monocytic cells.

We have also demonstrated that Vpr-induced apoptosis was mediated by the down-regulation of antiapoptotic Bcl2 gene. Bcl2 and its closest homologues, BclxL and Bclw, potently inhibit apoptosis in response to many signaling pathways (35, 51). Bcl2 has been shown to protect cells from Vpr-induced apoptosis most likely by regulating inner and outer MMP through interactions with major components of permeability transition pore complex such as adenine nucleotide translocator in the inner membrane and the voltage-dependent anion channels in the outer membrane and/or through autonomous channel-forming activities (8, 9, 19). Vpr was shown to physically and functionally interact with the ANT-(104–116) site in adenine nucleotide translocator, consequently converting adenine nucleotide translocator into a nonspecific pore, leading to inner MMP that triggers matrix swelling, outer membrane rupture, and permeability to cytochrome c (9, 19).


Figure 6
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  		FIGURE 6.
Vpr-(52–96)-activated JNK induces down-regulation of antiapoptotic Bcl2 and c-IAP1 genes. A, Vpr-(52–96) peptide inhibited Bcl2 expression as determined by RPA. THP-1 cells (5 x 106/ml) were stimulated with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptides for 0–6 h. RNA was extracted as described under "Experimental Procedures." RPA was performed by using the BD Riboquant package kit with a multiprobe template set of anti- and proapoptotic genes of the Bcl2 family and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32 as internal controls. The results shown are a representative of three different experiments. B, densitometric analysis of the Bcl2 gene family following Vpr-(52–96) treatment from the results shown in A. C, Vpr-(52–96) peptide induced down-regulation of Bcl2 and c-IAP1 genes through JNK activation. Cells (5 x 106/ml) were stimulated with 1.5 µM Vpr-(52–96) peptides for 3 h in the presence or the absence of SP600125 (SP), DXM, or SB202190 (SB) followed by semiquantitative RT-PCR analysis for Bcl2 and c-IAP1 expression. D and E, cells (1 x 106/ml) were pretreated with either SP600125, DXM, or SB202190 for 2 h followed by stimulation with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptides for 24 h. Cell lysates were subjected to Western blot analysis for expression of c-IAP1 (D) and Bcl2 and phospho-Bcl2 (E) using anti-RIAP1, anti-Bcl2, and anti-phospho-Bcl2 antibodies, respectively. The membranes were reprobed with anti-beta-actin antibodies. The results shown are representative of three different experiments.

 
There is substantial agreement that antiapoptotic c-IAP1 and c-IAP2 genes endow cells with protection against a number of apoptotic stimuli (52). Recently we demonstrated a critical role for c-IAP2 in conferring resistance to staurosporine-induced apoptosis in human monocytic cells (28). Herein we show that Vpr-induced apoptosis in monocytic cells may be mediated by inhibition of c-IAP1 expression in addition to that of Bcl2. How IAPs confer antiapoptotic signals has been investigated recently. IAPs regulate the caspase cascade by binding to caspases 3 and 7 and by inhibiting the activation of procaspases 8 and 9 (52). c-IAP1 was also shown to inhibit apoptosis through caspase-independent mechanisms such as via activation of NF{kappa}B and JNK MAPKs (53). In addition, c-IAPs were suggested to obstruct apoptosis through binding to the IAP antagonists such as second mitochondria-derived activator of caspase (54) or interfering with the signaling pathway initiated by tumor necrosis factor-{alpha} by interacting with tumor necrosis factor receptor-associated factor through the baculoviral IAP repeat 1 (BIR1) domain (55).

JNK can phosphorylate (Ser/Thr)-Pro motifs in the activation domains of various transcription factors such as c-Jun, Jun-B, Jun-D, activating transcription factor-2, PU.1, Sp1, and Ets-2 (22, 46, 56). Our results suggest that Vpr-(52–96)-activated JNK causes down-regulation of transcription factors involved in the regulation of Bcl2 and c-IAP1 gene transcription. Bcl2 transcription is regulated by NF{kappa}B, Sp1, and CREB (32), whereas c-IAP1 transcription was shown to involve activation of NF{kappa}B and CREB transcription factors (39, 40). Although both peptides activated all three members of the MAPK family, activation of JNK by the Vpr-(52–96) peptides resulted in inhibition of binding of corresponding NF{kappa}B and CREB transcription factors to their respective binding sites, and inhibition of JNK activity restored this binding and subsequent transcription of Bcl2 and c-IAP1 genes. These observations suggest specific interaction of Vpr-(52–96) peptide with member(s) of the JNK MAPKs family or with any of these transcription factors either alone or as a complex in the cytoplasm to prevent their translocation into the nuclei. The identity of the JNK family member and/or the transcription factor involved in binding with Vpr-(52–96) peptide remains to be investigated. Nonetheless Vpr has been shown to engage with a number of host cellular proteins to promote cell cycle arrest such as Vpr-bound protein, a protein believed to interfere with Vpr-mediated arrest by sequestering Vpr in the cytoplasm (57), and the human homologue of MOV34, a proteosomal subunit (58).


Figure 7
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  		FIGURE 7.
Vpr-(52–96)-activated JNK inhibits Bcl2 and cIAP-1 transcription by down-regulating the binding of respective transcription factors. Cells (5 x 106/ml) were treated with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptides for 2 h in the presence or the absence of SP600125 (SP), DXM, or SB202190 (SB). Nuclear proteins (5 µg) were incubated with 32P-labeled oligonucleotide probes corresponding to NF{kappa}B (A), CREB (B), or Sp1 (C) sequences derived from the Bcl2 promoter and NF{kappa}B (D) or CREB (E) sequences derived from c-IAP1 promoter. The specificity of NF{kappa}B, CREB, and Sp1 binding was determined by incubating nuclear proteins with unlabeled NF{kappa}B, CREB, and Sp1 or nonspecific oligonucleotides. The supershift analysis was performed by treating the nuclear proteins with oligonucleotide probes in the presence or the absence of anti-p50, anti-p65 NF{kappa}B, anti-CREB, or anti-Sp1 antibodies. The results shown are representative of three different experiments. Sp. CC, specific cold competitor; NS CC, nonspecific cold competitor.

 
Recently a glucocorticoid receptor complex was identified as an intracellular target for Vpr (58, 59) suggesting their possible involvement in Vpr-(52–96)-induced apoptosis. The observations that both glucocorticoids (60) and Vpr inhibited NF{kappa}B induction suggest a role for glucocorticoid receptors in Vpr-mediated apoptosis. However, unlike glucocorticoids that are known to inhibit JNK activity (22, 61), Vpr-(52–96) induced JNK phosphorylation in our study. Therefore, it seems unlikely that Vpr-(52–96) induces apoptosis in monocytic cells through glucocorticoid receptors.

In summary, we demonstrate for the first time that JNK activated selectively by the HIV Vpr peptide plays a critical role in the induction of cell death in primary monocytes and leukemic THP-1 cells. Furthermore our results showing JNK activation associated with inhibition of Bcl2 and c-IAP1 expression provide a broad basic mechanism for Vpr-induced apoptosis (Fig. 9). In view of studies suggesting HIV Vpr as a potential therapeutic antiproliferative agent against malignancies (3, 4), our findings suggest that targeted delivery of Vpr-(52–96) peptide or specific cIAP-1/Bcl2 antagonists to monocytic leukemic cells and possibly HIV-infected monocytic cells and other viral reservoirs of monocytic lineage may induce apoptosis in monocytic leukemic cells and prove to be a potentially therapeutic agent to eliminate virus-infected cells. Because deregulation of the JNK pathway has been implicated in cancer and other diseases including Alzheimer and Parkinson diseases (24, 50), investigation of the molecular mechanisms that govern the role of the JNK pathway in apoptosis should provide insight into its biological functions and strategies to target this pathway for prevention and treatment of human diseases and cancer.


Figure 8
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  		FIGURE 8.
Vpr-(52–96)-activated JNK induces apoptosis by inhibiting the expression of Bcl2 and c-IAP1 in normal human monocytes. A, Vpr-(52–96)-induced apoptosis in human monocytes. Monocytes (0.5 x 106/ml) were stimulated with various concentrations of either Vpr-(52–96) or Vpr-(1–45) peptides for 12 h followed by staining with intracellular PI. The results shown are representative of three different experiments. B, Vpr-(52–96) and Vpr-(1–45) peptides activate JNK mitogen-activated protein kinases in normal human monocytes. Left panel, monocytes (3.0 x 106/ml) were treated with a 1.5 µM concentration of either Vpr-(52–96) or Vpr-(1–45) peptides for 0–60 min. Right panel, monocytes were pretreated with SP600125 (0–20 µM) for 2 h prior to stimulation with Vpr-(52–96) or Vpr-(1–45) peptides. Total proteins (30 µg) were subjected to Western blot analysis using anti-phospho-JNK antibodies followed by reprobing the membranes with anti-JNK antibodies. The results shown are representative of three different experiments. C, Vpr-(52–96)-activated JNK induces apoptosis in human monocytes. Cells (0.5 x 106/ml) were pretreated with SP600125 (0–20 µM) for 2 h prior to stimulation with 1.5 µM Vpr-(52–96) or Vpr-(1–45) peptide. After 12 h, cells were analyzed for apoptosis by intracellular PI staining (upper panel) and annexin/PI (lower panel). The results shown are representative of three independent experiments. D, monocytes (5 x 106/ml) were stimulated with 1.5 µM Vpr-(52–96) peptides for 2 h in the presence or the absence of SP600125 followed by semiquantitative RT-PCR analysis for Bcl2 and c-IAP1 expression. E and F, monocytes were pretreated with SP600125 for 2 h followed by stimulation with 1.5 µM Vpr-(52–96) or Vpr-(1–45) for 12 h. Cell lysates were subjected to Western blot analysis for expression of Bcl2 and phospho-Bcl2 (E) and c-IAP1 (F) using anti-Bcl2, anti-phospho-Bcl2, and anti-RIAP1 antibodies, respectively, followed by reprobing the membranes with anti-beta-actin antibodies. The results shown are representative of three independent experiments. con., concentration; SB, SB202190; SP, SP600125.

 


Figure 9
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  		FIGURE 9.
Schematic diagram showing the signaling pathways involved in Vpr-mediated apoptosis. Entry of Vpr into the monocytic cells induces JNK activation resulting in transcriptional inhibition of c-IAP1 and Bcl2 expression, activation of caspases, and apoptosis. Vpr is also known to activate the ATR DNA damage pathway that acts on Cdc25c through Hus1 and Rad17. This process leads to inactivation of cyclin B and cell cycle arrest (42–45).

 

   FOOTNOTES
 
* This work was supported in part by grants from the Canadian Institutes of Health Research (to A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the Ontario Graduate Scholarship in Science and Technology and Ontario Graduate Scholarship. Back

2 Recipient of the Career Scientist Award from the Ontario HIV Treatment Network. To whom correspondence should be addressed: Division of Virology, Research Inst., Children’s Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-737-7600 (ext. 3920); Fax: 613-738-4825; E-mail: akumar{at}uottawa.ca.

3 The abbreviations used are: Vpr, viral protein R; DXM, dexamethasone; ERK, extracellular signal-regulated kinase; HIV, human immunodeficiency virus; IAP, inhibitor of apoptotic protein; c-IAP1, cellular inhibitor of apoptotic protein 1; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, mitochondrial membrane potential; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; Vpr-(1–45), N-terminal amino acids 1–45 peptide; Vpr-(52–96), C-terminal amino acids 52–96 peptide; RPA, RNase protection assay; CREB, cyclic AMP-response element-binding protein; siRNA, small interfering RNA; IMDM, Iscove’s modified Dulbecco’s medium; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; ATR, ataxia-telangiectasia and Rad3-related. Back


   ACKNOWLEDGMENTS
 
Dr. M. Kryworuchko and Dr. A. Badley are acknowledged for critically reading the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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