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Originally published In Press as doi:10.1074/jbc.M512109200 on August 18, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31647-31658, October 20, 2006
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Intracellular HIV-Tat Expression Induces IL-10 Synthesis by the CREB-1 Transcription Factor through Ser133 Phosphorylation and Its Regulation by the ERK1/2 MAPK in Human Monocytic Cells*

Katrina Gee{ddagger}§1, Jonathan B. Angel{ddagger}2, Wei Ma, Sasmita Mishra3, Niranjala Gajanayaka§, Karl Parato{ddagger}, and Ashok Kumar§||24

From the Departments of ||Pathology and Laboratory Medicine and Biochemistry, Microbiology, and Immunology, §Division of Virology and Molecular Immunology, Research Institute, Children's Hospital of Eastern Ontario, Ottawa K1H 8L1 and the {ddagger}Ottawa Health Research Institute and the Division of Infectious Diseases, Department of Medicine, Ottawa Hospital General Campus, University of Ottawa, Ottawa, Ontario K1H 8L6, Canada

Received for publication, November 10, 2005 , and in revised form, July 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus (HIV)-Tat plays an important role in virus replication and in various aspects of host immune responses, including dysregulation of cytokine production. IL-10, an anti-inflammatory cytokine, is up-regulated during the course of HIV infection representing an important pathway by which HIV may induce immunodeficiency. Here we show that extracellular as well as intracellular Tat induced IL-10 expression in normal human monocytes and promonocytic THP-1 cells. The signaling pathways involved in the regulation of IL-10 production by endogenous Tat remain unknown. To understand the molecular mechanism underlying intracellular Tat-induced IL-10 transcription, we employed a retroviral expression system to investigate the role of MAPKs and the transcription factor(s) involved. Our results suggest that an inhibitor specific for the ERK1/2, PD98059, selectively blocked intracellular Tat-induced IL-10 expression in THP-1 cells. Furthermore, intracellular Tat activated the CREB-1 transcription factor through Ser133 phosphorylation that was regulated by ERK MAPK as determined by IL-10 promoter analysis and gel shift assays. Overall, our results suggest that intracellular HIV-Tat induces IL-10 transcription by ERK MAPK-dependent CREB-1 transcription factor activation through Ser133 phosphorylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-10 is a pleiotropic cytokine whose effects primarily include the inhibition of antigen-presenting cell-dependent cytokine synthesis by Th1 cells and associated autoimmune and inflammatory responses (1, 2). IL-10 is produced by a wide variety of cell types, including CD4+ Th0 and Th2 cells, CD8+ T cells, regulatory T cells, B cells, and monocytic cells (1, 2). Typically, IL-10 inhibits antigen-driven activity of both Th1 and Th2 subsets (3) and hence is not strictly a Th2-type cytokine, although it facilitates the induction of Th2 cell types. IL-10 is also known to down-regulate the release of reactive oxygen and nitrogen intermediates resulting in macrophage deactivation, which may allow the growth of tumor cells and intracellular microbes (4). The potent inhibitory action of IL-10 on macrophages, particularly at the level of cytokine production, supports an important role for IL-10 in the regulation of T cell responses, acute inflammation, and autoimmune responses. Additionally, IL-10 exhibits stimulatory functions such as CD14 induction on human monocytic cells and B cell growth and differentiation (5-7).

IL-10 has also been suggested to play a vital role in the immunopathogenesis of a number of infectious diseases, including HIV5/AIDS (8-12). IL-10 is produced constitutively during HIV infection, and its levels increase with disease progression and gradually decrease with the use of effective anti-retroviral therapy (12-15). It is now well established that monocytic cells serve as long term viral reservoirs in chronically infected HIV patients and therefore play a key role in the natural history of HIV infection (8, 16). We and others have demonstrated that HIV infection of monocytic cells in vitro results in enhanced IL-10 production (9, 12, 17), which may be of significance because of the ability of IL-10 to induce immune unresponsiveness (18), to inhibit HIV replication (19), and to limit viral entry as a result of its inhibitory effects on the expression of chemokine receptors on T cells (20). IL-10 was also shown to enhance the expression of CXCR-4 on dendritic cells as well as enhance HIV replication in these cells (21). Therefore, exposure of dendritic cells to IL-10 may favor the emergence of X4 strains of HIV, promote the development of HIV reservoirs, and may provide a mechanism for the virus to evade host immune responses.

The enhanced IL-10 production in HIV infection has been attributed to the HIV regulatory proteins, including the HIV accessory protein, Tat, in a number of cell types such as monocytes/macrophages and T cells (22-24). HIV-Tat, a 14-16-kDa protein, is a transactivating molecule critical to the replication of HIV during active infection (25). Tat accumulates in the nucleus of infected cells, and it is also secreted into the plasma of HIV-infected patients where it can exert its effects on uninfected bystander cells (25, 26). It is expressed early in HIV infection where it binds a stable RNA hairpin structure called the Tat activation region at the 5' end of HIV RNA. By doing so, it functions to recruit host transcription factors in order to initiate viral replication (27). In addition, Tat affects several host cellular processes contributing to immune system malfunction such as induction of T cell apoptosis (28, 29), inhibition of major histocompatibility complex expression (30, 31), and cytokine (IL-10, IL-12, tumor necrosis factor-{alpha}, and transforming growth factor-beta) production (22-24, 29, 32-35).

The mechanism by which HIV-Tat influences a wide variety of biological functions has been investigated. There is evidence that extracellular Tat can be taken up by uninfected cells and reach the nucleus rapidly where it can activate a number of transcription factors, including AP-1, Sp1, CCAAT/enhancer-binding protein (C/EBP)-beta, cAMP-responsive element binding protein (CREB), and NF{kappa}B (34-39). The signaling proteins responsible for the induction of these transcription factors have not been elucidated; however, it has been shown that extracellular Tat-induced signaling involves the activation of mitogen-activated protein kinase (MAPK), including c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase, as well as calcium signaling pathways (22, 23, 37, 40, 41). These signaling cascades are believed to be activated following interaction of extracellular Tat with a number of cell surface receptors, including integrin receptors, members of the vascular endothelial growth factor receptor family, and the CXCR4 chemokine receptors (12, 42-44).

Recently, we and others have investigated the intracellular signaling events following LPS-induced activation of the CD14-Toll-like receptor-4 complex resulting in human and murine IL-10 production (45, 46). We demonstrated that in human monocytic cells, LPS-induced IL-10 production was regulated by the Sp-1 transcription factor through the activation of p38 MAPK (45). In addition, STAT-3 and C/EBPbeta were implicated in the regulation of IL-10 production in different cell systems (47, 48). There is evidence to suggest that extracellular recombinant HIV-Tat induces IL-10 in human monocytic cells through the activation of PKC-betaII- and {delta}-dependent pathways (22, 23, 40). However, the molecular mechanism by which endogenously expressed Tat regulates IL-10 production is not known. Here studies conducted to understand the molecular mechanism involved suggested for the first time that intracellularly expressed HIV-Tat induces IL-10 transcription by the CREB transcription factor through the activation of p42/44 ERK MAPK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture 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). Cells were cultured in Iscove's modified Dulbecco's medium (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 MAP/ERK kinase-1 (Calbiochem), selectively blocks the activity of ERK and has no effect on the activity of other serine/threonine protein kinases, including p38 or JNK MAPK (49). The pyridinylimidazole SB202190 (Calbiochem), a potent inhibitor of p38 MAPK, has no significant effect on the activity of ERK or JNK MAPK subgroups (50). SP600125, a specific JNK inhibitor (Biomol, Plymouth meeting, PA), is a reversible ATP competitive inhibitor with more than 300-fold selectivity versus related MAPK, including ERK1 and p38, and protein kinase A and the inhibitor of NF{kappa}B kinase, I{kappa}B kinase 2 (51). Recombinant HIV-Tat was obtained from the NIH AIDS Research and Reference Reagent Program. To ensure that the Tat was endotoxin-free, the Tat preparation was treated with polymyxin B-coated beads (Sigma). The endotoxin levels were tested by the Limulus amebocyte lysate assay (BioWhittaker) and were found to be less than 0.06 enzyme units/ml.

Isolation of Monocytes—Purified, nonactivated monocytes were obtained by a negative selection procedure involving depletion of T cells and B cells using magnetic polystyrene M-450 Dynabeads (Dynal) coated with antibodies specific for CD2 (T cells) or CD19 (B cells), as described earlier (45, 52). Briefly, PBMCs (10 x 106/ml) isolated as described above were resuspended with CD2 and CD19 Dynabeads and were incubated for 30 min on ice with constant mixing. Cells were incubated at 37 °C for 2 h following which nonadherent cells were removed. The adherent mononuclear cells obtained contained less than 1% CD2+ T cells and CD19+ B cells, as determined by flow cytometry.

Generation of pTat and pLXIN Retroviruses—The exon 1 of Tat was amplified by PCR from pSV2tat72 (AIDS Research and Reference Reagent Program, National Institutes of Health) using the following primers: sense, 5'-TTGGAGGCCTAGGCTTTTG-3'; antisense, 5'-TGTAGGTAGTTTGTCCAATTATGTCA-3'. EcoRI restriction sites were inserted by employing the pGEM®-T Easy Vector System (Promega). The amplified Tat fragment thus generated was ligated into an EcoRI restriction digest of the retroviral backbone pLXIN (BD Biosciences) and designated as the pTat vector. The amphotrophic packaging cell line PT67 (BD Biosciences) was transfected with pTat and pLXIN vectors using FuGENE. Briefly, PT67 cells were plated at 1.5 x 106 cells/ml in 6-well plates (Falcon), and cells were transfected with 5 µg/ml each of pTat or pLXIN as per the manufacturer's instructions. Stable cell lines producing pTat and pLXIN viruses were made by culturing the transfected cells in 400 µg/ml of geneticin for 2 days. Virus-containing supernatants were collected at the time of passaging the cells. Cells were passaged a maximum of five times.

Determination of Tat Biological Activity in Cells Infected with pTat Retrovirus—HLM1 cells (AIDS Research and Reference Reagent Program, National Institutes of Health), a HeLaT4+ cell line transduced with defective mutant Tat-containing HIV, were propagated in Dulbecco's modified Eagle's medium (Invitrogen) plus 5% fetal bovine serum (containing 100 µg/ml of geneticin) and cultured for 7 days with pTat or pLXIN retroviruses or sodium butyrate (NaB) (10 mM; Sigma), a nonviral activator of transcription (53) as a positive control. On day 7 post-infection, HIV-1 production by HLM1 cells was determined by p24 ELISA (Immunodiagnostics Inc.).

Infection of THP-1 Cells with Retroviruses—THP-1 cells were cultured in virus-containing supernatant collected from the packaging cell line for 16-48 h. Polybrene (Sigma; 1 µg/ml) was added at the time of infection in order to enable virus entry into the cells. After 24 h, THP-1 cells were washed and infected a second time by resuspending cells in fresh retrovirus-containing supernatant. Cells were then harvested 16-24 h after the second infection.

Intracellular IL-10 Expression by Flow Cytometry—Intracellular IL-10 expression was determined by flow cytometry as described earlier (9). Briefly, cells were washed once at the time of harvesting with PBS, 0.1% sodium azide. Cells were then incubated in Perm2 buffer (BD Biosciences) for 10 min followed by washing with PBS, 0.1% sodium azide. Cells were stained with PE-conjugated anti-IL-10 monoclonal antibodies (BD Biosciences), and isotype-matched control antibodies (BD Biosciences) were also included. Data were acquired on a BD FACScan flow cytometer and analyzed using the WinMDI version 2.8 software package (J. Trotter, Scripps Institute, San Diego). Validity of comparisons in the expression levels of IL-10 between different samples was ensured through the use of CalibriteTM Beads (BD Biosciences).

Measurement of Phospho-ERK by ELISA—THP-1 cells were infected with either pTat or pLXIN retroviruses for various times followed by collection of cell pellets. The phospho-ERK ELISA (R & D Systems) was performed essentially as described in the manufacturer's instructions. Briefly, cell pellets were incubated in lysis buffer (1 mM EDTA, 0.5% Triton X, 5 nM NaF, 6 M urea, 25 µg/ml leupeptin, 25 µg/ml pepstatin, 100 µM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 2.5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate) for 1 h at room temperature. Lysates were centrifuged for 20 min at 14,000 x g, and protein estimation was performed using the Bradford method (Bio-Rad). Cell lysates were serially diluted in buffer (1 mM EDTA, 0.5% Triton X, 5 nM NaF) to concentrations ranging from 10 to 100 ng/ml. ELISA plates (Nunc) were prepared by coating with primary anti-phospho-ERK antibodies overnight at 4 °C. The plates were washed in PBS, 1% bovine serum albumin followed by incubation of the cell lysates overnight at 4 °C. Phospho-ERK standard from the kit was used for standard curve determination. The plates were washed again, and secondary biotinylated anti-phospho-ERK antibodies were added for another2hat room temperature. Streptavidin-peroxidase was used at a final concentration of 1:1000 (Jackson ImmunoResearch). The color reaction was developed by o-phenylenediamine (Sigma) and hydrogen peroxide, and the absorbance was read at 450 nm.

Construction of Luciferase Reporter Gene Vectors—A series of hIL-10 promoter fragments (-890 to +120; Gen-BankTM accession X78437 [GenBank] ) were amplified from genomic DNA by PCR as described earlier (45). The primers with restriction sites used to amplify the hIL-10 promoter fragments from genomic DNA are shown in Table 1. The amplification consisted of denaturation at 95 °C for 2 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 1 min, and extension at 72 °C for 2 min, and a final elongation at 72 °C for 10 min. The amplified promoter products were subcloned into the PCRII-TOPO vector, and the sequences were confirmed. They were then subcloned into the XhoI polylinker site of pGL3B, the basic luciferase reporter plasmid, and confirmed again by sequencing. All DNA sequencing was performed by the Biotechnology Research Institute (University of Ottawa). A site-directed mutation of the CRE sequence (ACGTCA) was generated by PCR using mutagenic primers (Table 1) to substitute cytosine with guanine at -631 and cytosine with adenine at -636 (see Fig. 5A). The fragment containing the CRE mutation (-430 to +120 bp) was inserted into the pGL3B reporter vector.


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  		TABLE 1
Primers for amplification of IL-10 promoter fragments

 
Transient Transfection of Cells and Measurement of Luciferase Activity—Cells were transfected with plasmids containing various IL-10 promoter fragments using Lipofectamine reagent (Invitrogen) following the manufacturer's instructions and as described earlier (45). 10 µg of the test plasmid and 5 µg of pSV-beta-galactosidase internal control vector (Promega) were incubated for 45 min with 10 µl of Lipofectamine reagent in 200 µl of Opti-MEM I reduced serum medium (Invitrogen) to allow formation of DNA-liposome complexes. These complexes were added to the cell suspension in each well, and cells were cultured for 24 h. Cells were harvested and then assayed for luciferase and beta-galactosidase activity by using luciferase and beta-galactosidase assay kits (Promega), respectively, in a Bio Orbit 1250 luminometer (Fisher).


Figure 1
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  		FIGURE 1.
Exogenous HIV-Tat induces IL-10 production in primary monocytes. Primary human monocytes (0.5 x 106/ml) isolated from PBMCs of HIV-negative individuals were incubated with recombinant HIV-Tat at concentrations ranging from 10 to 500 ng/ml for 24 h. A, the cell supernatants were analyzed for IL-10 expression by ELISA. Results shown are a mean ± S.D. from five different individuals. B, monocytes (0.5 x 106/ml) treated with Tat (200 ng/ml) for 24 h were analyzed for intracellular IL-10 expression by flow cytometry as described under "Experimental Procedures." Results shown are a representative experiment from five different individuals.

 
RNA Isolation and Quantitative RT-PCR for IL-10 and HIV-Tat—Total RNA was extracted using the RNeasy Plus® mini kit (Qiagen). Total RNA (1 µg) was reverse-transcribed by using the high capacity cDNA archive kit (Applied Biosystems). 2.5 µl of the cDNA was used as a template in each PCR with PCR TaqMan® universal master mix containing 1x PCR buffer and the appropriate concentrations of gene-specific primers, TaqMan® probe, 5 mM MgCl2, 0.025 unit of AmpliTaq Gold, and 0.2 mM dNTPs combined in a total volume of 25 µl. IL-10 and beta-actin primers were purchased from Applied Biosystems. HIV-Tat custom-made primers were obtained using the Primer Express® software from Applied Biosystems. The cycling parameters were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min on a 7500 real time PCR system (Applied Biosystems). The cycle threshold (Ct) values corresponding to the PCR cycle number at which fluorescence emission in real time reaches a threshold above the base-line emission were determined. Fold increase for IL-10 or HIV-Tat normalized to beta-actin expression levels was determined using the {Delta}{Delta}Ct method. Results are depicted as mean fold change ± S.D.

Electrophoretic Mobility Shift Assays (EMSA)—EMSAs were performed as described earlier (45, 52). Briefly, cells were infected with either pLXIN or pTat in the presence or the absence of various inhibitors. The nuclear proteins (5 µg) were mixed with 32P-labeled CREB oligonucleotide probes for 20 min, and the resulting complexes were separated on a 5% nondenaturing gel. The oligonucleotide probes containing sequences corresponding to the CREB-binding site were as follows: wild type, 5'-CAA TTT GTC CAC GTC ACT GTG ACC-3'; mutant, 5'-CAA TTT GTC CAGATC GCT GTG ACC-3'. Mutant nucleotides are indicated in boldface type. To determine the specificity of the proteins binding the CREB probe, parallel EMSA reactions were incubated with 50-200-fold excess of unlabeled specific and mutant oligonucleotide probes for 20 min prior to the addition of labeled probe. Supershift experiments were also performed by using mouse anti-CREB-1 monoclonal antibodies (Santa Cruz Biotechnology).

Western Blotting—Cell pellets were lysed for 30 min with lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium vanadate, and 1 mM EGTA, pH 7.7). Total protein lysates were subjected to electrophoresis on 12% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad), and the membranes were probed for phosphorylated CREB anti-Ser133 phosphotyrosine CREB or anti-CREB antibodies (Cell Signaling Technologies). The immunoblots were developed by ECL (Santa Cruz Biotechnology) as per the manufacturer's instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Extracellular and Intracellular HIV-Tat Induce IL-10 Expression in Purified Human Monocytes and THP-1 Cells—HIV-Tat has been shown to induce the expression of IL-10 in primary human monocytes (22-24, 40). We confirmed these observations by treating human monocytes with highly purified endotoxin-free recombinant Tat for 24 h followed by detection of IL-10 production by ELISA and intracellular staining by flow cytometry. The HIV-Tat induced IL-10 production in primary human monocytes in a dose-dependent manner as determined by ELISA (Fig. 1A). HIV-Tat at a concentration of 200 ng/ml induced a moderate shift for intracellular IL-10 detection by flow cytometry (Fig. 1B). It may be noted that the same cells when analyzed by ELISA produced ~200 pg/ml of IL-10.

To examine the intracellular signaling pathways involved in HIV-Tat-induced IL-10 production in monocytic cells, we examined several human monocytic cell lines including THP-1, U937, and HL-60 cells as model systems for the production of IL-10 in response to exogenous Tat. None of the cell lines examined produced IL-10 in response to exogenous Tat as determined by ELISA (data not shown). Subsequently, we investigated whether intracellularly expressed HIV-Tat in monocytic cells has the capacity to induce IL-10 production. To investigate the effect of intracellular HIV-Tat on IL-10 production, we engineered a retroviral Tat (pTat) expression system using the pLXIN replication-deficient retroviral vector as the backbone.

Retrovirally pTat-infected THP-1 cells expressed intracellular Tat message as determined by RT-PCR analysis by 12 h after infection (data not shown). To ensure maximal Tat expression, a second round of infection 24 h after the primary infection was performed. HIV-Tat RNA was measured at various times following the second infection. HIVTat expression was observed 4 h after the second infection that was maintained until 24 h as determined by semi-quantitative RT-PCR analysis (data not shown). HIV-Tat RNA was also measured by quantitative RT-PCR. Maximum HIV-Tat RNA was expressed at 16 h after the second infection (Fig. 2A). The biological activity of HIV-Tat expressed from pTat retrovirus was determined by performing a p24 ELISA-based LTR assay. Treatment of HLM1 cells with two different preparations of pTat retroviruses, pTat1 and pTat2, significantly enhanced p24 production compared with the cells infected with a control pLXIN vector (Fig. 2B). Furthermore, p24 production following infection with pTat1/pTat2 was equivalent to the cells activated by a nonviral transcription inducer, NaB, as a positive control (Fig. 2B). Because large volumes of media were required to perform pTat retroviral infection, the level of IL-10 produced was diluted and thus difficult to detect by ELISA. To circumvent this problem, pTat-induced intracellular IL-10 was measured by flow cytometry. Infection of THP-1 cells with the pTat retrovirus resulted in increased expression of IL-10 when compared with cells infected with control pLXIN retrovirus. IL-10 expression was induced as early as 16 h after the first infection (data not shown); however, maximal IL-10 induction was observed 16 h post-secondary infection. The level of IL-10 production following infection of THP-1 cells with the pTat retrovirus as determined by flow cytometry was comparable with ~200 pg/ml of IL-10 produced by LPS-stimulated THP-1 cells as determined by ELISA (Fig. 3, A and B). Furthermore, there was no difference in IL-10 expression between uninfected and pLXIN-infected THP-1 cells (data not shown). It may be noted that treatment of pLXIN-infected cells with the exogenous Tat did not induce IL-10 production, suggesting that infection of cells with the control pLXIN virus did not affect its ability to respond to recombinant Tat.


Figure 2
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  		FIGURE 2.
HIV-Tat is expressed in THP-1 cells following in vitro infection with pTat retrovirus. A, THP-1 cells (0.5 x 106/ml) were infected twice with either pTat or the control vector pLXIN. The pTat-infected cells were harvested at various times post-infection. Cells infected with pLXIN were collected 16 h post-infection. HIV-Tat expression was measured by quantitative RT-PCR analysis as described under "Experimental Procedures." Relative percent Tat expression was calculated as a percentage of Tat expressed relative to the level expressed in cells harvested 16 h after infection with the pTat virus. Results shown are the mean ± S.D. from three different infections in THP-1 cells. B, HIV-Tat expressed following infection with the pTat retrovirus is biologically active. HLM1 cells (0.5 x 106/ml) were left uninfected, infected twice with either pTat preparations pTat1 or pTat2 or pLXIN retroviruses, or were treated with NaB as a control for induction of transcription. HIV-Tat-induced LTR activity was measured by the ability of Tat to direct HIV replication as measured by a p24 ELISA as described under "Experimental Procedures." Results shown are a mean ± S.D. from three different experiments.

 


Figure 3
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  		FIGURE 3.
Intracellular HIV-Tat induces IL-10 expression in THP-1 cells as determined by flow cytometry. A, THP-1 cells (0.5 x 106/ml) were infected twice with either pTat (dark line) or pLXIN (shaded histogram) retroviruses. Cells were harvested at 16 h post-second infection. B, THP-1 cells (0.5 x 106/ml) were either left untreated (shaded histogram) or treated with LPS (1 µg/ml) (dark line) for 24 h. IL-10 expression was measured by intracellular staining and flow cytometric analysis. The histograms shown are a representative of five separate experiments.

 
Tat protein is secreted from the HIV-infected cells. Therefore, it is likely that the Tat may be secreted from the pTat-infected cells, which could in turn induce IL-10 production following interaction with the neighboring cells in our system. To further characterize that IL-10 expression in pTat-infected cells is because of endogenous Tat, we determined whether Tat was secreted in the supernatants of THP-1 cells infected with pTat retroviruses by analyzing its biological activity by p24 ELISA-based LTR assay in HLM-1 cells containing defective mutant HIV-Tat. Cells were infected with the pTat or pLXIN viruses, and the supernatants were collected 24 h after the second infection. The HLM-1 cells were cultured with the supernatants for 3-7 days followed by determination of p24 production by ELISA as described under "Experimental Procedures." The supernatants failed to induce p24 production (data not shown) indicating the absence of soluble Tat in the pTat-infected cells. Because recombinant exogenous Tat failed to induce IL-10 production in THP-1 cells and the fact that the pTat-infected cells did not secrete Tat in their supernatants suggest that the IL-10 produced by the pTat-infected THP-1 cells was because of endogenous Tat.


Figure 4
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  		FIGURE 4.
HIV-Tat-induced IL-10 expression is regulated by ERK MAPK activation. A, THP-1 cells (0.5 x 106/ml) were incubated with either of the MAPK inhibitors SB202190, SP600125, or PD98059 for 2 h prior to the second infection with the pTat or pLXIN retroviruses. Intracellular induction of IL-10 was measured by flow cytometry as described under "Experimental Procedures." Dark line, pTat-infected cells; shaded histogram, pLXIN-infected cells; colored lines, cells treated with various concentrations of p38 (left panel), JNK (middle panel), or ERK (right panel) inhibitors (25 µM, red line;50 µM, blue line). The histograms shown are representative of five separate experiments. B, intracellular HIV-Tat induces the phosphorylation of ERK MAPK that is inhibited by PD98059 in THP-1 cells. THP-1 cells (0.5 x 106/ml) were treated with 10 µM of PD98059 for 2 h prior to the second infection with either pTat or pLXIN retroviruses. Levels of phospho-p42/44 ERK MAPKs were measured by a phospho-p42/44 ELISA kit (R & D Systems), as described under "Experimental Procedures." Results shown are a mean ± S.D. from three different experiments. C, PD98059 treatment down-regulates IL-10 mRNA expression but does not down-regulate HIV-Tat expression in THP-1 cells. THP-1 cells (0.5 x 106/ml) were treated with varying doses (0-25 µM) of PD98059 prior to the second infection with pTat retrovirus. Cells were harvested 16 h post-infection. Quantitative RT-PCR analysis was performed using primers specific for IL-10, HIV-Tat, and beta-actin as described under "Experimental Procedures." The result shown is a mean ± S.D. fold change in IL-10 or Tat expression calculated from three separate experiments.

 
Involvement of p42/44 ERK MAPK in HIV-Tat-induced IL-10 Production—We and others have previously identified a critical role for p38 MAPK in the regulation of LPS-induced IL-10 expression in murine and human monocytic cells (45, 54). It is known that recombinant HIV-Tat can induce the phosphorylation of p38, JNK, and p42/44 ERK MAPKs in various cell types (22, 37, 40). We sought to determine whether any of the MAPK, and in particular p38 MAPK, was involved in the induction of IL-10 by endogenously expressed HIV-Tat. The role of MAPKs has been extensively studied by employing pharmacological inhibitors specific for various MAPKs. SB202190, PD98059, and SP600125 have been used to specifically inhibit the activation of p38, ERK, and JNK MAPKs, respectively (45, 49-52). THP-1 cells were treated with various concentrations of SB202190, PD98059, or SP600125 for 2 h prior to the first and second infection with pTat retrovirus. Pretreatment of cells with the p38 or the JNK inhibitors did not significantly affect HIV-Tat-induced IL-10 expression at any concentration. Interestingly, pretreatment of cells with the p42/44 MAPK inhibitor, PD98059, even at low concentrations of 10 µM significantly reduced IL-10 expression following the first (data not shown) and the second infection of THP-1 cells with the pTat virus (Fig. 4A). To confirm that intracellular HIV-Tat was able to induce p42/44 MAPK activation, THP-1 cells infected with the pTat retrovirus were examined for p42/44 phosphorylation by employing anti-phospho-p42/44-specific antibodies in a novel commercially available ELISA-based assay. Treatment of cells with 10 µM PD98059 prior to the second infection with pTat retrovirus completely inhibited p42/44 phosphorylation as examined at various times following infection. In contrast, treatment of cells infected with the control pLXIN retrovirus did not induce p42/44 phosphorylation (Fig. 4B). To confirm that PD98059 did not inhibit pTat retroviral entry or Tat expression, cells treated with various doses of PD98059 were infected with pTat retrovirus, and Tat mRNA expression was measured by quantitative RT-PCR. PD98059 did not inhibit Tat expression, rather an increase in Tat expression was observed in cells treated with various concentrations of PD98059 (Fig. 4C). As expected, PD98059 significantly inhibited IL-10 expression (p < 0.064 at 5 µM, 0.01 at 10 µM, and 0.003 at 25 µM of PD98059) as determined by quantitative RT-PCR analysis of the same samples (Fig. 4C). The biological activity of SB202190 and SP600125 was determined by their ability to inhibit LPS-induced p38 and JNK phosphorylation in THP-1 cells in a dose-dependent manner (data not shown) (45, 52). Therefore, the lack of IL-10 expression observed in the presence of PD98059 cannot be attributed to nonspecific effects of the inhibitor or absence of retroviral entry, infection, or Tat expression. These results suggest a previously unknown role for the p42/44 ERK MAPK in the IL-10 regulation in human monocytic cells following infection with the pTat retrovirus.

Determination of DNA Sequences in the IL-10 Promoter Region Required for IL-10 Transcription in Response to Intracellularly Expressed HIV-Tat—The above results showing the involvement of ERK and not of p38 MAPK in the regulation of pTat-induced IL-10 expression suggested that intracellular Tat may regulate IL-10 production through a novel signaling pathway. IL-10 has been shown to be regulated by three transcription factors, namely Sp-1, STAT-3, and C/EBPbeta, in different cell systems (45-48). To understand the regulation of IL-10 transcription in THP-1 cells expressing intracellular Tat, a full-length human IL-10 promoter fragment encompassing residues from -890 to +120 bp relative to the transcription start site was amplified by PCR from the IL-10 promoter region, sequenced, and subcloned into the XhoI polylinker site of the luciferase reporter plasmid, pGL3B, as described earlier (45). THP-1 cells infected with the pTat retrovirus for various times were transfected with the full-length IL-10-promoter reporter construct (pIL-10Pr-GL3B) and measured for luciferase activity (Fig. 5A). The maximum increase in luciferase activity ranged from 6- to 8-fold relative to the control plasmid, pGL3B (Fig. 5A), and was observed at 24 h after the second infection with the pTat retrovirus. There was no difference in the luciferase activities in cells infected with the control pLXIN retrovirus and transfected with either pIL-10Pr-GL3B or the control pGL3B (Fig. 5A). These results suggest that the expression of the tat gene in THP-1 cells is capable of transactivating the human IL-10 promoter.

To identify the transcription factors implicated in Tatinduced IL-10 transcription, a series of IL-10 promoter fragments encompassing residues from -890 to + 120 bp relative to the transcription start site were amplified, sequenced, and subcloned into the XhoI polylinker site of pGL3B as described earlier (45). The exact size of the amplified product and the location of consensus sequences for the transcription factor-binding sites within the IL-10 promoter are shown in Fig. 5B. THP-1 cells infected with the pTat retrovirus were transfected with various IL-10 promoter deletion constructs. Examination of luciferase activity induced by the pTat retrovirus of the sequential 5' deletion mutants of the IL-10 promoter region revealed that deletion of the sequences from -890 to -432 had no effect on the pTat-induced luciferase activity compared with the full-length pIL-10Pr-GL3B (Fig. 5B). Deletion of sequences up to -384 bp abrogated the pTat-induced luciferase activity compared with the cells transfected with pIL-10Pr-GL3B. The luciferase activity in these cells was comparable with the cells transfected with the pGL3B plasmid (Fig. 5B). It may be pointed out that transfection of cells infected with the pTat retrovirus with an IL-10 promoter deletion construct (-432 bp) devoid of the Sp-1 (-636 bp) and STAT-3 (-736 bp) binding sequences did not affect luciferase activity, suggesting that Sp-1 and STAT-3, unlike in LPS-induced IL-10 transcription (45, 46), are not involved in Tat-induced IL-10 transcription. These results suggest that the sequences located between -432 and -384 bp are necessary for the induction of IL-10 transcription by intracellular Tat.

The CREB Transcription Factor-binding Site within the IL-10 Promoter Is Sufficient for the Inductive Effects of Intracellular Tat on IL-10 Transcription—Examination of the DNA sequence between -432 and -384 bp of the IL-10 promoter using Matinspector software revealed the presence of a consensus CRE sequence for the CREB transcription factor (5'-ACGTCA-3' at -410 to -404 bp). Because Tat has been suggested to activate CREB (39), we hypothesized that intracellular Tat may regulate IL-10 transcription through the activation of the CREB transcription factor. To investigate the role of CREB, we generated a mutant CRE site in the IL-10 promoter construct (mCRE) by site-directed mutagenesis, substituting guanine with cystine at position -410 bp (Fig. 5). Transfection of the pTat retrovirus-infected cells with the mCRE construct resulted in a complete loss of luciferase activity to basal levels as compared with the cells transfected with the wild type CRE containing vector, suggesting that CRE sequence was critical to the intracellular HIV-Tat-induced IL-10 transcription (Fig. 5B).

The involvement of the CRE sequence in IL-10 transcription by intracellular HIV-Tat was also confirmed by transfecting THP-1 cells simultaneously with the HIV-Tat plasmid (pSV2tat72) and the full-length pIL-10Pr-GL3B constructs for 24 h followed by analysis of luciferase activity (Fig. 5C). Similar to the results obtained above with THP-1 cells infected with the pTat retrovirus, cotransfection of THP-1 cells with the pSV2tat72 and pIL-10Pr-GL3B resulted in enhanced luciferase activity. This increase in luciferase activity was found to be 3-5-fold compared with the cells cotransfected with the control vector (Fig. 5C). In addition, examination of the same IL-10 promoter constructs revealed that deletion of sequences from -890 to -432 had no effect on luciferase activity compared with the cells cotransfected with pSV2tat72 and pIL-10Pr-GL3B (Fig. 5C). However, deletion of sequences up to -384 bp abrogated luciferase activity compared with the cells cotransfected with pSV2tat72 and pIL-10Pr-GL3B. The luciferase activity in cells cotransfected with HIV-Tat and the IL-10 promoter construct containing sequences from -384 to +120 bp was comparable with the cells transfected with the pGL3B alone (Fig. 5C). To further confirm the involvement of the CRE in HIV-Tat-induced IL-10 transcription, THP-1 cells were cotransfected with the pSV2tat72 and the mCRE IL-10 promoter construct. Cotransfection of cells with pSV2tat72 and the mCRE containing the IL-10 promoter construct resulted in complete loss of luciferase activity to the basal level as compared with the cells transfected with the wild type CRE construct (Fig. 5C). These observations further suggested that the CRE sequence was critical to HIV-Tat-induced IL-10 transcription in THP-1 cells.


Figure 5
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  		FIGURE 5.
HIV-Tat induces IL-10 transcription in THP-1 cells via the CREB-binding sequence in the IL-10 promoter. A, pTat induces luciferase activity in THP-1 cells transfected with p-IL-10Pr-GL3B. THP-1 cells (0.5 x 106/ml) were infected twice with either pTat or pLXIN retroviruses for 24 h followed by transient cotransfection either with p-IL-10Pr-GL3B or the pGL3B control vector, and with 5 µg of pSV-beta-galactosidase plasmid. Cells were cultured for another 24 or 48 h followed by measurement of luciferase and beta-galactosidase activities in the cell lysates. B, upper panel, line diagram of the nucleotide sequence of the 5'-flanking promoter region of the human IL-10 gene (GenBankTM accession number X78437). The putative cis-regulatory elements are indicated. Lower panel, THP-1 cells (0.5 x 106/ml) were infected twice with either pTat or pLXIN retroviruses for 24 h followed by transient cotransfection either with various hIL-10 promoter deletion constructs, IL-10 promoter constructs containing either wild type CRE or mutant CREB (mCRE) binding sequences or the pGL3B control vector, and with 5 µg of pSV-beta-galactosidase plasmid. Cells were cultured for another 24 h followed by measurement of luciferase and beta-galactosidase activities in the cell lysates. The exact location of various transcription factor-binding sites is indicated. C, THP-1 cells (0.5 x 106/ml) were transiently cotransfected with pSV2tat72 and either with various hIL-10 promoter deletion constructs, IL-10 promoter constructs containing either wild type (wt CRE) or mutant CREB (mCRE) binding sequences, or the pGL3B control vector, and with 5 µg of pSV-beta-galactosidase plasmid. The transfected cells were cultured for 24 h followed by analysis for luciferase activity. Luciferase activity was normalized with beta-galactosidase activity, and the fold increase in luciferase activity was determined. The results shown are a mean ± S.D. of four experiments performed in triplicate.

 
Inhibition of Tat-induced p42/44 ERK MAPK Inhibits Luciferase Activity of IL-10 Promoter—The above results suggest the involvement of ERK MAPK and the CRE sequences in intracellular HIV-Tat-induced IL-10 expression. Therefore, it was of interest to determine whether intracellular HIV-Tat regulates IL-10 expression by regulating the activity of CRE through the activation of ERK MAPK. To address this question, THP-1 cells infected with pTat retrovirus were treated with inhibitors specific for p38, ERK, or JNK MAPKs for 2 h prior to transfection with the IL-10 promoter deletion constructs. As observed above, the full-length IL-10 promoter construct as well as the IL-10 promoter deletion constructs spanning -432 to -890 bp resulted in a 6-8-fold increase in luciferase activity in pTat-infected cells compared with the cells transfected with the control plasmid (Fig. 6). Pretreatment of cells infected with pTat retrovirus with PD98059 abrogated the luciferase activity in cells transfected with all IL-10 promoter constructs that contained the wild type CRE sequence. In contrast, pretreatment of same cells with either SB202190 or SP600125 for 2 h prior to transfection with any of the IL-10 promoter constructs that contained the CRE sequence did not affect luciferase activity compared with the untreated cells (Fig. 6). These results indicate that IL-10 induction by intracellular Tat may be mediated by the CRE activity through ERK MAPKs.


Figure 6
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  		FIGURE 6.
HIV-Tat-induced IL-10 promoter activity is dependent on ERK MAPK activity. THP-1 cells (0.5 x 106/ml) were infected with either pTat or pLXIN retroviruses for 24 h. Two h prior to the second infection with the retroviruses, cells were treated with either SB202190 (25 µM), PD98059 (25 µM), or SP600125 (25 µM) following which cells were transiently cotransfected with 10 µg of either hIL-10 promoter construct containing wild type CREB-binding sequence or the pGL3B control vector, and with 5 µg of pSV-beta-galactosidase plasmid. Cells were cultured for another 24 h followed by measurement of luciferase and beta-galactosidase activities in the cell lysates. Luciferase activity was normalized with beta-galactosidase activity, and the fold increase in luciferase activity was determined. The results shown are a mean ± S.D. of four experiments performed in triplicate.

 


Figure 7
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  		FIGURE 7.
HIV-Tat induced the activation of CREB-1 binding to the CREB-binding sequence located in the IL-10 promoter. A, THP-1 cells (0.5 x 106/ml) were infected with either pTat (T) or pLXIN (P) retroviruses, and cells were harvested at various times post-second infection. Nuclear extracts were isolated and analyzed for binding to 32P-labeled oligonucleotide probes corresponding to the CREB-binding site in the human IL-10 promoter (left panel). Specificity for the probe was confirmed by cold competition (CC) with either excess unlabeled CREB probe, a mutated (M) CREB probe, or with a nonspecific (NS) probe for NF{kappa}B (middle panel). Additionally, nuclear extracts were incubated with various concentrations of antibodies specific for the CREB-1 transcription factor and observed for slower migration of DNA-protein complexes by supershift analysis (right panel). Arrows indicate three distinct CREB-1 bands. B, HIV-Tat-induced CREB-1 is regulated by the activation of ERK MAPK. THP-1 cells (0.5 x 106/ml) were infected with pTat retrovirus for 24 h. Two h prior to the second infection with the retrovirus, cells were treated with either SB202190, PD98059, or SP600125 following which cells were harvested 16 h post-second infection, and the nuclear extracts were analyzed for binding to the CREB oligonucleotide probe as described above in the legend to A. The results shown are representative of three separate experiments.

 
HIV-Tat Induces Specific Binding of CREB-1 to the CRE in the IL-10 Promoter—The CREB transcription factor is known to bind the CRE sequence and has also been shown to be activated by HIV-Tat (55). To confirm that intracellular Tat-induced IL-10 is regulated by the activation of CREB, we studied whether intracellular Tat induced the activation and binding of CREB to the CRE sequence on the IL-10 promoter by EMSA. THP-1 cells were infected with pTat or control pLXIN retroviruses for 0-24 h followed by gel shift analysis for binding of nuclear proteins to the CRE oligonucleotide probe. The results revealed binding of CREB to the CRE as early as 4 h with maximal binding at 16 h following infection with pTat retrovirus compared with the cells infected with the control retrovirus (Fig. 7A, left panel). We observed three distinct DNA-CREB protein complexes that were completely blocked by the unlabeled CRE oligonucleotides but not the nonspecific oligonucleotides (NF{kappa}B), indicative of CRE specificity (Fig. 7A, middle panel). There is a nonspecific band above the free probe with a similar intensity indicating equal loading in each lane. Furthermore, nuclear extracts obtained from cells infected for 16 h with pTat retrovirus were incubated with various concentrations of anti-CREB-1 antibodies in order to perform supershift analyses (Fig. 7A, right panel). A distinct high molecular weight migration of the DNA-CREB protein complex was observed following incubation with anti-CREB-1, suggesting that the activation of the CREB-1 transcription factor is induced by intracellular HIV-Tat in THP-1 cells. However, the lower two bands of Fig. 7A, right panel, are of light intensity, which may not have been supershifted. It is likely that these lower bands may contain other transactivators/coactivators, such as CBP and C/EPB-beta that may cooperate with CREB-1; however, these transcription factors cannot be precisely identified under the present experimental conditions.

Because CREB activation has been shown to occur through the activation of MAPK in other cell systems (56), we investigated whether intracellular Tat regulates IL-10 transcription by CREB through the activation of ERK MAPK. THP-1 cells were treated with specific inhibitors for either p38, JNK, or p42/44 ERK MAPKs for 2 h prior to the second infection with pTat retrovirus for 16 h and analysis of CREB binding to its probe corresponding to the CRE sequence in the IL-10 promoter. The results show that pretreatment of infected THP-1 cells with PD98059 resulted in a dramatic inhibition of HIV-Tat-induced DNA-CREB-1 protein binding, whereas pretreatment with either SB202190 or SP600125 had no such effect even when used at concentrations as high as 50 µM (Fig. 7B).


Figure 8
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  		FIGURE 8.
HIV-Tat-induced Ser133 phosphorylation of CREB is regulated by the activation of ERK MAPK. THP-1 cells were pretreated with PD98059 at the concentrations indicated for 2 h prior to the second infection with pTat retrovirus. Cells were harvested 16 h post-second infection, and the protein extracts were analyzed for Ser133 phosphorylation of CREB by Western blotting using a specific anti-phospho-Ser133 CREB antibody. The membrane was stripped and reprobed with anti-CREB as an equal loading control. The antiphospho-Ser133 CREB antibody cross-reacts with the ATF-2 transcription factor and thus exhibits two bands corresponding to phosphorylated CREB and ATF-2 in Western blot analysis. The result shown is a representative of four different experiments.

 
Intracellular Tat Induces Ser133 Phosphorylation of CREB-1 That Is Regulated by ERK MAPK—The CREB transcription factor has been shown to be activated by phosphorylation at Ser133 (39). Therefore, it was interesting to determine whether intracellular Tat induces IL-10 transcription through CREB-1 phosphorylation at Ser133 and whether this phosphorylation is also regulated by the ERK MAPK. For this, pTat retrovirus-infected THP-1 cells were pretreated with PD98059 and examined for Ser133 phosphorylation of CREB by Western blot analysis by using anti-Ser133-phospho-CREB antibodies. This antibody cross-reacts with the ATF-2 transcription factor and exhibits two bands corresponding to the phospho-CREB and phospho-ATF-2 transcription factors in Western blot analysis. The results show that intracellular Tat induced CREB phosphorylation at Ser133 compared with the basal levels and that this phosphorylation was inhibited by PD98059 in a dose-dependent manner (Fig. 8). Interestingly, intracellular Tat induced the phosphorylation of ATF-2 that was also inhibited to the basal levels by PD98059 in a dose-dependent manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding the regulation of IL-10 expression in HIV infection is important in deciphering the dysregulation of cytokine function induced by HIV and more broadly how HIV infection induces a state of immunodeficiency. The results from this study and from other laboratories suggest that the extracellular HIV accessory protein Tat enhances IL-10 production in human monocytic cells (22, 40). By employing first exon-deleted mutants and anti-Tat antibodies specific for a distinct region of Tat, Badou et al. (22) demonstrated that the critical region responsible for stimulation was located within residues 1-45. The mechanism by which extracellular Tat regulates IL-10 expression has been reported to be mediated by the PKC isozymes (22, 23). However, there is little information on how intracellular Tat regulates host cellular gene products. Specifically, the mechanism by which intracellular Tat enhances IL-10 expression remains unknown. Here we provide evidence for the first time that intracellular HIV-Tat induces IL-10 transcription by the activation of the CREB-1 transcription factor through Ser133 phosphorylation and its regulation by the p42/44 ERK MAP kinase.

It is well established that extracellular Tat displays several pleiotropic activities on the functions of bystander uninfected T cells and monocytes. For example, Tat has been shown to induce the expression of cytokines (IL-6, tumor necrosis factor-{alpha}, IL-10, IL-2, and IL-12), cytokine receptors (IL-4R and IL-2R), and adhesion molecules, and it stimulates growth as well as causes apoptosis depending on the cell type (22-24, 28-35). Hence, understanding of the molecular mechanisms underlying Tat-mediated biological effects remains a central issue in AIDS pathogenesis. There is evidence that extracellular Tat, following interactions with a variety of cell surface receptors, activates a cascade of signaling pathways, including MAPKs, PKC, phosphoinositide 3-kinase, and p56lck tyrosine kinases, and transcription factors such as NF{kappa}B and CREB (22, 23, 34-41). The results of this study show that exogenous Tat induced IL-10 expression in primary human monocytes; however, it was unable to induce IL-10 expression in THP-1 cells. It has been suggested that extracellular Tat is not internalized in some monocytic cell types, including U937 cells (29). Because endogenously expressed Tat resulted in the induction of IL-10 in THP-1 cells, the absence of IL-10 induction by extracellular Tat may be due to its inability to enter THP-1 cells. Therefore, to precisely identify the signaling molecules and the transcription factors regulating Tat-induced IL-10 expression, we generated a retroviral expression vector that was able to express Tat upon its infection of THP-1 cells and induce IL-10 expression.

We and others have demonstrated previously a selective role for p38 MAPK in the regulation of LPS-induced IL-10 expression in monocytic cells (45, 54). In this study, however, p38 and JNK MAPKs were not involved in IL-10 induction in cells infected with HIV-Tat retrovirus. Although extracellular Tat is known to activate MAPKs in various cell types (22, 37, 40), this is the first study showing the activation of ERK by intracellular Tat in monocytic cells. How endogenously expressed Tat activates ERK is not clear at present. ERK activation is mediated by the activation of the G-protein Ras, which induces the activation of Raf family members. Subsequently, the MAPK kinases MEK1 and MEK3 are activated, which induce the phosphorylation of ERK1/2. It is possible that Tat may induce ERK phosphorylation either directly following its interaction with ERK or indirectly through its interaction with upstream kinase(s) in the ERK signaling pathway. Extracellular Tat has also been shown to regulate IL-10 induction through the activation of PKC betaII and {delta} isozymes (22, 23, 40). Whether intracellular Tat induces IL-10 through PKC activation remains to be investigated. Because MAPKs are downstream targets for PKC (57), intracellular Tat may regulate IL-10 induction by ERK through the activation of distinct members of the PKC family.

CREB is a critical transcription factor involved in the regulation of several cellular genes. cAMP is generally believed to mediate binding of CREB to a conserved CRE sequence present in the promoter of several cAMP-responsive genes (58, 59). The CREB protein consists of two transactivation domains, namely the glutamine-rich Q2 and the kinase-inducible domain (60). The glutamine-rich Q2 domain interacts with the general transcription factor TAFII130/135 and recruits a functional RNA polymerase II complex for basal transcriptional activity. In contrast, the kinase-inducible domain is involved in CREB-mediated transcription of cellular genes. CREB was originally shown to become phosphorylated at Ser133 by activated protein kinase A (61). Subsequently, it was shown to be phosphorylated at other sites, including Ser142, Ser98, and Ser117, by a number of upstream kinases (58). CREB may also exert its biological effects by recruiting a number of transactivators or coactivators such as CBP, p300, C/EBP-beta, and Oct-1 (58, 60, 61). To be active, CREB requires post-translational modifications, such as phosphorylation, glycosylation, or alteration in the CREB-CBP interactions (61).

Here we identified a CRE sequence ACGTCA in the IL-10 promoter region for CREB binding (-410 to -404 bp) as playing a key role in intracellular Tat-induced IL-10 transcription. Site-directed mutation of the CRE sequence abrogated luciferase activity in HIV-Tat retrovirus-infected cells as well as in cells cotransfected with Tat and mutant CRE IL-10 promoter plasmids. In addition, Tat induced the activation of CREB-1 as determined by the binding of CREB-1 to its binding site in the IL-10 promoter. Furthermore, intracellular Tat, like the extracellular Tat as shown previously (39), induced Ser133 phosphorylation of CREB. Whether intracellular Tat directly activates CREB is not clear at present. Recently, Tat-interacting protein, Tip60, which is expressed ubiquitously in various cell types, has been shown to bind CREB (61). It is possible that Tat activates CREB following its interaction with Tip60; however, this remains to be determined.

It is interesting to observe that Tat-induced IL-10 transcription seems to be mediated by a single transcription factor. Although LPS-induced IL-10 transcription in monocytic cells has been shown previously to be regulated by at least three transcription factors, namely Sp-1, STAT-3, and C/EBPbeta (45-48), our results suggest that deletion of Sp-1 (-636 bp) and of STAT-3 (-736 and -120 bp) sequences failed to modulate Tat-induced effect on luciferase activity. The observations that mutation of the CRE sequence in the IL-10 promoter abrogated intracellular Tat-induced luciferase activity suggest that Tatinduced IL-10 transcription may be primarily regulated by CREB. Even though Tat was reported to induce IL-10 expression through NF-{kappa}B activation (22), the IL-10 promoter does not contain an NF{kappa}B-binding site, and hence it is unlikely to be involved in Tat-induced IL-10 transcription. Although our results suggest that ATF-2, Sp-1, or STAT-3 may not be involved in Tat-mediated IL-10 transcription in monocytic cells, we cannot rule out the involvement of other transactivators/coactivators, such as CBP and C/EPB-beta, that may cooperate with CREB-1 in Tat-mediated IL-10 transcription in monocytic cells or by a novel transcription factor in other cell types. This assumes significance in view of our observations that intracellular Tat induced phosphorylation of ATF-2 in addition to that of CREB-1, both of which were regulated by the ERK MAPK pathway.

Investigations for the involvement of upstream ERK MAPK in Tat-induced CREB activation revealed that Tat-induced IL-10 transcription may be mediated by CREB through ERK activation. The ERK inhibitor PD98059 inhibited Tat-induced CREB activation as determined by luciferase activity and EMSA. The CREB transcription factor is known to act as a substrate for several kinases, including protein kinase A, protein kinase G, PKC, ERK, and p38 MAPKs (56, 59, 60). The ERK MAPK has also been shown to induce a more prolonged CREB phosphorylation profile compared with other kinases such as calmodulin-dependent protein kinase-II (60, 62). It will be interesting to determine the involvement of Tat-activated upstream kinases other than ERK MAPK that may phosphorylate CREB-1 resulting in eventual IL-10 transcription.

The observation that Tat enhances IL-10 production in monocytic cells has several implications. First, it suggests that HIV employs Tat to enhance interactions between monocytic cells and T cells during HIV-1 infection. Because Tat is indispensable for HIV replication (25, 26), and IL-10 is known to inhibit HIV replication in monocytic cells (19, 63), Tat-induced IL-10 may prevent excessive HIV replication as a negative feedback regulator in monocytic cells. Because IL-10 is also known to prevent apoptosis in human monocytic cells (64), Tat may be a factor to prevent apoptosis of monocytic cells and thus promote the development of monocytic cells as viral reservoirs.

In summary, by using a retrovirus to express intracellular Tat, our results clearly show for the first time the involvement of the CREB-1 transcription factor and its activation through Ser133 phosphorylation in the regulation of IL-10 transcription in human monocytic cells. Furthermore, ERK MAPK was shown to regulate intracellular T at-induced CREB-1 Ser133 phosphorylation and IL-10 transcription. Keeping in view our previous observations involving the activation of p38 MAPK and the Sp-1 transcription factor in the regulation of LPS-induced IL-10 gene transcription (45), our results suggest the differential involvement of various transcription factors and signaling molecules depending upon the cell type and the signaling stimulus. Identification of this signaling pathway provides important information regarding the mechanism of HIV-mediated immunodeficiencies and more broadly may provide strategies to restore immunological status in HIV-infected individuals.


   FOOTNOTES
 
* This work was supported in part by grants from the Ontario HIV Treatment Network and 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 a fellowship from the Ontario HIV Treatment Network. Back

2 Recipient of the Career Scientist Award from the Ontario HIV Treatment Network. Back

3 Supported by a fellowship from the Ontario Graduate Scholarship program and the Ontario Graduate Scholarships in Science and Technology program. Back

4 To whom correspondence should be addressed: Division of Virology, Dept. of Pathology and Laboratory Medicine, Research Institute, Children's Hospital of Eastern Ontario, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-737-7600 (Ext. 3920); Fax: 613-738-4825; E-mail: address: akumar{at}uottawa.ca.

5 The abbreviations used are: HIV, human immunodeficiency virus; CREB-1, cAMP-responsive element-binding protein-1; C/EBP-beta, CCAAT/enhancer-binding protein-beta; ERK, extracellular signal-regulated kinase; JNK, c-Jun N terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NaB, sodium butyrate; PKC, protein kinase C; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; EMSA, electrophoretic mobility shift assays; m, mutant; h, human; PBS, phosphate-buffered saline; LTR, long terminal repeat; CRE, cAMP-responsive element; CBP, CREB-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Kryworuchko for critically reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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