A role for mitogen-activated protein kinase and Ets-1 in the induction of interleukin-10 transcription by human immunodeficiency virus-1 Tat

Abstract

The human immunodeficiency virus (HIV) Tat protein has multiple regulatory roles, including trans-activation of the HIV genome and regulation of immune signalling processes, including kinase activation and cytokine expression. We recently demonstrated that HIV-1 Tat induces the expression of interleukin (IL)-10 via p38 mitogen-activated protein kinase (MAPK) activation. We further delineated that the Tat-responsive element of the IL-10 promoter was located within 625 to 595 bp upstream from the transcription start site. Using electrophoretic mobility shift assays, the transcription factors Ets-1 and Sp-1 were shown to bind to the IL-10 promoter to activate transcription of the gene. Furthermore, sequential deletional mutations of the Ets-1- and Sp-1-binding sites in the −625/−595 region reduced the DNA binding and transcription activity of the IL-10 promoter. Our results also showed that both the Tat-induced and Ets-1-regulated IL-10 promoter-driven luciferase activity can be abrogated by inhibitors of the p38 MAPK activity. In conclusion, the coordinated activities of p38 MAPK and the transcription factors, Ets-1 and Sp-1, may play an important role in the HIV-1 Tat-induced IL-10 transcription.

Introduction

The human immunodeficiency virus (HIV) trans-activator (Tat) protein plays an important role in the development of acquired immune-deficiency syndrome (AIDS) and its associated diseases. The primary function of Tat is to activate the HIV-long terminal report (LTR) to enhance the replication of HIV in the virus-infected cells. In previous studies, Tat has also been shown to have the ability to induce apoptosis in neurons, or to stimulate the microglia and macrophages to release neurotoxins that are related to the HIV-associated dementia.¹

In immune dysregulation, Tat is a strong inducer of tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-10 production, which may play a key role in the development of B-cell lymphoma, Kaposi's sarcoma and AIDS-associated non-Hodgkin's lymphoma in HIV-infected individuals.² High levels of IL-10 can be found in the sera of HIV patients.^3,4 Further investigations suggested that HIV and its viral proteins, such as Tat, play a crucial role in the production of IL-10.^5,6 IL-10 is known to be an anti-inflammatory cytokine responsible for suppression of the immune response, thus preventing the development of chronic inflammation. The functions of IL-10 are to inhibit specific cytokines produced by T helper 1 (Th1) and natural killer cells, as well as macrophages.^7–9 IL-10 is capable of suppressing the production of IL-6 or TNF-α, which are induced during phagocytosis. It can also inhibit the induction of IL-8, IL-12 or even IL-10. In HIV infection, previous studies showed that the high levels of IL-10 may be related to the development of AIDS-associated B-cell lymphoma.¹⁰ The lymphoma and its surrounding cells produce IL-10 to serve as a B-cell stimulatory factor to support the growth of the malignant cells. Genetic studies of the IL-10 promoter also suggested that some of the individuals with genotypes that have the propensity to produce high levels of IL-10 have faster progression of AIDS.¹¹

Regulating IL-10 gene transcription by different inducers requires the activation of specific transcription factors. Although several transcription factor-binding sites have been described in the IL-10 promoter region, the function of each of these putative binding sites has not been clearly defined. In the lipopolysaccharide (LPS)-induced IL-10 transcriptional activation in a monocytic cell line, transcription factors Stat3, Sp-1 and mitogen-activated protein kinase (MAPK) appear to play important roles.^12,13 In B cells, the binding sites of IFN regulatory factor 1 (IRF-1) and Stat3 have been shown to be involved in the interferon-α (IFN-α)-induced IL-10 mRNA synthesis.¹⁴

Cross-talk pathways of cytokine induction and complexity in the promoter regions involving multiple transcription factor-binding sites are advantageous in the host immune response. The transcriptional activation of a specific gene usually involves the co-operation of several transcription factors. This complex system of regulation provides a platform for the distinctive immune responses inducible by different stimuli.

The transcription factor Ets-1, a key member of the Ets family, binds to specific DNA sequences containing GGAA/T with additional flanking nucleotides. In regulating transcription, it seems that individual Ets family members serve as cofactors with other transcription factors. For example, during HIV-1 replication, activation of the HIV-1 LTR is regulated by the interactions among Ets-1, E-box-binding protein (USF-1) and nuclear factor-κB (NF-κB).^15,16 On the other hand, the synergistic effects between Ets-1, AP-1 and NF-κB also play an important role in granulocyte–macrophage colony-stimulating factor (GM-CSF) transcription.¹⁷ It appears that the functional role of individual Ets family members is to interact with various transcription factors expressed in different cell types, thus serving its collaborative function in the regulation of cell-type specific genes.

Ets-1 regulates several biological processes, including the immune response, apoptosis, growth and developmental differentiation, and ontogenesis.^18–20 For example, Ets-1 induces the expression of bax or bid for the initiation of apoptosis in endothelial cells. However, activation of apoptosis in T cells can be observed in Ets-1-deficient cells.²¹ These reports suggest that the function of Ets-1 to induce or to prevent apoptosis is dependent on the activity of other cellular factors, as well as its own expression levels. Thus, recent reports have proposed the potential role of Ets-1 in oncogenesis.

In light of the critical role of IL-10 in AIDS development, the molecular mechanisms of Tat-induced IL-10 gene transcription were investigated. We have identified a Tat-responsive region in the IL-10 promoter that regulates IL-10 transcription. Furthermore, Ets-1 was identified as a transcriptional regulator of the IL-10 gene in the Tat-induced process. In delineating the Tat-induced signalling pathway, we showed that activation of the IL-10 promoter by Ets-1 is related to the activity of the cellular kinase, p38 MAPK.

Materials and methods

Cell cultures

The human monocytic THP-1 and cervical adenocarcinoma HeLa cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA). The cell lines were maintained in RPMI-1640 and minimum essential medium, respectively (Invitrogen, Carlsbad, CA) at 37° in a humid atmosphere of 5% CO₂.

Recombinant Tat protein

Recombinant HIV Tat protein was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, MD. This Tat protein contains amino acid residues encoded by the first and the second exons. The protein was reconstituted in phosphate-buffered saline (PBS) containing 1 mg/ml of bovine serum albumin (BSA) and 0·1 mm dithiothreitol (DTT) (Mock reagent) prior to the experiments. The levels of endotoxin were measured by the Pyrochrome® Chromogenic kit (Associates of Cape Cod, Falmouth, MA) and were shown to be less than 0·025 EU/µg in these samples. The biological activity of Tat was confirmed by its induction of the luciferase activity in cells transfected with a plasmid encoding the HIV-LTR-driven luciferase cDNA (results not shown).

Isolation of RNA and the reverse transcription–polymerase chain reaction

Total cellular RNA extracted by TRIzol (Invitrogen) was reverse transcribed into cDNA by the SuperScript II system (Invitrogen), according to the manufacturer's instructions.

A typical polymerase chain reaction (PCR) was performed in a 25-µl reaction mixture containing 1·5 mm MgCl₂, 0·2 mm of each deoxynucleoside triphosphate, 0·25 µm each primer, 2 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ) and 1 µl of cDNA. The reactions were cycled 35 times under the following parameters: 95° for 30 seconds, 60° for 30 seconds and 72° for 60 seconds.

Construction of plasmids

A series of IL-10 promoter fragments was amplified by PCR from human genomic DNA using different primer sets (Tables 1 and 2). The PCR products were analysed by agarose-gel electrophoresis. The purified DNA, containing fragments of the IL-10 promoter, was cloned into the pDrive (Qiagen, GmbH, Germany) or pCR2.1-TOPO vectors (Invitrogen), according to the manufacturers' instructions. The sequences of the IL-10 promoter fragments were confirmed by DNA sequencing. The IL-10 promoter fragments in the plasmids were subsequently digested by KpnI and BglII and then inserted into the firefly luciferase reporter plasmid, pGL3-Basic (Promega, Madision, WI). A Renilla luciferase reporter vector, pRL-TK, was used as a control to correct for the transfection efficiency in each series of experiments.

Table 1.

Sequences of the primers for the generation of luciferase plasmids

Primer name	Sequences	IL-10 promoter region amplified1
IL-10 promoter −85 #	5′-ATG AAG AGG CCT CCC TGA G-3′	−85/+163
IL-10 promoter GR #	5′-TTACTCGAGGAATGAGAACCCACAGCTG-3′	−354/+163
IL-10 promoter YY #	5′-AGCTCGAGAGTTGGCACTGGTGTACC-3	−860/+163
IL-10 promoter ST #	5′-AACCTCGAGCAGCAAGTGCAGACTAC-3′	−738/+163
IL-10 promoter −625 sense #	5′-CTGGAACACATCCTGTGACCCCGCCTGTCCT-3′	−625/+163
IL-10 promoter −625-A	5′-CTGGAACACATACTGTGACCCCGCCTGTCCT-3′	−625/+163
IL-10 promoter Sp-1 mutant #	5′-ACACATCCTGTGACCCAGCGTGTCCTGTAGGAAGC-3′	−620/+163
IL-10 promoter −625 Ets-M sense	5′-CTGGAACACATAATGTGACCCCGCCTGTCCT-3′	−625/+163
IL-10 promoter −625 Sp-1-m sense #	5′-CTGGAACACATGCTGTGACCCAGCGTGTCCT-3′	−625/+163
IL-10 promoter −610 #	5′-GGGGTACCCCTGACCCCGCCTGTCCTGTAG-3 ‘	−610/+163
IL-10 promoter +168 antisense	5′-GGAGATCTCGAAGCATGTTAGGCAG-3′

¹The +1 position refers to the transcriptional start site of X78437 in GenBank.

^#Primer sequences were obtained from ref. 13.

IL, interleukin.

Table 2.

Luciferase constructs used in this study

Plasmid names	IL-10 promoter region amplified1
p[−85]	−85/+163
p[−354]	−354/+163
p[−860]	−860/+163
p[−738]	−738/+163
p[−610]	−610/+163
p[−625]	−625/+163
p[−625A]	−625/+163
p[−620]	−620/+163
p[−625]Ets-M	−625/+163
p[−625]Sp-1-M	−625/+163

¹The +1 position refers to the transcriptional start site of X78437 in GenBank.

IL, interleukin.

The full-length Ets-1 cDNA was amplified by PCR from the mRNA of THP-1 cells with the forward primer 5′-ATGAAGGCGGCCGTCGATCT-3′ and a reverse primer 5′-TCACTCGTCGGCATCTGGCT-3′ using the Expand High Fidelity PLUS PCR system (Roche, Indianapolis, IN). The PCR products were purified and inserted into the pCR2.1-TOPO vector. The pcDNA3-ets-1 plasmid, containing the ets-1 PCR fragment, was generated by cutting with KpnI and BglII, and then the fragment was inserted into the corresponding sites in the pcDNA3 vector.

Transient transfection and dual luciferase assay

THP-1 cells were transiently cotransfected with 2 µg of one of the IL-10 promoter luciferase reporter plasmids and with 100 ng of pRL-TK by using the FuGene 6 reagent (Roche), according to the manufacturer's instructions. Transient transfections of HeLa cells with 1 µg of full-length Ets-1 plasmid and 2 µg of IL-10 promoter luciferase reporter plasmids were performed using the LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturer's protocol.

For the luciferase assay, the protein extracts were collected using Passive Lysis Buffer (Promega). The transfected cells, in 24-well plates or 12-well plates, were washed in cold 1 × PBS and then incubated with 100 µl of Passive Lysis Buffer. The cells were incubated for 15 min on a shaker table. The lysates were stored at −70°. The luciferase activities were measured by the Dual-Luciferase® Reporter Assay System (Promega). The luciferase activities were detected by the Fusion-Alpha Universal Microplate Reader (PerkinElmer, Boston, MA).

Sequence analysis

The human IL-10 gene sequence was retrieved from the NCBI GenBank™, accession number X78437.2. The Transcription Element Search System (tess) (version 2·0), using transfec (version 4·0) databases, was used for predicting the transcription factor binding elements in the IL-10 promoter. The positions in the promoter are relative to the transcription start site of the IL-10 gene (+1).

Preparation of cellular extracts

Protein extract was collected as previously described.²² In brief, the cells were washed with PBS and resuspended in buffer A for 15 min followed by the addition of 10% Nonidet P-40 to collect the cytoplasmic portion. The nuclear pellet was resuspended in buffer C for 15 min on ice to complete the lysis of nuclear membrane.

For collection of the whole-cell lysate, cells were washed with cold PBS and incubated in cold lysis buffer (50 mm Tris–HCl, pH 7·4; 150 mm NaCl; 50 mm NaF; 10 mmβ-glycerophosphate; 0·1 mm EDTA; 10% glycerol; 1% Triton X-100; 1 mm phenylmethylsulphonyl fluoride; 1 mm sodium orthovanadate; 2 µg/ml of pepstatin A; 2 µg/ml of aprotinin and 2 µg/ml of leupeptin) for 20 min. The lysate was then centrifuged at 4° for 20 min. The supernatant was collected and stored at −70°.

Western blot analysis

Cytoplasmic proteins (20 µg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes for probing overnight with antibodies specific for p38 or phosphorylated p38 MAPK, which were purchased from Cell Signaling Technology (Beverly, MA). The membranes were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase (BD Transduction Laboratory, San Jose, CA). The signal was visualized by using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Electrophoretic mobility shift assays

The oligodeoxynucleotide sequences used for different transcription factor assays are summarized in Table 3. The labeling of the oligodeoxynucleotides, the DNA–protein binding reactions and electrophoretic mobility shift assay (EMSA) were performed as described previously.²² For the supershift assay, antibodies specific for Ets-1 or normal goat serum were incubated with the nuclear extracts for 30 min on ice before the addition of radioactively labelled oligodeoxyribonucleotides.

Table 3.

Sequences of oligodeoxynucleotide probes for electrophoretic mobility shift assays

Name	Sequence
Sp-1 EMSA	5′-ATTCGATCGGGGCGGGGCGAGC-3′
AP-1 EMSA	5′-CGCTTGATGACTCAGCCGGAA-3′
Ets-1 EMSA	5′-GATCTCGAGCAGGAAGTTCGA-3′
Ets-1 mutant (EMSA)∧	5′- GATCTCGAGCAAGAAGTTCGA-3′
NF-IL-6	5′-GATCGGACGTCACATTGCACAATCTTAATAAT-3′
NF-IL-6 mutant∧	5′-GGACGTCACACTACAAACTCTTAATAA-3′
IL-10 promoter −655	5′-CTGGAACACATCCTGTGACCCCGCCTGTCCT-3′
IL-10 promoter −655 Ets-M∧	5′-CTGGAACACATAATGTGACCCCGCCTGTCCT-3′
IL-10 promoter −655 Sp-1-M∧	5′-CTGGAACACATCCTGTGACCCAGCGTGTCCT-3′

EMSA, electrophoretic mobility shift assay; IL, interleukin; NF, nuclear factor.

^∧Indicates mutant probes. Mutated nucleotides are underlined.

Chromatin immunoprecipitation assay

THP-1 cells in culture media were first treated with mock reagent or HIV Tat protein for 45 min. This was followed by fixation with 1% formaldehyde for 10 min at 37° for DNA–protein cross-linking. The cells were incubated in SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8·1) for 10 min on ice. The chromatin was obtained by sonication at power level 4 of the Branson 150 sonicator (Branson Ultrasonic, Danbury, CT) for 10 seconds for three cycles, and placed on ice between intervals. The samples were immunoprecipitated with antibodies specific for Ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or normal goat serum at 4° overnight. The control was prepared, as described above, except that no antibodies were added to the samples. The DNA–protein crosslink was reversed from the immunoprecipitated sample by incubation with 5 m NaCl for 4 hr. Furthermore, the DNA was eluted by elution buffer (1% SDS, 0·1 m NaHCO₃) for 15 min at room temperature. The DNA obtained was purified by phenol–chloroform extraction followed by ethanol precipitation. The DNA underwent PCR amplification using the primer set IL-10 promoter −85/IL-10 promoter +163 antisense, as listed in Table 1.

Results

Induction of IL-10 transcription by Tat

We initially investigated the induction levels of IL-10 mRNA by Tat in the human monocytic cell line, THP-1. To determine the optimal time for Tat protein to induce IL-10, THP-1 cells were treated with 100 ng/ml of Tat protein for 2 to 24 hr, or with mock reagent for 24 hr. The IL-10 mRNA expression was detected in the Tat- treated cells in a time-dependent manner (Fig. 1).

Figure 1.

THP-1 cells were treated with 100 ng/ml of Tat protein for different time periods. Total cellular RNA was extracted for real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis of interleukin (IL)-10. The cells without treatment (UT) or treated with mock reagent (M), as described in the Materials and methods, were used as controls (lanes 1 and 2). The results (mean ± standard deviation) have been normalized with the measurement of 18S mRNA levels. The experiment shown is representative of three independent experiments. The statistical significance of the fold induction compared with untreated/mock is indicated as follows: *P < 0·05.

Determination of IL-10 promoter activities in THP-1 cells

To define the minimal IL-10 promoter region responsive to Tat induction, a series of 5′ IL-10 firefly luciferase reporter constructs, containing progressive deletions of the IL-10 5′ flanking region, was generated. All plasmid constructs were defined in relation to the transcription start codon. The constructs were transiently cotransfected into THP-1 cells with the internal control Renilla luciferase vector (pRL-TK) for 24 hr in order to determine the transfection efficiencies. The transfected cells were incubated with or without 100 ng of recombinant Tat for another 24 hr before measuring the luciferase activities. Measurement of the firefly luciferase activities of the reporter constructs was normalized with measurement of the Renilla luciferase vector. Stimulation with Tat did not induce the luciferase activity of pGL3B alone. In contrast, the luciferase activity was increased in the Tat-treated cells when the p[−738] promoter construct was used (Fig. 2a). For the plasmid with deletion of the 5′ end to the site −625 nucleotide (nt), p[−625], the luciferase activity was similar to, but slightly lower than, that of the construct p[−738]. In sharp contrast, the luciferase activity of the construct p[−610] showed a dramatic decrease in the Tat- induced expression compared with that of the construct p[−738]. Because the luciferase activity of the construct p[−610] was 80% lower than that of the longer construct p[−625], this suggested that the region between nucleotides −625 and −610 plays an important role in regulating the Tat-induced IL-10 transcription in THP-1 cells. After this region, progressive deletion to the site −85 nts showed a minimal level of inducible luciferase activity, similar to that of the parental plasmid pGL3B (Fig. 2a).

Figure 2.

Identification of the core region of human interleukin (IL)-10 promoter. (a) THP-1 cells were transiently cotransfected with plasmids (pGL3-basic) encoding the luciferase reporter linked to a series of IL-10 promoter fragments and pRL-TK plasmid. Twenty-four hours following transfection, the cells were treated with 100 ng of Tat. The firefly luciferase activities of the construct were measured after an additional 24 hr of incubation. The activities were normalized using Renilla luciferase activity as a transfection control. The values were then normalized to the cells expressing the same construct without Tat induction. The luciferase activities shown here represent the mean ± standard deviation (obtained from three independent experiments) of the percentage of the enzyme activities in the cells transfected with the p[−738] construct. The statistical significance of the percentage difference is indicated as follows: *P < 0·05. (b) A luciferase reporter plasmid linked to the IL-10 promoter region that contained the C to A mutation at position −592 was constructed and named p[−625A] (with adenine at position −592). THP-1 cells were transfected with individual plasmids including p[−738] (IL-10 promoter region −738 to +163), p[−625] (cytosine at position −592), p[−625A] (adenine at position −592) and pGL3B (parental luciferase plasmid without IL-10 inserts). The cells were incubated for 24 hr and followed by Tat treatment (100 ng) for another 24 hr. The respective luciferase activities of the transfected cells were measured as in (a). The data presented represent the mean of three independent experiments. The statistical significance is indicated as follows: *P < 0·05. (c) THP-1 cells were transfected with IL-10 promoter-encoded plasmids, including p[−738], p[−625], p[−610], p[−85] or pGL3B. Twenty-four hours after transfection with the individual plasmids, cells were treated with lipopolysaccharide (LPS) (1 µg/ml) for another 24 hr, and luciferase activities were measured as in (a). The statistical significance of the percentage difference is indicated as follows: *P < 0·05. (d) Detailed sequences of the transcription factor-binding elements in the region −595 to −625 of the IL-10 promoter.

Previous studies showed that the single nucleotide polymorphism (SNP) at −592(C/A) correlated with the progression of AIDS. We investigated whether this point mutation has any effects on IL-10 production in Tat-induced THP-1 cells. Cells transfected with the respective IL-10 promoter-encoded plasmids (with a C or A SNP at position −592) were treated with Tat in a manner similar to that described above. The results demonstrated that there were no significant differences in the Tat-induced IL-10 promoter activity between the C/C and A/A genotypes (Fig. 2b).

We also investigated another physiological stimulus, LPS, on the IL-10 promoter activities. LPS is a cell wall component of Gram-negative bacteria. The host immune system targets this lipopolysaccharide for the recognition of bacterial infection. Cells transfected with the IL-10 plasmids were incubated with LPS (1 µg/ml) for 24 hr and then the luciferase activity was measured. As shown, the cells were responsive to LPS with significant induction of IL-10 promoter-driven luciferase activities in cells transfected with the p[−738] and p[−625] promoter constructs (Fig. 2c). These results implied that the promoter region responsible for Tat-induced IL-10 expression may have significant overlaps with the LPS-responsive elements. Taken together, the region was functional when stimulated by HIV proteins and bacterial products.

As the region −625/−610 nts was responsible for the Tat-induced IL-10 promoter activity, we investigated which transcription factor-binding elements within this region were involved. Computer-based analysis of this region using the tess program highlighted several putative binding sites for different transcription factors, including NF-IL-6, Ets-1, Sp-1 and AP-1. The binding site for AP-1, in fact, overlaps with those of Ets-1 and Sp-1 (Fig. 2d).

DNA-binding activity of the IL-10 promoter region, −625/−610 nts, in the Tat-induced THP-1 cells

To demonstrate the inclusion of functional transcription factor-binding elements in the IL-10 promoter region −625/−610 nts, EMSA of the Tat-induced cells were performed. The nuclear extracts were isolated from THP-1 cells treated with different concentrations of Tat for 30 min and then incubated with ³²P-labelled double-stranded oligodeoxynucleotides spanning the −625/−610 nt region. A major band was observed in all nuclear extracts of Tat-induced cells (Fig. 3a; DNA–protein complex). The DNA-binding activity of the −625/−610 nt region increased gradually with increasing doses of recombinant Tat in the treatment of THP-1 cells (Fig. 3a, lanes 2–4). The EMSA results also showed that the Tat-induced DNA-binding activity of −625/−610 nts was a time-dependent process (Fig. 3b). When followed over a period of 90 min, the binding activity in the Tat-treated THP-1 cells peaked at 60 min (Fig. 3b, lane 3) before declining to the basal level at 90 min (Fig. 3b, lane 4). To indicate the specificity of the binding reaction, 50-fold excess amounts of unlabelled oligodeoxynucleotides were added to the reaction mixture before addition of the radiolabelled probe. (Fig. 3a,b, lane 5).

Figure 3.

Electrophoretic mobility shift assay (EMSA) of the DNA-binding activity of the indicated interleukin (IL)-10 promoter region. (a) THP-1 cells were treated with the indicated concentrations of recombinant Tat protein for 30 min. Nuclear extracts (2 µg) were incubated with ³²P-labelled oligodeoxynucleotides for 30 min and then electrophoresed on a 5% Tris-glycine polyacrylamide gel. The addition of 50× unlabelled oligodeoxynucleotide as a competitor to the nuclear extracts from the cells treated with Tat for 30 min is indicated in lane 5. (b) Tat (100 ng) was added to the THP-1 cells for the indicated time periods. EMSA was performed as described in (a). The addition of 50× unlabelled oligodeoxynucleotide as a competitor to the nuclear extracts from the cells treated with Tat for 30 min is indicated in lane 5. (c) A band shift analysis of THP-1 cells (treated with Tat for 30 min) with the indicated competitor oligodeoxynucleotides in 50× excess. Nuclear extracts (2 µg) were incubated with ³²P-labelled oligodeoxynucleotides specific for the indicated region (nucleotides −625 to −595) for 30 min. In each set of experiments, the unlabelled consensus sequence of the indicated transcription factor was used as a competitor. The nuclear extracts were then electrophoresed on a 5% Tris-glycine polyacrylamide gel. (d) Supershift EMSA analysis was performed using the Ets-1 antibodies or normal goat serum. The Ets-1 antibodies (1 µg) or normal goat serum were incubated with the nuclear extract (2 µg) from cells treated with 100 or 200 ng of Tat for 30 min before the addition of ³²P-labelled oligodeoxynucleotides. The results presented are representative of three independent experiments. (e) Chromatin immunoprecipitation (ChIP) was performed using the Ets-1 antibodies or normal goat serum with mock-treated or Tat-treated THP-1 cell lysate. Mock reagent (lane 1) or 100 ng of Tat (lane 2) were incubated with the THP-1 cells for 45 min before protein–DNA isolation. The total cellular protein was immunoprecipitated with Ets-1 antibodies or normal goat serum. The DNA bound to the chromatin was then extracted and analysed by polymerase chain reaction (PCR). The input control represents 2% of the total lysate. The results presented are representative of three independent experiments.

Identification of Ets-1- and Sp-1-binding sites as specific inducible responsive elements

In order to identify the functional binding elements, competition experiments were carried out, where a 50-fold molar excess of unlabelled probes with the indicated nucleotide sequences were incubated with the ³²P-labelled double-stranded oligodeoxynucleotide of the −625/−610 region. These unlabelled non-radioactive probes were the consensus oligodeoxynucleotide sequences of the wild-type NF-IL-6-, Ets-1-, Sp-1-, AP-1- and NF-κB-binding sites. Our results showed that the addition of excess amounts of consensus Ets-1 or Sp-1 elements inhibited the formation of the complex (Fig. 3c, lanes 2 and 4), whereas the addition of NF-IL-6, AP-1 or NF-κB consensus elements did not show any reduction in band intensity when compared with that of the competitor sample (Fig. 3c, lanes 1, 3 and 5). These results suggest that the Ets-1 or Sp-1 sequence competes with its own respective binding element in the −625/−610 nt region for binding with the activated factors. Supershift assay using anti-Ets-1 was used to confirm further the binding of Ets-1 to the promoter region. The band shifted when anti-Ets-1 was added to the reaction mixture (Fig. 3d, Shifted Complex I), whereas the migrating band was weakened (Fig. 3d, DNA–protein complex). The EMSA experiments demonstrated that Ets-1 and Sp-1 did bind to the IL-10 promoter region −625/−610 nts, which may play a role in the transcriptional activation of IL-10. To study the binding of Ets-1 to the endogenous IL-10 promoter in greater detail, chromatin immunoprecipitation (ChIP) assays were performed. In the Tat-treated THP-1 cells, more IL-10 promoter DNA was immunoprecipitated by using anti-Ets-1 compared with the mock agent-treated cells (Fig. 3E, lane 1 versus lane 2). These results showed that Ets-1 binds to the endogenous IL-10 promoter to enhance transcription.

In attempts to confirm the regulatory role of the Ets-1- and Sp-1-binding elements in the IL-10 promoter region, EMSA and luciferase reporter assays were used. Nuclear extracts of the Tat-treated THP-1 cells were incubated with radiolabelled wild-type −625/−610 oligodeoxynucleotides, or the corresponding mutant oligodeoxynucleotides encoding mutated Ets-1- or Sp-1-binding elements (Fig. 4a). The results illustrated that there were no DNA-binding activities with mutants encoding either the Ets-1 or Sp-1 mutated binding elements (Fig. 4b, lanes 3 and 4). Additionally, luciferase assays were used to measure the transcription activities of the respective Ets-1- or Sp-1-mutated IL-10 promoter. Point mutations were introduced into the putative Ets-1 or Sp-1 elements of p[−625] by PCR cloning. When two cytosines (−614 and −613 nts) of the Ets-1 site were substituted by adenines, the luciferase activity was reduced to the basal level, as demonstrated by the pGL3B plasmid (Fig. 4c). Similar results of reduced luciferase activity were obtained in the cells transfected with the Sp-1 mutated luciferase plasmid (Fig. 4c). These findings illustrated that the Ets-1- and Sp-1-binding elements in the −625/−610 nt region were required for the IL-10 promoter activity.

Figure 4.

The regulatory role of Ets-1 and Sp-1 in the interleukin (IL)-10 promoter. (a) Nucleotides that were altered in each mutant probe are underlined. The bracketed region indicates the transcription factor-binding site. (b) Radiolabelled wild-type and mutant probes were used for electrophoretic mobility shift assay (EMSA) with 2 µg of nuclear extracts from THP-1 cells treated with Tat for 30 min. (c) THP-1 cells were transiently cotransfected with the luciferase reporter plasmids (encoding the wild type, mutated Ets-1 or mutated Sp-1 site of the IL-10 promoter fragments) and pRL-TK plasmid. Parental pGL3B was used as a plasmid control. Cells were stimulated with 100 ng of Tat for 24 hr followed by luciferase activity measurement. The activities were normalized using Renilla luciferase activity as a transfection control. The values were then normalized to the same construct without the Tat induction. Luciferase activities were shown as percentage of p[−625] ± standard deviation, on the basis of three independent experiments. The statistical significance of percentage difference is indicated as follows: *P < 0·05. (d) Activation of Ets-1 in Tat-treated THP-1 cells. THP-1 cells were treated with the indicated concentrations of recombinant Tat protein for 30 min. Nuclear extracts (2 µg) were incubated with ³²P-labelled Ets-1 consensus oligodeoxynucleotides for 30 min and then electrophoresed on a 5% Tris-glycine polyacrylamide gel. A 50× molar excess of unlabelled Ets-1 sequences was used as a control of binding specificity. The results presented are representative of three independent experiments.

To investigate the activation of Ets-1 in Tat-induced THP-1 cells, we collected the nuclear extracts for incubation with the ³²P-labelled double-stranded consensus Ets-1 oligodeoxynucleotides to perform EMSA. The results showed that Tat induced the DNA-binding activity of Ets-1 compared with that of the untreated cells (Fig. 4d, lanes 2 and 1, respectively). With the addition of a 50-fold excess of wild-type unlabelled oligodeoxynucleotides, the labelled band was not observed (Fig. 4d, lane 3), demonstrating its specificity.

Ets-1 is essential for Tat-induced IL-10 promoter activation

After confirming the binding of Ets-1 in the Tat-induced THP-1 cells, the effects of Ets-1 in regulating IL-10 promoter activity were examined. Full-length Ets-1 plasmid (pcDNA3-ets-1) was generated and transiently transfected into HeLa cells, with or without the IL-10 promoter luciferase plasmids. The immunoblots showed that the expression of Ets-1 was similar in the cotransfected cells (i.e. with the Ets-1 expression and IL-10 promoter plasmids) or in cells transfected with pcDNA3-ets-1 alone (Fig. 5a, lanes 3–5). Having confirmed the levels of Ets-1 protein in the cells, the luciferase activities of the reporter plasmids were examined. The luciferase activities of the almost full-length IL-10 promoter constructs, p[−860], p[−738] and p[−625], in the Ets-1-expressing HeLa cells, were induced eightfold more than the shorter IL-10 promoter construct, p[−85], and the parental plasmid, pGL3B (Fig. 5b). The fold of induction here has been compared to cells without exogenous Ets-1 overexpression. In order to confirm the role of Ets-1 in the regulation of IL-10 gene transcription, we measured the mRNA level of IL-10. The results showed that the IL-10 mRNA level was higher in pcDNA3-ets-1 overexpressing cells than in the control cells with pcDNA3 transfection (Fig. 5c). These results suggested that Ets-1 plays a significant role in regulating the IL-10 gene regulation.

Figure 5.

Interleukin (IL)-10 promoter activation is regulated by Ets-1. (a) HeLa cells were transiently transfected with the indicated plasmids. Total cell lysates were transferred to nitrocellulose membranes and then immunoblotted against specific Ets-1 antibodies. The experiment shown is representative of three independent experiments. (b) HeLa cells were transiently cotransfected with the pcDNA3-Ets-1 and the indicated IL-10 promoter luciferase reporter plasmid. The luciferase activities were measured 24 hr post-transfection and normalized using Renilla luciferase activity as a transfection control. Luciferase activities were shown as the mean of induction ± standard deviation (SD), on the basis of three independent experiments. The statistical significance of the fold induction compared with pGL3B is indicated as follows: *P < 0·05. (c) HeLa cells were transiently transfected with the indicated plasmids. Total cellular RNA was extracted 24 hr post-transfection for reverse transcription–polymerase chain reaction (RT–PCR) analysis of IL-10 transcription. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RT–PCR was performed as an internal loading control of RNA. The results presented are representative of three independent experiments. (d) Activation of p38 mitogen-activated protein kinase (MAPK) in Tat-treated THP-1 cells. THP-1 cells were incubated with different concentrations of Tat protein for 60 min. Twenty micrograms of cytoplasmic protein were collected for the immunoblot assays with anti-p38 or anti-phosphorylated p38 MAPK immunoglobulin. The results shown are representative of three independent experiments. (e) Effects of kinase inhibitor on the Tat-induced IL-10 promoter activities. THP-1 cells were transiently cotransfected with p[−738] luciferase reporter plasmid and pRL-TK plasmid. Cells were incubated for 1 hr with 1 µm SB203580 (SB) before the addition of 100 ng of Tat for 24 hr. The activity was normalized using Renilla luciferase activity as a transfection control. The values were then compared to cells transfected with the same construct, but without subsequent Tat treatment. Luciferase activities are presented as mean percentage differences compared with Tat-treated cells (± SD) on the basis of three independent experiments. The statistical significance of the percentage differences compared with Tat-only treated cells are indicated as follows: *P < 0·05. (f) Effects of kinase inhibitor on the IL-10 promoter activities in Ets-1 overexpressing HeLa cells. HeLa cells were transiently transfected with the indicated expression plasmids with the pcDNA3-Ets-1, p[−738] luciferase reporter plasmid and the pRL-TK plasmid. Cells were incubated with 1 µm SB before the 24-hr transfection. The luciferase activities were measured after 24 hr of transfection and normalized to Renilla luciferase activity, which was used as a transfection control. Luciferase activities are presented as percentage differences compared with Ets-1 overexpressing cells (± SD) on the basis of three independent experiments. The statistical significance of the percentage difference compared with cells expressing Ets-1 only is indicated as follows: *P < 0·05.

The role of p38 MAPK in regulating IL-10 transcription

We have recently reported that Tat activates p38 MAPK to induce IL-10 in primary blood monocytes.²² Because the −625/−610 nt region of the IL-10 promoter may play a regulatory role in the Tat-induced IL-10 transcription, we investigated whether p38 MAPK is involved in regulating the Tat-induced cellular events at these sites. For studying the role of p38 MAPK, we measured the levels of activated p38 MAPK in Tat-treated THP-1 cells. The THP-1 cells were incubated with different concentrations of Tat for 60 min, followed by protein isolation. The cytoplasmic portion of the Tat-treated cells was separated by SDS–PAGE and transferred to nitrocellulose membranes. The membranes were probed with antibodies specific for p38 or the phosphorylated form of p38 MAPK, respectively. The results demonstrated that Tat induced phosphorylation of the p38 MAPK in a dose-dependent manner (Fig. 5d). In another set of experiments, cells were transiently transfected with the indicated IL-10 promoter-linked luciferase reporter p[−738] for 24 hr, and then incubated, with or without SB203580 (SB), for 1 hr before the addition of Tat. SB has been well documented to be the small-molecule kinase inhibitor for p38 MAPK. The level of cellular toxicity is reported in the literature.²² We have also tested and confirmed the level of cellular toxicity at this concentration. The results showed that pretreatment of the cells with SB decreased the luciferase activity induced by Tat (Fig. 5e). The concentrations of SB used have been well documented to be non-toxic to the cells in previous studies.²³ Other kinase inhibitors, including PD98059 and SP600125 [for extracellular signal-regulated kinase-1/2 (ERK1/2) and Jun N-terminal kinase, respectively], had no effects on the Tat-induced events (results not shown). These results suggest that p38 MAPK is involved in regulating the activity of the IL-10 promoter.

We further examined the effects of p38 MAPK in the Ets-1 regulated IL-10 promoter activity. HeLa cells transfected with pcDNA3-ets-1 and p[−738] promoter plasmids were treated with SB for 24 hr. The luciferase activities in the cells incubated with SB were reduced by 45% compared with that of the cells without the inhibitor treatment (Fig. 5f). Taken together, these results indicated that p38 MAPK plays a significant role in modulating the Ets-1-regulated IL-10 promoter activity.

Discussion

In this study, we identified the promoter region of the human IL-10 gene to be responsive to HIV-1 Tat induction. The responsive element was located between −625 and −610 nts upstream of the IL-10 transcription start codon. By using computer analysis, several putative transcription factor-binding sites were initially suggested. By using EMSA of the nuclear extract, and assays for luciferase activities of the IL-10 promoter, we demonstrated that the Ets-1- and Sp-1-binding sites within the −625 and the −610 regions were responsible for regulating the IL-10 promoter activity in response to Tat activation. We further examined the signalling pathways leading to IL-10 gene transcription and demonstrated that p38 MAPK is responsible for mediating the Tat-induced IL-10 gene transcription.

As the first line of defence to foreign antigens or pathogens, the response of monocytes and macrophages must be sufficiently expedient to generate intercellular signals to mobilize multiple branches of the immune system. Secretion of cytokines by monocytes and macrophages is ideal for rapid deployment of immune activation. However, such efficient recruitment of the immune system can be exploited by highly adaptable viruses, such as HIV. This retrovirus has been successful in evading immunity via the activity of its proteins, including Tat. The HIV-1 Tat protein is one of the regulatory proteins produced during the early phase of HIV-1 infection. It is secreted from the infected cell into the circulatory system. In culture supernatants of HIV-infected cells, ≈ 0·1–1 ng/ml of Tat is produced. This concentration is similar to the level of cytokines or chemokines produced in stimulated or infected cells. Additionally, the levels of Tat in the patient's sera appear to be proportional to the viral titres found in the circulation. Thus, Tat production has been postulated to be related to the progression of AIDS.²⁴

Different doses of extracellular Tat protein (10 ng/ml to 1 µg/ml) have been shown to induce cytokines in monocytes and macrophages in previous studies, including ours.^6,22,25–30 The mechanisms of the transcriptional activation of IL-6 were examined and demonstrated by chloramphenicol acetyl transferase (CAT) assay and EMSA.²⁶ Our research group also performed experiments, using recombinant Tat immobilized on culture well, as a model to examine the extracellular effects of Tat on immune cells. The results showed that immobilized Tat induced the expression of IL-6, IL-10 and TNF-α (data not shown). In the present study, we further investigated the molecular mechanisms of the Tat-induced IL-10 transcription using the monocytic THP-1 cell line.

In order to understand the components for transcriptional activation, transcription factor-binding elements in the IL-10 promoter were analysed. The results showed that several different binding elements, including Sp-1, AP-1, Stats, NF-κB, NF-IL-6 and Ets-1, are located in the promoter region. In previous reports, Sp-1 has been shown to play an important role in the LPS-induced IL-10 transcription in both human and mouse cell models.^13,31 Other transcription factor-binding elements, including CREB/ATF and C/EBP, have also been suggested to be of importance in tissue-specific and differentiation-dependent IL-10 transcription.³² These reports suggested that the regulatory mechanism of IL-10 transcription is cell type- and stimulant-specific.

In our study, on Tat-induced IL-10 transcription, the transcription factors involved in the regulation of IL-10 were investigated. It is possible that Ets-1, Sp-1 and AP-1 are the regulatory transcription factors responsible for Tat effects because their binding regions are situated between −625 and −595 nts. From the EMSA results, it is surprising that the AP-1 consensus oligodeoxynucleotide cannot abrogate the DNA-binding activity of the −625/−595 region, because the AP-1-binding site is situated from −612 to −601 nts, overlapping with the responsive region (Fig. 3c). It is possible that the binding sequence of AP-1 in the IL-10 promoter has three nucleotides different from that of the consensus sequences of the AP-1-binding site. The binding affinity of the IL-10 sequence may not be as strong as that of the consensus AP-1 sequence. An alternative mechanism of action could be that the IL-10 promoter site is indeed occupied by AP1 family members that are repressors of transcription. Tat stimulation would lead to both removal of AP1-related factors from the promoter and replacement with Sp1 and Ets-1 for initiation of transcription. The exact mechanisms of how AP-1 mediates Tat-induced IL-10 expression still have to be elucidated.

In the −625/−595 nt region, the Ets-1-binding element is adjacent to the Sp-1 element (Fig. 2b). This provides an insight into the importance of the co-operation between Ets-1 and Sp-1 for the activity of the −625/−595 nt region. By eliminating either one of the binding elements, the activity of the region was abrogated (Fig. 4c). These results further illustrate that activation of the IL-10 promoter is coregulated by Ets-1 and Sp-1. In previous studies, the activation of Sp-1 by LPS in regulating IL-10 gene transcription has been illustrated.¹³ Our study also showed that Sp-1 was involved in the Tat-induced IL-10 gene transcription.

Here, we showed that Tat could also induce the DNA-binding activity of the transcription factor, Ets-1. We further showed that Ets-1 has the ability to mediate the Tat-induced IL-10 transcription by using the Ets-1 overexpression effects on IL-10 promoter activity. It is known that the activation of Ets-1 can be regulated by several pathways. The first is the activity of calmodulin-dependent kinase II (CaMKII), which inhibits Ets-1 transcriptional activity.^33,34 The other pathway involves the myosin light-chain kinase (MLCK), which represses the Ets-1 transcription activity by phosphorylation of the exon VII domain. An additional way to mediate the activity of Ets-1 is by activating its protein activity. Protein kinase C-α (PKCα) is one of the activators of Ets-1. The activation of Ets-1 by PKCα is also targeted at the exon VII domain, but it is not mediated by calcium. In contrast, the activation of CaMKII and MLCK is a calcium-dependent process.^35,36 Another activation mechanism is through the phosphorylation of threonine-38 of Ets-1. MAPKs, including ERK1 and ERK2, together with Ras, are responsible for mediating the activation of threonine-38. This cascade of activation is observed in the Ras-responsive genes that are known to have several Ets-1/AP1-binding elements.^37,38

In regulating gene transcription, the binding of the activated transcription factors to their respective cognate DNA-binding elements plays an important role. With the mutation on the binding elements, the activity of the promoter could be diminished. This postulate can be substantiated by several recently identified SNPs in the human IL-10 promoter region. The three common SNPs are located at the −1082(G/A), −819(C/T) and −592(C/A) positions of the promoter. By different combinations of these SNPs, the levels of IL-10 production can be altered.^39–41 There is a strong correlation of the high level of IL-10 production with the SNP at −592(C/C).¹¹ Patients with AIDS-associated diseases, such as non-Hodgkin's B-cell lymphoma, have been shown to have the high IL-10-expressing (−592 C/C) genotype. Also, patients with another SNP (−592A/A or C/A) were found to have rapid development of AIDS. According to our results, the IL-10 promoter activity of the −592 (A/A) genotype is similar to that of the −592 (C/C) counterpart in the Tat-treated cells. This implies that the dysregulation of IL-10 production in different genotypes may not be related to the effects of Tat.

In this project, we showed that p38 MAPK may play an important role in regulating the Tat-induced IL-10 promoter activation. The synthesis of IL-10 in Tat-induced monocytes and macrophages has already been proposed.⁶ In previous studies, the induction of IL-10 by Tat occurs through the activation of PKC isozymes β_II and δ in monocytes. Together with our results, it appears that Tat activates different pathways to up-regulate the production of IL-10. This diverse activation system is beneficial for replication of the virus and its consequent effects on disease progression. Our results provided an in-depth analysis of the transcription factors and the role of enhancer sites involved in the IL-10 promoter. Here, the role of Ets-1 in regulating the promoter activity was delineated. By using a p38 MAPK inhibitor, the IL-10 promoter activities were reduced despite an overexpression of Ets-1 (Fig. 5f) These results open an intriguing question as to whether p38 MAPK plays a general role in controlling the Ets-1-regulated activities in other signalling pathways.

The primary function of Tat is to induce several cellular and viral genes by modulating the activation of transcription factors.^42,43 The resultant activation or repression of these genes occurs via the interactions of Tat with these transcription factors. Previous reports showed that Tat induces the activation of NF-κB, NF-IL-6 and Sp-1.^26,44,45 Here, we showed that Tat induces the activity of Ets-1, which is known to serve as a co-operator with other transcription factors. This would provide an added advantage for Tat to induce several pathways of cellular processes, including viral replication and cytokine induction. The findings here described differential signalling mechanisms for Tat to modulate its pleiotropic effects.

In conclusion, we have delineated the molecular mechanisms of HIV-1 Tat-induced IL-10 gene transcription. This activation is mediated by p38 MAPK, resulting in the regulation of Ets-1 and Sp-1 activation. We also showed that HIV-1 Tat induces the p38 MAPK-mediated activation of Ets-1, which may be important for the differential signalling cascades.

Abbreviations

AIDS

acquired immune-deficiency syndrome

BSA

bovine serum albumin

CaMKII

calmodulin-dependent kinase II

ChIP

chromatin immunoprecipitation

DTT

dithiothreitol

EMSA

electrophoretic mobility shift assay

ERK-1/2

extracellular signal-regulated kinase-1/2

GM-CSF

granulocyte–macrophage colony-stimulating factor

HIV

human immunodeficiency virus

IFN-γ

interferon-γ

IL

interleukin

IRF-1

IFN regulatory factor 1

LPS

lipopolysaccharide

LTR

long terminal report

MAPK

mitogen-activated protein kinase

MLCK

myosin light-chain kinase

NF-κB

nuclear factor-κB

nt

nucleotide

nts

nucleotides

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

PKCα

protein kinase C-α

RT–PCR

reverse transcription–polymerase chain reaction

SB

SB203580

SDS–PAGE

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

SNP

single nucleotide polymorphism

Th1

T helper 1

TNF-α

tumour necrosis factor-α