The p38 Mitogen-activated Kinase Pathway Regulates the HumanInterleukin-10 Promoter via the Activation of Sp1 TranscriptionFactor in Lipopolysaccharide-stimulated Human Macrophages*

Received for publication, December 12, 2000, and in revised form, January 24, 2001Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M011157200

Wei Ma‡§, Wilfred Lim¶, Katrina Gee¶i, Susan Aucoin‡, Devki Nandan**, Maya Kozlowski‡‡,Francisco Diaz-Mitoma‡¶§§, and Ashok Kumar‡¶§§¶¶

From the Departments of §§Pediatrics, and ¶Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa,Ontario K1H 8L1, Canada, the ‡Division of Virology and Molecular Immunology, Research Institute, Children’s Hospitalof Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, the **Division of Infectious Diseases, Department of Medicine,Vancouver General Hospital, University of British Columbia, Vancouver, British Columbia V5Z 3J5, Canada, and‡‡Health Canada, Therapeutic Products Programme, Research Services Division, Ottawa, Ontario K1A 0L2, Canada

Interleukin-10 (IL-10), a pleiotropic cytokine that in-hibits inflammatory and cell-mediated immune re-sponses, is produced by a wide variety of cell types in-cluding T and B cells and monocytes/macrophages.Regulation of pro- and anti-inflammatory cytokines hasbeen suggested to involve distinct signaling pathways.In this study, we investigated the regulation of the hu-man IL-10 (hIL-10) promoter in the human monocyticcell line THP-1 following activation with lipopolysac-charide (LPS). Analysis of hIL-10 promoter sequencesrevealed that DNA sequences located between basepairs 2652 and 2571 are necessary for IL-10 transcrip-tion. A computer analysis of the promoter sequence be-tween base pairs 2652 and 2571 revealed the existenceof consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr virus-specific nuclear antigen-2 (EBNA-2)-liketranscription factors. THP-1 cells transfected with a plas-mid containing mutant Sp1 abrogated the promoter activ-ity, whereas plasmids containing the sequences for PEA1,YY1, and EBNA-2-like transcription factors did not influ-ence hIL-10 promoter activity. To understand the eventsupstream of Sp1 activation, we investigated the role ofp38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibi-tors. SB202190 and SB203580, the p38-specific inhibitors,inhibited LPS-induced IL-10 production. In contrast,PD98059, a specific inhibitor of extracellular signal-regu-lated kinase kinases, failed to modulate IL-10 production.Furthermore, SB203580 inhibited LPS-induced activationof Sp1, as well as the promoter activity in cells transfectedwith a plasmid containing the Sp1 consensus sequence.These results suggest that p38 mitogen-activated proteinkinase regulates LPS-induced activation of Sp1, which inturn regulates transcription of the hIL-10 gene.

An appropriate balance between pro- and anti-inflammatoryinfluences in the immune response is critical in the resolutionof many pathological conditions. Interleukin-10 (IL-10),1 a cy-tokine that inhibits inflammatory and cell-mediated immuneresponses (1), has enormous potential for the treatment ofinflammatory and autoimmune disorders. Human IL-10 (hIL-10), a nonglycosylated 178-amino acid polypeptide, is encodedby a gene located on chromosome 1q and has more than 73%amino acid sequence homology with murine IL-10 (mIL-10)(1–3). IL-10 is a pleiotropic molecule that is produced by a widevariety of cell types, including CD41 Th0 and Th2 cells, CD81

T cells (4), B cells (1, 5, 6), and monocytes/macrophages (7). Themajor biological effects of IL-10 include inhibition of antigen-presenting cell-dependent cytokine synthesis by Th1 cells, co-stimulation of mast cell growth, and costimulation of thymo-cyte growth in the presence of IL-2 and/or IL-4 (1, 8). IL-10inhibits antigen-driven activity of both Th1 and Th2 subsets (1,4, 8), although it facilitates the induction of Th2 cell types.IL-10 exhibits stimulatory effects on B cell growth and differ-entiation (6, 9, 10) and acts as an autocrine growth factor forLy-11 B cells, which are important in murine models of auto-immune disease (11).

The potent action of IL-10 on macrophages, particularly atthe level of monokine production (1, 7, 8), supports an impor-tant role for IL-10 not only in the regulation of T cell responses(1, 4, 8) but also in acute inflammatory and autoimmune re-sponses (12–14). In mice, IL-10 administration has been shownto inhibit a number of immunological effects, including delayedtype hypersensitivity, alterations in vascular permeability, andincreases in footpad cytokine production (15). Conversely,IL-10 transgenic mice were shown to be unable to limit thegrowth of immunogenic tumor cells (16). In contrast, IL-10knockout mice demonstrate a state of chronic inflammation(12), severe disease in experimental allergic encephalomyelitis(13), and bronchopulmonary aspergillosis (14), suggesting thatIL-10 plays a beneficial role in controlling the harmful inflam-matory response in these conditions. In humans, high levels ofIL-10 have been shown to be produced in patients with HIVinfection (17–19) and in septic shock (20). There is also evi-dence to suggest that polymorphism in the hIL-10 promoter

* This work was supported by grants from the Ministry of Health,Ontario, Canada, the Research Institute, Children’s Hospital of EasternOntario, and the Canadian Foundation for AIDS Research (A. K.). Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

§ Supported by the Ontario HIV Treatment Network.i Supported by a fellowship from the Medical Research Council of

Canada and the Strategic Areas of Development from the University ofOttawa, Ottawa, Ontario.

¶¶ To whom correspondence should be addressed: Division of Virol-ogy, Research Institute, Children’s Hospital of Eastern Ontario, Uni-versity of Ottawa, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada.Tel.: 613-738-3920; Fax: 613-738-4819; E-mail address: akumar@med.uottawa.ca.

1 The abbreviations used are: IL, interleukin; hIL, human interleu-kin; mIL, murine interleukin; EBNA-2, Epstein-Barr virus-specific nu-clear antigen-2; ERK, extracellular signal-regulated kinase; JNK, c-JunN terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activatedprotein; MAPK, MAP kinase; TNF, tumor necrosis factor; bp, basepair(s); PCR, polymerase chain reaction; TLR, Toll-like receptor(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 17, Issue of April 27, pp. 13664–13674, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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region is associated with altered IL-10 expression in autoim-mune diseases including multiple sclerosis, rheumatoid arthri-tis, and systemic lupus erythematosus (21).

The molecular mechanisms underlying the regulation of cy-tokine synthesis in mononuclear phagocytes are not fullyknown. LPS is perhaps the best characterized monocytic mito-gen, which, following interaction with its receptor CD14, in-duces first proinflammatory (IL-1, TNF-a, etc.) and then anti-inflammatory (IL-10, sTNF-R, and IL-1R antagonist) cytokines(7, 22). LPS-induced cell signaling is known to activate protein-tyrosine kinases (23) and the MAP kinases p38, p42/44 extra-cellular signal-regulated kinase (ERK), and p54 (stress-activat-ed protein kinase/c-Jun N-terminal kinase (JNK)) (24–27). Arecent report has indicated that IL-10 production is dependenton protein-tyrosine kinases and protein kinase C activation ina murine cell line (28). In addition, factors that elevate cAMPhave been suggested to be involved in the regulation of mono-cytic IL-10 synthesis, primarily at the mRNA level (29, 30).Recently, it has been suggested that p38 MAPK is involved inthe regulation of IL-10 production (31).

Regulation of gene expression for several pro- and anti-in-flammatory cytokines has been studied. Transcription factorsincluding Rel, C/RBP, AP-1, and NF-kB have been implicatedin the regulation of proinflammatory cytokine genes (32–37).However, very little is known about the regulation of thehIL-10 gene and the involvement of MAP kinases in this proc-ess. To gain insight into hIL-10 gene regulation, we have em-ployed promonocytic THP-1 cells that produce IL-10, IL-12, andTNF-a following LPS stimulation as do normal human mono-cytes. Using mutagenesis, we analyzed the promoter sequenceof the hIL-10 gene, and we present evidence that an elementlocated at 2650 bp, encompassing a Sp1 consensus sequence isinvolved in the transcription of the hIL-10 gene. This wasfurther demonstrated by introducing a mutation in the Sp1consensus sequence that abrogated IL-10 promoter activity.Furthermore, the Sp1 transcription factor is induced by LPSstimulation and is selectively regulated by the p38 MAPkinase.

EXPERIMENTAL PROCEDURES

Cell Lines, Cell Culture, and Reagents—THP-1, a promonocytic cellline, was obtained from the American Type Culture Collection (ATCC;Manassas, VA). 5–15% of these cells express CD14 on their surface.THP-1 cells transfected with a plasmid containing CD14 cDNA se-quences (THP-1/CD14) were kindly provided by Dr. Richard Ulevitch(The Scripps Research Institute, La Jolla, CA) (Fig. 1). Cells werecultured in Iscove’s modified Dulbecco’s medium (Sigma) supplementedwith 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/mlgentamicin, 10 mM HEPES, and 2 mM glutamine. PD98059 (Calbio-chem), an inhibitor of mitogen-activated protein kinase/extracellularsignal-regulated kinase kinase-1 kinase, selectively blocks the activityof ERK MAP kinase and has no effect on the activity of other serinethreonine protein kinases including Raf-1, p38, and JNK MAP kinases,protein kinase C, and protein kinase A (24, 38). The pyridinyl imida-zoles SB202190 and SB203580 (Calbiochem), potent inhibitors of p38and p38b MAP kinases, have no significant effect on the activity of theERK or JNK MAP kinase subgroups (24, 39). LPS was purchased fromSigma.

Cell Stimulation, Collection of Culture Supernatants, and Measure-ment of IL-10 by ELISA—THP-1 cells were cultured at concentrationsof 0.5 3 106 cells/ml in 24-well culture plates (Falcon, Becton-Dickinson,Franklin Lakes, NJ). Cells were left unstimulated or were treated for48 h with various agents for different periods of time, following whichthey were stimulated with 1 mg/ml LPS. The supernatants were frozenat 270 °C and thawed at the time of analysis. IL-10 levels were measuredby enzyme-linked immunosorbent assay by using two different mono-clonal antibodies that recognize distinct epitopes, as described (17, 40).

RNA Isolation and Semiquantitative Reverse Transcriptase-basedPolymerase Chain Reaction (PCR) for IL-10—Total RNA was extractedas described (40) using a monophase solution containing guanidinethiocyanate and phenol (Tri Reagent solution; Molecular Research Cen-

ter, Inc., Cincinnati, OH). Total RNA (1 mg) was reverse transcribed byusing Moloney murine leukemia virus reverse transcriptase(PerkinElmer Life Sciences). Equal aliquots (5 ml) of cDNA equivalentto 100 ng of RNA were subsequently amplified for IL-10 and b-actin.The oligonucleotide primer sequences for IL-10 and b-actin (Stratagene,La Jolla, CA) are as follows: IL-10 sense (59-GCC TAA CAT GCT TCGAGA TC-39); IL-10 antisense (59-TGA TGT CTG GGT CTT GGT TC-39);b-actin sense (59-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-39); b-actin antisense (59-CTA GAA GCA TTT GCG GTG GAC GATGGA GGG-39). The amplification conditions for IL-10 and b-actin havebeen described (40). The amplified products IL-10 (204 bp) and b-actin(610 bp) were resolved by electrophoresis on 1.2% agarose gels andvisualized by ethidium bromide staining.

Flow Cytometric Analysis—Cells were subjected to flow cytometricanalysis as described (17, 40). Briefly, cells were stained with 3 ml offluorescein isothiocyanate-labeled anti-CD14 monoclonal antibodies(Becton Dickinson) along with isotype (IgG2b)-matched control antibod-ies (Becton Dickinson). The gates were set in accordance with gatesobtained with the isotype-matched control antibodies. Population datawere acquired on a Becton Dickinson FACScan flow cytometer, andfigures were generated using the WinMDI software package (J. Trotter,Scripps Institute, San Diego, CA).

Gel Mobility Shift Assays—Cells (107) were harvested in Tris-EDTA-saline (TES) buffer, pH 7.8, and centrifuged at 200 3 g for 5 min. Thecells were lysed for 10 min at 4 °C with buffer A (10 mM HEPES, 10 mM

KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, pH 7.9) containing 0.1% Nonidet P-40. The lysates were cen-trifuged at 20,000 3 g for 10 min. The pellet containing the nuclei wassuspended in buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2mM EDTA, and 25% glycerol) at 4 °C for 15 min. The supernatantcontaining the nuclear proteins was collected and frozen at 280 °C.Both buffers A and B contained the proteolytic inhibitors dithiothreitol,phenylmethylsulfonyl fluoride, and spermidine at 0.5 mM as well as0.15 mM spermine and 5 mg/ml each of aprotinin, leupeptin, and pep-statin. Nuclear proteins (5 mg) were mixed for 20 min at room temper-ature with 32P-labeled oligonucleotide probe Sp1, and the complexeswere subjected to nondenaturing 17% polyacrylamide gel electrophore-sis for 90 min. The gel was dried and exposed to x-ray film. Theoligonucleotide sequences for Sp1 are as follows: 59-d(ATT CGA TCGGGG CGG GGC GAG C)-39 and 39-d(TAA GCT AGC CCC GCC CCGCTC G)-59 (Promega Corp., Madison, WI).

Construction of Luciferase Reporter Gene Vectors—A series of hIL-10promoter fragments (see Fig. 3; fragment 2890 to 1120; GenBankTM

accession number X78437) were amplified from genomic DNA by PCR.The primers with restriction sites used to amplify the hIL-10 promoterfragments from genomic DNA are shown in Table I. The amplificationconsisted of denaturation at 95 °C for 2 min, 30 cycles of denaturationat 95 °C for 30 s, annealing at 58 °C for 1 min and extension at 72 °C for2 min, and final elongation at 72 °C for 10 min. The amplified promoterproducts were subcloned into the PCRII-TOPO vector, and the se-quences were confirmed. The correct insertions were subcloned into theXhoI polylinker site of pGL3B, the basic luciferase reporter plasmid,and sequences were confirmed again. All DNA sequencing was per-formed by the Biotechnology Research Institute (University of Ottawa).A site-directed mutation of the Sp1-binding sequence (cccgcc) was gen-erated by PCR using mutagenic primers (Table I) to substitute cytosinewith guanine at 2631 and cytosine with adenine at 2636 (see Fig. 6A).The fragment containing the Sp1 mutation (2650 to 1120 bp) wasinserted into the pGL3B reporter vector.

Transient Transfection of Cells and Measurement of Luciferase Ac-tivity—Transfection of THP-1 and CD14-transfected THP-1 (THP-1/CD14) cells with plasmids containing various IL-10 promoter fragmentswas performed using LipofectAMINE Reagent (Life Technologies, Inc.)following the manufacturer’s instructions. 10 mg of the test plasmid and5 mg of pSV-b-galactosidase internal control vector (Promega) wereincubated for 45 min with 10 ml of LipofectAMINE reagent in 200 ml ofOPTI-MEM I Reduced Serum Medium (Life Technologies) to allowformation of DNA-liposome complexes. These complexes were added tothe cell suspension in each well, and cells were cultured for 24 h.Following incubation, cells were stimulated with 1 mg/ml LPS and werecultured for another 24 h. Cells were harvested and then assayed forluciferase and b-galactosidase activity by using a luciferase assay kitand b-galactosidase assay kit purchased from Promega in a Bio Orbit1250 Luminometer (Fisher).

Immunoprecipitation and Western Blot Analysis—Cell pellets werelysed 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 (2 mg)

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were precleared with protein A-Sepharose 4B beads (Amersham Phar-macia Biotech) for 1 h at 4 °C followed by incubation for 2 h at 4 °C withprotein A-Sepharose beads and antibodies as indicated in the figurelegends. Anti-p38 and anti-p42/44 rabbit polyclonal antibodies werepurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Im-mune complexes were washed three times with the lysis buffer, boiledfor 5 min in SDS-polyacrylamide gel electrophoresis buffer, and sub-jected to electrophoresis on 8% polyacrylamide SDS gels. Proteins weretransferred to ImmobilonTM-P membranes (Millipore Corp., Bedford,MA), and the membranes were probed for phosphorylated p38 andp42/44 proteins using anti-phosphotyrosine 4G10 (UBL) antibodies.The immunoblots were developed by ECL (Amersham Pharmacia Bio-tech) as per the manufacturer’s instructions.

Statistical Analysis—All transfection studies were performed in trip-licate dishes in 3–5 separate experiments. The results are expressed asmean 6 S.D.

RESULTS

THP-1 Cells Produce IL-10 in Response to LPS—To under-stand LPS-induced IL-10 regulation in human monocytes, weemployed two promonocytic cell lines, THP-1 and THP-1/CD14.CD14 was found to be expressed on ;10–15% of THP-1 cells,and this number increased to more than 50% after stimulationwith LPS (Fig. 1). Since production of cytokines by LPS-stim-ulated monocytic cells lines is critically dependent on the levelof CD14 expression, we employed THP-1 cells transfected withCD14 (THP-1/CD14) for analysis of the regulation of IL-10production. All THP-1/CD14 cells constitutively expressed veryhigh levels of CD14 on their surfaces compared with untrans-fected cells (15%) (Fig. 1). Stimulation of THP-1/CD14 cells

FIG. 1. Flow cytometric analysis ofCD14 expression on THP-1 andTHP-1/CD14 cells. THP-1 and THP-1/CD14 cells were stimulated with LPS (1mg/ml) for 24 h and analyzed by flow cy-tometry for CD14 surface expression.

TABLE IPrimers for amplification of IL-10 promoter fragments and sizes of the PCR products generated from genomic DNA

Primer name Primer sequence Regionamplified

Productlength

bp bp

Sense primersIL-10 PromGR 59-TTACTCGAGGAATGAGAACCCACAGCTG-39 2384/1120 504IL-10 PromcA 59-TTCCTCGAGGGCAATTTGTCCACGTC-39 2432/1120 552IL-10 PromNF 59-ATCCTCGAGGAAGTCTTGGGTATTC-39 2490/1120 610IL-10 PromGM 59-AACCTCGAGGGATATTTAGCCCAC-39 2571/1120 691IL-10 PromEB 59-ATCCTCGAGGCTGCTTGGGAACTTTGAG-39 2589/1120 709IL-10 PromSpM 59-ACACATCCTGTGACCCAGCGTGTCCTGTAGGAAGC-39 (Sp1 mutant) 2650/1120 770IL-10 PromSp 59-ATTCTCGAGGAACACATCCTGTGACC-39 2652/1120 772IL-10 PromST 59-AACCTCGAGCAGCAAGTGCAGACTAC-39 2768/1120 888IL-10 PromYY 59-AGCTCGAGAGTTGGCACTGGTGTACC-39 2890/1120 1100

Antisense primer 59-ACTTCGAAGTTAGGCAGGTTGCCTG-39

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with LPS induced IL-10 expression as determined by enzyme-linked immunosorbent assay (Fig. 2A) and reverse tran-scriptase-based PCR analysis (Fig. 2B, C). Maximal levels ofIL-10 mRNA were detected within 4 h after stimulation ofTHP-1 (Fig. 2B) and THP-1/CD14 (Fig. 2C) cells. Production ofIL-10 protein was elevated in THP-1/CD14 cells compared withTHP-1 cells, an observation that correlated with CD14 surfaceexpression. Production of IL-10 was dependent on the concen-tration of LPS used for cell stimulation, and 1 mg of LPSproduced maximal levels of IL-10 (data not shown).

Determination of DNA Sequences in the IL-10 Promoter Re-gion Required for IL-10 Transcription—Inducible genes, in-cluding cytokine genes, contain DNA sequences within theirpromoter region that are responsible for regulating transcrip-tion. The human IL-10 promoter was recently cloned and char-acterized (41). To understand the regulation of IL-10 genetranscription in LPS-stimulated THP-1 cells, we used PCR toclone the hIL-10 promoter fragment encompassing nucleotideresidues from 2890 to 1120 bp relative to the 11 transcriptionsite (Fig. 3). The amplified promoter fragment was subclonedinto the XhoI polylinker site of the luciferase reporter plasmid,pGL3B. THP-1/CD14 cells were transiently transfected with

the IL-10-promoter/luciferase reporter construct (pIL-10Pr-GL3B). After 24 h of transfection, the cells were stimulatedwith LPS for varying periods of time ranging from 6 to 36 h,following which relative luciferase activity was assessed. Theresults show that luciferase activity could be detected by 12 hand peaked at 24 h following stimulation with LPS (Fig. 4A).The maximum increase in luciferase activity ranged from 6- to8-fold relative to the unstimulated cells. The cells transfectedwith the promoterless plasmid pGL3B did not show any in-crease in luciferase activity following stimulation with LPS(Fig. 4B). Similar results were obtained for THP-1 cells, al-though the increase in luciferase activity was relatively lowerthan for the THP-1/CD14 cells transfected with the pGL3Bcontaining the IL-10 promoter (Fig. 4B).

To determine the DNA sequences in the hIL-10 promoterthat are required for IL-10 transcription, a series of hIL-10promoter fragments (from 59 2890 to 39 1120 bp relative to the11 transcription site of the hIL-10 gene) were produced bygenerating successive deletions starting from the 59-end. Var-ious hIL-10 promoter fragments were amplified from thehIL-10 promoter region, sequenced, and inserted into the lucif-erase expression plasmid (pGL3B). The exact size of the am-plified product and the location of consensus sequences forvarious transcription factors identified within the hIL-10 pro-moter (Fig. 3) are depicted in Fig. 5 (left panel). Examination ofthe DNA sequences within the hIL-10 promoter region contain-ing various deletions revealed that deletion of sequences from2890 to 2652 bp had no effect on luciferase activity comparedwith the plasmid containing the complete promoter sequence.However, deletion of sequences from 2571 bp and beyond com-pletely abrogated luciferase activity compared with cells trans-formed with unmutagenized promoter sequences. Further-more, the luciferase activities of these constructs wascomparable with the activity observed in unstimulated cellsand in cells transfected with the control plasmid (pGL3B) (Fig.5). Similar results were obtained for both THP-1 (Fig. 5, middlepanel) and THP-1/CD14 (Fig. 5, right panel) cells. The resultsshown are a mean of four experiments performed with each ofthe THP-1 and THP-1/CD14 cells transfected with the hIL-10promoter constructs containing 59-deletions. These results sug-gest that DNA sequences located between 2571 and 2652 bprelative to the 11 transcription site are necessary for hIL-10transcription in THP-1 and THP-1/CD14 cells following LPSstimulation.

Sp1 Binding Site in the hIL-10 Promoter Is Sufficient toInduce IL-10 Production—A computer-aided analysis of thehIL-10 promoter sequence between 2652 and 2571 bp revealedthe existence of consensus sequences for four transcriptionfactors, including Sp1 (59-cccgc-39 at 2636 to 2631 bp), PEA1(59-aggaag-39 at 2622 to 2617 bp), YY1 (59-aaaatggaa-39 at2600 to 2592), and EBNA-2-like factor (59-cttgggaactt-39 at2585 to 2575) (Fig. 3). This suggests that any one or more ofthe above mentioned transcription factors may be involved inregulating transcription of the hIL-10 gene. To investigate therole of the Sp1 transcription factor in hIL-10 gene transcrip-tion, we used PCR to introduce site-directed mutations in theSp1 consensus sequence by substituting cytosine with guanineat position 2631 bp and cytosine with adenine at position 2636bp (Fig. 6A). The fragment containing the Sp1 mutant sequencewas cloned into pGL3B. To understand the role of EBNA-2-liketranscription factor, we amplified another fragment spanning adistance from 2589 to 1120 bp and cloned it into pGL3B. Thisfragment was devoid of Sp1, PEA1, and YY1 transcriptionfactor-binding sites. THP-1 and THP-1/CD14 cells transfectedwith a plasmid containing an EBNA-2-like transcription factorsequence did not show any increase in luciferase activity (Fig.

FIG. 2. LPS stimulation induces the synthesis of IL-10. THP-1and THP-1/CD14 cells were stimulated with LPS (1 mg/ml) for varioustimes ranging from 2 to 48 h and analyzed by enzyme-linked immu-nosorbent assay for IL-10 production (A). THP-1 (B) and THP-1/CD14(C) cells were stimulated with LPS (1 mg/ml) for various times rangingfrom 2 to 24 h. Cells were harvested for mRNA isolation. IL-10 expres-sion was determined by semiquantitative reverse transcriptase-basedPCR analysis using b-actin as a standard control.

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6B), indicating that the EBNA-2-like transcription factor maynot play any role in hIL-10 gene transcription. However, THP-1and THP-1/CD14 cells transfected with a plasmid containingan Sp1 mutant sequence did not show luciferase activity com-pared with the plasmid containing the wild type Sp1 consensussequence. The results shown are a mean of four experimentsperformed with THP-1 (data not shown) and THP-1/CD14 cells(Fig. 6B). These results suggest that Sp1 plays a significantrole in transcription of the hIL-10 gene. Since the plasmidcontaining the Sp1 mutant sequence also contained consensussequences for PEA1, YY1, and EBNA-2-like transcription fac-tors, these results further suggest that PEA1, YY1, and EBNA-2-like factors may not be involved in the induction of hIL-10gene transcription by LPS.

Selective Role of p38 MAP Kinase in IL-10 Production byLPS-stimulated THP-1 Cells—It has been suggested that p38MAP kinase plays a vital role in hIL-10 synthesis followingstimulation of human monocytes with LPS (31). MAPKs areserine/threonine protein kinases, which include the p42/44ERKs, the p54 and p46 JNK1 and -2/stress-activated proteinkinase, and p38 MAPK (24). These three families of MAPKform three parallel signaling cascades activated by distinct andsometimes overlapping sets of stimuli (24). LPS-induced cellsignaling is known to involve activation of p38, p42/44 ERK,and JNK MAPK (25–27). We hypothesized, therefore, that theMAPK, and in particular the p38 MAP kinase, regulate IL-10synthesis through activation of Sp1 transcription factor in hu-man monocytic cells.

To investigate the role of MAPK in the regulation of hIL-10production in LPS-stimulated THP-1/CD14 cells, we examinedthe activation of ERK and p38 kinases. Cells were stimulatedwith LPS for 15 min and subjected to immunoprecipitationwith anti-p38 and anti-ERK antibodies, followed by Westernblot analysis with anti-phosphotyrosine antibodies. The resultsshow the LPS-induced tyrosine phosphorylation of p38 andERK kinases. To control for protein loading, the blots werestripped and reprobed with anti-p38 and anti-ERK antibodies(Fig. 7). To better understand the role of MAP kinases inLPS-mediated signaling, specific inhibitors of p38 (SB202190and SB203580) and p42/44 ERK kinases (PD98059) were em-ployed. SB202190 and SB203580 are selective and potent in-hibitors of p38 and p38b MAP kinases, respectively, and haveno significant effect on the activity of the ERK or JNK MAP

kinase subgroups (39, 42). Similarly, PD98059 is a potent andspecific inhibitor of ERK. PD98059 mediates its effects by bind-ing to and inactivating the ERK MAP kinase without affectingthe activity of either p38 or JNK (38, 42). To determine whetherSB202190, SB203580, and PD98059 specifically inhibit thephosphorylation of p38 and ERK kinases, respectively, THP-1cells were treated with these inhibitors at a concentration of 10nM for 2 h, followed by stimulation with LPS for 15 min. Theresults show that SB202190 (Fig. 7A) and SB203580 (data notshown) inhibited the phosphorylation of p38 and that PD98059inhibited the phosphorylation of ERK-2 MAP kinase (Fig. 7B).The concentration of inhibitors used in above experiments didnot affect cell viability (data not shown).

To determine the role of p38 and ERK MAP kinases in IL-10production, THP-1/CD14 cells were treated with specific inhib-itors for these MAP kinases for 2 h prior to stimulation withLPS. Cell supernatants were harvested at 48 h for analysis ofIL-10 production, since previous experiments had shown thatcytokine production peaked 48 h after LPS stimulation (datanot shown). The results show that treatment of THP-1/CD14cells with PD98059 at doses as high as 75 mM did not affectIL-10 production in a statistically significant manner (Fig. 8).Doses higher than 50 mM for PD98059 were not used in subse-quent experiments, since these concentrations were cytotoxicas determined by trypan blue exclusion (data not shown). Incontrast, treatment of THP-1/CD14 cells with SB202190 andSB203580 completely inhibited LPS-induced IL-10 productionin a dose-dependent manner (Fig. 8). These results clearlydemonstrate that LPS-induced IL-10 production is regulatedby p38 MAP kinases in THP-1/CD14 cells.

The Inhibitor of p38 MAP Kinase Abrogates Luciferase Ac-tivity of the Sp1-containing hIL-10 Promoter Construct—Tofurther investigate the role of p38 MAP kinase in the activationof Sp1 leading to IL-10 gene transcription, THP-1 and THP-1/CD14 cells were transfected with pGL3B containing a series ofsuccessive 59 deletions derived from 2890 to 1120 bp of thehIL-10 promoter sequence. The transfected cells were culturedfor 2 h in the presence and in the absence of p38 inhibitor,SB203580, prior to stimulation with LPS. Luciferase activitywas measured after 24 h. As observed above, deletion of se-quences spanning 2652 to 2890 bp from the hIL-10 promoterregion revealed an 8–10-fold increase in the luciferase activityin LPS-stimulated THP-1/CD14 cells, compared with the un-

FIG. 3. Nucleotide sequence of the5*-flanking promoter region of hIL-10gene (GenBankTM accession numberX78437). The translation start codon(ATG) is italicized. The putative cis-regu-latory elements are marked with solidlines below/above according to their orien-tation, respectively.

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stimulated cells or cells transfected with the control plasmid(Fig. 9). Treatment of the same cells with p38 inhibitorSB202190 completely abrogated the luciferase activity (Fig. 9),suggesting the involvement of p38 in the regulation of Sp1activity. As observed above, deletion of sequences between2571 and 1120 bp did not show any increase in luciferaseactivity that remained comparable with the activity observedin unstimulated or in cells transfected with the control plasmid(pGL3B). Similar results were obtained with THP-1 cells trans-fected with the above mentioned plasmids containing hIL-10promoter sequences with 59-end deletions and cultured in thepresence of p38 inhibitor (Fig. 9).

To confirm that p38 MAP kinase activates Sp1 transcriptionfactor, THP-1 and THP-1/CD14 cells were transfected with aplasmid containing a wild type or mutant Sp1 sequence andcultured in the presence or absence of SB202190. Treatmentwith SB202190 completely abrogated the luciferase activityobserved in LPS-stimulated cells transfected with the plasmidcontaining the wild type Sp1 sequence (Fig. 10). Similarly,treatment of LPS-stimulated cells transfected with plasmidscontaining either a mutated Sp1 sequence or an EBNA-2-like

transcription factor sequence with p38 inhibitor SB202190 didnot result in any change in luciferase activity (Fig. 10). Theresults shown are a mean of four experiments performed withboth THP-1 (data not shown) and THP-1/CD14 cells (Fig. 10).

Sp1 Binding to the IL-10 Promoter in LPS-stimulated CellsIs Regulated by p38 MAP Kinase—To further understandwhich signaling events downstream of MAP kinases may beinvolved in IL-10 transcription, we investigated the activationof p38 MAP kinase substrates. The above results suggest thatthe p38 MAP kinase and the Sp1 play a role in the regulationof hIL-10 transcription. Therefore, we investigated whetherLPS stimulation of THP-1 cells induced the binding of Sp1 tothe Sp1 binding site in the hIL-10 promoter. Cells were stim-ulated with LPS over a period of time ranging from 0 to 240min, and the nuclear extracts were analyzed in a gel shift assayfor binding to Sp1 oligonucleotide probes. The results revealedthat the maximum binding of Sp1 to the Sp1 oligonucleotidesequence of the hIL-10 promoter occurred 30–45 min followingstimulation of THP-1/CD14 cells with LPS (Fig. 11A). We ob-served three distinct Sp1 DNA-protein complex bands, namelyA, B, and C, in a gel shift assay that were completely blocked bycompetition with cold Sp1 oligonucleotides, indicating theirspecificities. It should be pointed out that bands A and B werealways induced by LPS. However, induction of band C was notobserved at all time points. The possible reasons for inconsis-tent induction of band C are not known. Similar results wereobtained with LPS-stimulated THP-1 cells (data not shown). Todetermine whether p38 MAP kinase delivers a signal via theactivation of Sp1 transcription factor, we investigated whetherSB202190, an inhibitor of p38 MAP kinase, inhibits binding ofSp1 to the Sp1-binding site of the IL-10 promoter. Incubation ofTHP-1/CD14 cells with SB202190 for 2 h prior to stimulationwith LPS resulted in the inhibition of Sp1 binding to theoligonucleotide containing the Sp1 sequence (Fig. 11B). Asabove, p38 inhibitors significantly reduced Sp1 binding to itsoligonucleotides in bands A and B. In contrast, PD98059 didnot affect Sp1 binding in LPS-stimulated cells (data notshown). These results suggest that p38 MAP kinase may pro-mote IL-10 expression by activating Sp1.

DISCUSSION

Bacterial endotoxin (LPS) is responsible for many of thecellular responses to Gram-negative bacterial infections (43).These responses may be induced after the association of LPSwith the LPS-binding plasma protein and the binding of thiscomplex with the CD14 receptor expressed on cells of monocyticlineage (44). LPS stimulates a variety of cytokines includingproinflammatory (IL-1, IL-6, TNF-a, etc.) and anti-inflamma-tory cytokines (e.g. IL-10) (7, 31, 33, 43). It is believed that LPSmay stimulate the expression of proinflammatory cytokinesthrough a common signaling pathway during inflammation.There is reasonably good evidence that production of TNF-a,IL-1b, and IL-6 can be regulated by the transcription factorNF-kB in various cell types (33–37). However, little is knownconcerning the regulation of hIL-10. The lack of kB bindingsites in the hIL-10 promoter makes it unlikely that the NF-kBis involved in IL-10 regulation (41). In this study, we investi-gated the regulation of the hIL-10 promoter in a humanpromonocytic cell line, THP-1, following activation with LPS.Extensive deletion analysis of hIL-10 promoter sequences re-vealed that an element encompassing the Sp1 transcriptionfactor-binding site is essential for IL-10 transcription. This wasconfirmed by transfecting THP-1 cells with a plasmid contain-ing a mutagenized Sp1 site, which was unable to drive theexpression of luciferase reporter. In addition, we analyzed theevents upstream of Sp1 activation. Our results clearly demon-strate that p38 MAP kinase regulates the LPS-induced activa-

FIG. 4. Luciferase activity in LPS-stimulated THP-1 andTHP-1/CD14 cells transfected with a hIL-10 promoter/luciferaseconstruct. A, time course of luciferase gene expression. THP-1 cells(1.5 3 106) were transiently cotransfected with 10 mg of either hIL-10promoter/luciferase reporter pGL3B construct or pGL3B vector controland with 5 mg of pSV-b-galactosidase control plasmid. Cells were grownfor 24 h followed by treatment with 1 mg/ml of LPS for 6, 12, 18, 24, and36 h. Luciferase and b-galactosidase activities were determined for thecell lysates. B, THP-1 and THP-1/CD14 cells (1.5 3 106) were tran-siently cotransfected with 10 mg of either hIL-10 promoter construct orvector control and with 5 mg of b-galactosidase control plasmid andallowed to grow for 24 h. The transfected cells were treated with 1 mg/mlof LPS for 24 h followed by measurement of luciferase and b-galacto-sidase activities. Cells transfected with vector pGL3B alone served as anegative control. Luciferase activity was normalized for b-galactosidaseactivity to give relative luciferase units (RLU). The results shown are amean 6 S.D. of four experiments performed in triplicate and normal-ized for b-galactosidase activity.

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tion of Sp1, which in turn regulates the transcription of thehIL-10 gene. These data suggest that the molecular regulationof pro- and anti-inflammatory cytokine genes is differentiallyregulated through diverse transcription factors (7, 8, 33, 34).

To better understand the LPS-induced signaling pathway inthe regulation of IL-10 synthesis, we employed two types ofTHP-1 cell lines that differ with respect to the level of CD14receptor expression on their surfaces. CD14 was expressed on5–15% of the THP-1 cells. To enhance LPS-mediated responsein these cells, THP-1/CD14 cells were used; these cells consti-tutively expressed CD14. Hence, transfection of THP-1/CD14cells with 59-deletion mutants of hIL-10 promoter linked to the

luciferase gene consistently revealed higher luciferase activitycompared with THP-1 cells transfected with the same con-structs. It should be pointed out that LPS stimulation of THP-1cells induces cytokine production in a manner similar to thatobserved with normal human monocytes. However, THP-1cells, unlike normal human monocytes, are free from negativefeedback regulation mediated by endogenously produced IL-10(data not shown).

Sp1 is a ubiquitous transcription factor that regulates theconstitutive activity of many genes studied. Sp1 plays a vitalrole in the regulation of transcription from TATA-less promot-ers that commonly encode housekeeping genes (45). Sp1 activ-

FIG. 5. Transcriptional activities of hIL-10 promoter in LPS-stimulated THP-1 and THP-1/CD14 cells. Left, the positions of thepotential regulatory elements in the hIL-10 promoter region (2890 to 1120 bp relative to the start site). The line diagram represents the extentof deletions within the hIL-10 promoter region in the seven DNA constructs used in the experiments. Putative binding sites for the transcriptionfactors glucocorticoid response element (GRE), cAMP, granulocyte-macrophage colony-stimulating factor (GM-CSF), Sp1, and signal transducersand activators of transcription (STAT) are shown. THP-1 (middle) and THP-1/CD14 (right) cells were cotransfected with 10 mg of either a promoterconstruct or vector control and with 5 mg of b-galactosidase control plasmid. After 24 h, cells were stimulated with LPS (1 mg/ml) for another 24 h.Cell lysates from unstimulated and LPS-stimulated cells were assayed for luciferase and b-galactosidase activities. Luciferase activity wasnormalized for b-galactosidase activity to give relative luciferase units (RLU). The results shown are a mean 6 S.D. of four experiments performedin triplicate.

FIG. 6. The effect of a mutant Sp1 binding site on hIL-10 promoter activity in LPS-stimulated THP-1/CD14 cells. A, a site-directedmutation of the Sp1 consensus sequence within the hIL-10 promoter is shown with respect to the wild type Sp1 sequence. The substitutednucleotides at positions 2631 and 2634 of the wild type consensus sequence are depicted in boldface letters. The amplified fragment (2650 to 1120bp) containing the mutations in the Sp1 sequence was cloned into pGL3B vector. B, THP-1/CD14 cells were cotransfected with either 10 mg of wildtype or mutant Sp1 construct and with 5 mg of b-galactosidase control vector. The transfected cells were treated with 1 mg/ml of LPS for 24 h.Luciferase activities of both unstimulated and LPS-stimulated cells were measured and are shown as a mean 6 S.D. of three experimentsperformed in triplicate and normalized for b-galactosidase activity. Putative binding sites for the transcription factors GRE, cAMP, and SP1 areshown in the left panel. STAT, signal transducers and activators of transcription; GM-CSF, granulocyte-macrophage colony-stimulating factor.

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ity and cellular content have been shown to be regulated duringdevelopment (46, 47), cellular proliferation (48), apoptosis (49),and other cellular processes (50, 51). Sp1 has been shown to beinvolved in mediating responses to various stimuli includinginduction of the TGF-b receptor gene (51, 52), epidermalgrowth factor-mediated expression of the gastrin gene (53), andcAMP-dependent induction of the CYP11A gene (54).

How Sp1 mediates its responses is presently not fully under-stood. Sp1 is a well characterized protein composed of 778amino acids. The amino-terminal portion of the molecule con-tains two glutamine-rich domains, each of which is associatedwith serine/threonine-rich regions (55). These domains are in-volved in transcriptional activation. The C-terminal region ofthe molecule contains the zinc finger DNA-recognition domain(55). Most of the gene regulation mediated by Sp1 requireseither post-translational modifications of Sp1, such as phos-phorylation and glycosylation (46, 56, 57) or alterations in theabundance of Sp1 protein (51). In addition, there are severalcoactivators of Sp1 such as CRSP, Rb, and hTAFII 130, whichallow Sp1 to stimulate transcription very effectively (45, 58).Translational modification of any of these coactivators may

FIG. 7. LPS stimulation induces p38 and p42/44 MAP kinaseactivity in THP-1/CD14 cells. To determine the effects of the p38MAP kinase-specific inhibitor (SB202190) and the p42/44 MAP kinase-specific inhibitor (PD98050) on LPS-induced activation of p38 or p42/44MAP kinase, respectively, cells were treated with SB202190 orPD98059 for 2 h. Cells were then stimulated with LPS (1 mg/ml) for 20min, followed by centrifugation and lysis of cell pellets. Proteins fromthe cell lysates were immunoprecipitated with anti-p38 (A) and anti-p42/44 (B) rabbit polyclonal antibodies (Santa Cruz Biotechnology). Theimmune complexes were subjected to SDS-polyacrylamide gel electro-phoresis followed by transfer of proteins onto the membranes. Themembranes were blotted with anti-phosphotyrosine antibodies (apy).To control for protein loading, the membranes were stripped and rep-robed with the same antibody used for immunoprecipitation. Ab, acontrol immunoprecipitation performed with antibody and Sepharosebeads in the absence of lysate. The experiment shown is representativeof three experiments.

FIG. 8. LPS-stimulated IL-10 production is selectively inhib-ited by inhibitors of p38 MAP kinases. To determine the effects ofthe p38 MAP kinase-specific inhibitors, SB203980 and SB202190, andthe p42/44 MAP kinase-specific inhibitor PD98050 on LPS-inducedIL-10 production, cells were treated with inhibitors for 2 h prior tostimulation with LPS (1 mg/ml). The supernatants were harvestedafter 48 h and analyzed by enzyme-linked immunosorbent assay forIL-10 production. The experiment shown is representative of threeexperiments.

FIG. 9. Effect of the p38 inhibitor SB202190 on LPS-induced hIL-10 promoter activation in THP-1/CD14 cells. Cells (1.5 3 106) weretransiently cotransfected with 10 mg of either hIL-10 wild type promoter or its deletion constructs and with 5 mg of b-galactosidase control vector.The transfected cells were pretreated with 15 mM SB202190 for 2 h followed by treatment with 1 mg/ml of LPS for 24 h. Unstimulated,LPS-stimulated, and LPS 1 SB202190-treated cells were harvested, and their lysates were assessed for luciferase and b-galactosidase activities.The results shown are means 6 S.D. of three experiments performed in triplicate and normalized by b-galactosidase activity.

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modulate the ability of Sp1 to regulate transcription. Molecularmechanisms by which Sp1 regulates IL-10 transcription re-main to be investigated.

It was surprising to find that only one transcription factor(Sp1) seems to play a prominent role in IL-10 regulation. Thisis in contrast to most of the cellular genes, and especiallycytokine genes, that are regulated by multiple transcriptionfactors. In view of established models of multiple transcriptionfactor involvement, it seems unlikely that other transcriptionfactors are not involved in the regulation of the hIL-10 gene.Our studies do not rule out the involvement of other transcrip-tion factors that may cooperate with Sp1 in hIL-10 transcrip-tion. It is likely that the transient transfection assay used inthe current study may not reveal the involvement of othertranscription factors. These factors may remain masked in ourexperimental system of transient transfection, a system that isknown to generate a high plasmid copy number or the accumu-lation of aberrant chromatin structure in these cells (59). Tocircumvent this possibility, a stable transfection approach maybe required. It is also possible that other transcription factorsinteracting with the hIL-10 promoter region beyond the 2890bp region participate in regulating the transcription of thehIL-10 gene. Further studies are required to address this pos-sibility. It is also likely that other transcription factors may beinvolved in IL-10 gene regulation in different IL-10-producingcell types and in response to distinct stimuli.

We have also investigated the upstream signaling eventsthat lead to Sp1 activation. We primarily investigated the roleof MAP kinases in this process and in IL-10 production viastimulation of CD14 receptors in THP-1 cells. LPS has beenshown to activate p38, p42/44 ERK, and JNK MAP kinases(24). These three types of MAP kinases can be activated indi-vidually or simultaneously, thereby suggesting their independ-ent signaling roles (24). The data presented in this study showthe selective involvement of p38 in IL-10 production in THP-1cells. Expression of the luciferase gene linked to the hIL-10promoter was mediated via p38 MAP kinase activation. A spe-cific inhibitor of the p42/44 ERK MAP kinases did not affectluciferase activity (data not shown). In addition, we also dem-onstrated that LPS induces the activation of Sp1 transcriptionfactor in a time-dependent manner. Maximum activity of Sp1activation was observed at 30–60 min poststimulation withLPS. Similarly, p38 inhibitors significantly reduced Sp1 bind-ing to its oligonucleotides. Furthermore, using Sp1 mutants ofthe hIL-10 promoter linked to the luciferase reporter gene, wedemonstrate for the first time that p38 MAP kinase plays adirect role in Sp1 activation and in inducing Sp1 binding to theIL-10 promoter.

The LPS-induced signaling pathway leading to the activa-tion of the p38 MAP kinase in monocytes/macrophages has

been investigated. LPS signaling through CD14 has beenshown to involve Toll-like receptors (TLR), specifically TLR-4,which associates with CD14 (60–62). LPS interaction withCD14 promotes dimerization of the TLR and subsequent re-cruitment of MyD88, a myeloid differentiation marker thatfunctions as an adaptor molecule (62, 63). MyD88 associatesvia its c-terminal toll homology domain with TLR and via itsN-terminal death domain with a serine-threonine protein ki-nase, IL-1R associated kinase (63). Upon interaction withMyD88, IL-1R-associated kinase is autophosphorylated andbinds to TRAF-6 (TNF-receptor associated factor-6). TRAF-6subsequently activates TAK-1 and mitogen-activated proteinkinase/extracellular signal-regulated kinase kinase kinase,members of the MAP kinase signaling cascade. This interactionactivates NF-kB (64, 65) complex in addition to the p38 MAPkinase (64). The molecular mechanism by which the p38 MAPkinase activates Sp1 to induce IL-10 gene transcription re-mains to be investigated. Nonetheless, the p38 MAP kinase hasbeen implicated in the activation of Sp1 in IL-1b-induced vas-cular endothelial cell growth factor gene expression (66) and inhyperosmotic stress-regulated cellular utilization of the serum-and glucocorticoid-inducible protein kinase (Sgk) (67).

Activation of the p38 MAP kinase has been shown to play acritical role in the regulation of several cellular and cytokinegenes in T cells and monocytes following their stimulation withvarious ligands. For example, T cell activation with specificantigen or staphylococcal antigens has been shown to inducep38 activation, resulting in TNF-a production (25). It has alsobeen demonstrated that p38 MAP kinase regulates IL-1 (25),IL-6 (68), TNF-a (25), IL-10 (31), and prostaglandin H syn-thase-2 (69) production in human monocytes through the acti-vation of the CD14 receptor. Functional roles for p38 have alsobeen described. The p38 MAP kinase is constitutively active inmouse thymocytes, suggesting a role in T cell survival (70, 71).Antigen receptor or Fas-mediated apoptosis of T and B cells isaccompanied by p38 activation (72, 73).

In summary, our results clearly show for the first time theinvolvement of the Sp1 transcription factor, and its activationvia p38 MAP kinase, in the regulation of hIL-10 gene transcrip-tion in human monocytic cell lines. While this work was inprogress, Brightbill et al. (74) demonstrated the involvement ofa nonconsensus Sp1-like sequence in mIL-10 expression usingRAW264.7, a murine macrophage cell line. Although thehIL-10 gene bears .80% nucleotide sequence homology and.73% amino acid sequence homology with mIL-10, the mIL-10and hIL-10 genes and their promoters are distinct (1–3). Incontrast to mIL-10, hIL-10 cDNA clones contain the insertionof Alu repetitive sequence elements in the 39-untranslated re-gion (2). Furthermore, the Sp1 consensus sequence is not pres-ent in the mIL-10 promoter (74). In contrast to our findings,

FIG. 10. The effect of the p38 inhib-itor SB202190 on wild type and Sp1mutant hIL-10 promoter activitiesin LPS-stimulated THP-1/CD14 cells.The cells were transiently cotransfectedwith 10 mg of either wild type or Sp1mutant hIL-10 promoter construct andwith 5 mg of b-galactosidase control plas-mid. The transfected cells were pre-treated with 15 mM SB202190 for 2 h fol-lowed by treatment with 1 mg/ml of LPSfor 24 h. Unstimulated, LPS-stimulated,and LPS 1 SB202190-treated cells wereharvested, and their lysates were as-sessed for luciferase and b-galactosidaseactivities. The results shown are ameans 6 S.D. of three experiments per-formed in triplicate and normalized byb-galactosidase activity.

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LPS stimulation of murine monocytic cells did not result in Sp1activation (74), indicating perhaps differential regulation ofIL-10 synthesis in murine and human monocytic cells. Takentogether, our results point to the key role of Sp1 and its acti-vation via p38 MAP kinase in the regulation of IL-10 transcrip-tion. These studies may provide a basis for the identification ofmolecular players in IL-10 regulation that may help in design-ing targeted drug therapy for inflammatory diseases.

Acknowledgments—Drs. Ken Dimock and Gina Graziani-Boweringare gratefully acknowledged for critically reading the manuscript.

REFERENCES

1. Moore, K. W., O’Garra, A., de Waal, M., Vieira, P., and Mosmann, T. R. (1993)Annu. Rev. Immunol. 11, 165–190

2. Eskdale, J., Kube, D., Tesch, H., and Gallagher, G. (1997) Immunogenetics 46,120–128

3. Vieira, P., de Waal-Malefyt, R., Dang, M. N., Johnson, K. E., Kastelein, R.,Fiorentino, D. F., deVries, J. E., Roncarolo, M. G., Mosmann, T. R., andMoore, K. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1172–1176

4. Del Prete, G., De Carli, M., Almerigogna, F., Giudizi, M. G., Biagiotti, R., andRomagnani, S. (1993) J. Immunol. 150, 353–360

5. Llorente, L., Zou, W., Levy, Y., Richaud-Patin, Y., Wijdenes, J., Alcocer-Varela,J., Morel-Fourrier, B., Brouet, J. C., Alarcon-Segovia, D., and Galanaud, P.(1995) J. Exp. Med. 181, 839–844

6. Rousset, F., Garcia, E., Defrance, T., Peronne, C., Vezzio, N., Hsu, D. H.,Kastelein, R., Moore, K. W., and Banchereau, J. (1992) Proc. Natl. Acad.Sci. U. S. A. 89, 1890–1893

7. de Waal, M., Abrams, J., Bennett, B., Figdor, C. G., and de Vries, J. E. (1991)J. Exp. Med. 174, 1209–1220

8. de Waal, M. R., Haanen, J., Spits, H., Roncarolo, M. G., te, V. A., Figdor, C.,Johnson, K., Kastelein, R., Yssel, H., and de Vries, J. E. (1991) J. Exp. Med.174, 915–924

9. Agematsu, K., Nagumo, H., Oguchi, Y., Nakazawa, T., Fukushima, K., Yasui,K., Ito, S., Kobata, T., Morimoto, C., and Komiyama, A. (1998) Blood 91,173–180

10. Briere, F., Bridon, J. M., Chevet, D., Souillet, G., Bienvenu, F., Guret, C.,Martinez-Valdez, H., and Banchereau, J. (1994) J. Clin. Invest. 94, 97–104

11. Ishida, H., Hastings, R., Kearney, J., and Howard, M. (1992) J. Exp. Med. 175,1213–1220

12. Yang, X., Gartner, J., Zhu, L., Wang, S., and Brunham, R. C. (1999) J. Immu-nol. 162, 1010–1017

13. Bettelli, E., Das, M. P., Howard, E. D., Weiner, H. L., Sobel, R. A., andKuchroo, V. K. (1998) J. Immunol. 161, 3299–3306

14. Grunig, G., Corry, D. B., Leach, M. W., Seymour, B. W., Kurup, V. P., andRennick, D. M. (1997) J. Exp. Med. 185, 1089–1099

15. Li, L., Elliott, J. F., and Mosmann, T. R. (1994) J. Immunol. 153, 3967–397816. Hagenbaugh, A., Sharma, S., Dubinett, S. M., Wei, S. H. Y., Aranda, R.,

Cheroutre, H., Fowell, D. J., Binder, S., Tsao, B., Locksley, R. M., Moore,K. W., and Kronenberg, M. (1997) J. Exp. Med. 185, 2101–2110

17. Kumar, A., Angel, J., Daftarian, M. P., Parato, K., Cameron, W. D., Filion, L.,and Diaz-Mitoma, F. (1998) Clin. Exp. Immunol. 114, 78–86

18. Muller, F., Aukrust, P., Nordoy, I., and Froland, S. S. (1998) Blood 92,3721–3729

19. Clerici, M., Wynn, T. A., Berzofsky, J. A., Blatt, S. P., Hendrix, C. W., Sher, A.,Coffman, R. L., and Shearer, G. M. (1994) J. Clin. Invest. 93, 768–775

20. Marchant, A., Deviere, J., Byl, B., De, G. D., Vincent, J. L., and Goldman, M.(1994) Lancet 343, 707–708

21. Turner, D. M., Williams, D. M., Sankaran, D., Lazarus, M., Sinnott, P. J., andHutchinson, I. V. (1997) Eur. J. Immunogenet. 24, 1–8

22. Hempel, L., Korholz, D., Bonig, H., Schneider, M., Klein-Vehne, A., Packeisen,J., Mauz-Korholz, C., and Burdach, S. (1995) Scand. J. Immunol. 41,462–466

23. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B., andHorak, I. D. (1993) J. Biol. Chem. 268, 20725–20728

24. Lewis, T. S., Shapiro, P. S., and Ahn, N. G. (1998) Adv. Cancer Res. 74, 49–13925. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green,

D., McNulty, D., Blumenthal, M. J., Heys, J. R., and Landvatter, S. W.(1994) Nature 372, 739–746

26. Liu, M. K., Herrera-Velit, P., Brownsey, R. W., and Reiner, N. E. (1994)J. Immunol. 153, 2642–2652

27. Hambleton, J., Weinstein, S. L., Lem, L., and DeFranco, A. L. (1996) Proc.Natl. Acad. Sci. U. S. A. 93, 2774–2778

28. Meisel, C., Vogt, K., Platzer, C., Randow, F., Liebenthal, C., and Volk, H. D.(1996) Eur. J. Immunol. 26, 1580–1586

29. Kambayashi, T., Jacob, C. O., Zhou, D., Mazurek, N., Fong, M., andStrassmann, G. (1995) J. Immunol. 155, 4909–4916

30. Platzer, C., Meisel, C., Vogt, K., Platzer, M., and Volk, H. D. (1995) Int.Immunol. 7, 517–523

31. Foey, A. D., Parry, S. L., Williams, L. M., Feldmann, M., Foxwell, B. M., andBrennan, F. M. (1998) J. Immunol. 160, 920–928

32. Lindroos, P. M., Rice, A. B., Wang, Y. Z., and Bonner, J. C. (1998) J. Immunol.161, 3464–3468

33. Bondeson, J., Browne, K. A., Brennan, F. M., Foxwell, B. M., and Feldmann,M. (1999) J. Immunol. 162, 2939–2945

34. Makarov, S. S., Johnston, W. N., Olsen, J. C., Watson, J. M., Mondal, K.,Rinehart, C., and Haskill, J. S. (1997) Gene Ther. 4, 846–852

35. Shakhov, A. N., Collart, M. A., Vassalli, P., Nedospasov, S. A., and Jongeneel,C. V. (1990) J. Exp. Med. 171, 35–47

FIG. 11. LPS stimulation activates Sp1 transcription factor ina time-dependent manner (A), and Sp1 activation is inhibited byinhibitors of p38 MAP kinase (B). A, THP-1/CD14 cells were stim-ulated with LPS (1 mg/ml) for various times ranging from 15 min to 4 hfollowed by centrifugation and collection of cell pellets. B, to determinethe effects of the p38 MAP kinase-specific inhibitor, SB202190, onLPS-induced Sp1 activation, cells were treated with SB202190 for 2 hprior to stimulation with LPS (1 mg/ml). To perform the gel shift assay,nuclear extracts were harvested from the cell pellets obtained at eachtime point. Nuclear extracts containing 5 mg of proteins were incubatedfor 1 h with 32P-labeled oligonucleotides corresponding to the consensussequence for Sp1. To determine the specificity of Sp1 transcriptionfactor binding, the nuclear extracts were incubated with either unla-beled oligonucleotides, corresponding to the consensus sequence forSp1, or with the control base pair-matched irrelevant oligonucleotide.The complexes were subjected to electrophoresis followed by autora-diography. Three distinct Sp1 DNA-protein complex bands, namely A,B, and C, were completely blocked by competition with cold Sp1 oligo-nucleotides, indicating their specificities.

Regulation of hIL-10 Promoter by Sp1 and p38 MAPK 13673

by guest on January 2, 2020http://w

ww

.jbc.org/D

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36. Hiscott, J., Marois, J., Garoufalis, J., D’Addario, M., Roulston, A., Kwan, I.,Pepin, N., Lacoste, J., Nguyen, H., and Bensi, G. (1993) Mol. Cell. Biol. 13,6231–6240

37. Libermann, T. A., and Baltimore, D. (1990) Mol. Cell. Biol. 10, 2327–233438. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995)

Proc. Natl. Acad. Sci. U. S. A. 92, 7686–768939. Lee, J. C., and Young, P. R. (1996) J. Leukocyte Biol. 59, 152–15740. Daftarian, P. M., Kumar, A., Kryworuchko, M., and Diaz-Mitoma, F. (1996)

J. Immunol. 157, 12–2041. Kube, D., Platzer, C., von Knethen, A., Straub, H., Bohlen, H., Hafner, M., and

Tesch, H. (1995) Cytokine 7, 1–742. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843–1484643. Morrison, D. C., and Ryan, J. L. (1979) Adv. Immunol. 28, 293–45044. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C.

(1990) Science 249, 1431–143345. Pugh, B. F., and Tjian, R. (1990) Cell 61, 1187–119746. Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995) J. Biol.

Chem. 270, 25879–2588447. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Mol. Cell. Biol. 11,

2189–219948. Mortensen, E. R., Marks, P. A., Shiotani, A., and Merchant, J. L. (1997) J. Biol.

Chem. 272, 16540–1654749. Piedrafita, F. J., and Pfahl, M. (1997) Mol. Cell. Biol. 17, 6348–635850. Vinals, F., Fandos, C., Santalucia, T., Ferre, J., Testar, X., Palacin, M., and

Zorzano, A. (1997) J. Biol. Chem. 272, 12913–1292151. Ammanamanchi, S., Kim, S. J., Sun, L. Z., and Brattain, M. G. (1998) J. Biol.

Chem. 273, 16527–1653452. Inagaki, Y., Truter, S., and Ramirez, F. (1994) J. Biol. Chem. 269,

14828–1483453. Merchant, J. L., Shiotani, A., Mortensen, E. R., Shumaker, D. K., and

Abraczinskas, D. R. (1995) J. Biol. Chem. 270, 6314–631954. Venepally, P., and Waterman, M. R. (1995) J. Biol. Chem. 270, 25402–2541055. Kadonaga, J. T., Courey, A. J., Ladika, J., and Tjian, R. (1988) Science 242,

1566–157056. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 63,

155–16557. Jackson, S. P., and Tjian, R. (1988) Cell 55, 125–13358. Naar, A. M., Ryu, S., and Tjian, R. (1998) Cold Spring Harbor Symp. Quant.

Biol. 63, 189–19959. Smith, C. L., and Hager, G. L. (1997) J. Biol. Chem. 272, 27493–2749660. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T.,

Takeda, K., and Akira, S. (1999) Immunity 11, 443–45161. Aderem, A., and Ulevitch, R. J. (2000) Nature 406, 782–78762. Takeuchi, O., Hoshino, K., and Akira, S. (2000) J. Immunol. 165, 5392–539663. Muzio, M., Natoli, G., Saccani, S., Levrero, M., and Mantovani, A. (1998) J.

Exp. Med. 187, 2097–210164. Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and

Matsumoto, K. (1999) Nature 398, 252–25665. Yang, R. B., Mark, M. R., Gurney, A. L., and Godowski, P. J. (1999) J. Immu-

nol. 163, 639–64366. Tanaka, T., Kanai, H., Sekiguchi, K., Aihara, Y., Yokoyama, T., Arai, M.,

Kanda, T., Nagai, R., and Kurabayashi, M. (2000) J. Mol. Cell Cardiol. 32,1955–1967

67. Bell, L. M., Leong, M. L., Kim, B., Wang, E., Park, J., Hemmings, B. A., andFirestone, G. L. (2000) J. Biol. Chem. 275, 25262–25272

68. Beyaert, R., Cuenda, A., Vanden Berghe, W., Plaisance, S., Lee, J. C.,Haegeman, G., Cohen, P., and Fiers, W. (1996) EMBO J. 15, 1914–1923

69. Pouliot, M., Baillargeon, J., Lee, J. C., Cleland, L. G., and James, M. J. (1997)J. Immunol. 158, 4930–4937

70. Sen, J., Kapeller, R., Fragoso, R., Sen, R., Zon, L. I., and Burakoff, S. J. (1996)J. Immunol. 156, 4535–4538

71. Salmon, R. A., Foltz, I. N., Young, P. R., and Schrader, J. W. (1997) J. Immu-nol. 159, 5309–5317

72. Juo, P., Kuo, C. J., Reynolds, S. E., Konz, R. F., Raingeaud, J., Davis, R. J.,Biemann, H. P., and Blenis, J. (1997) Mol. Cell. Biol. 17, 24–35

73. Graves, J. D., Draves, K. E., Craxton, A., Saklatvala, J., Krebs, E. G., andClark, E. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13814–13818

74. Brightbill, H. D., Plevy, S. E., Modlin, R. L., and Smale, S. T. (2000) J. Immu-nol. 164, 1940–1951

Regulation of hIL-10 Promoter by Sp1 and p38 MAPK13674

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Francisco Diaz-Mitoma and Ashok KumarWei Ma, Wilfred Lim, Katrina Gee, Susan Aucoin, Devki Nandan, Maya Kozlowski,

Lipopolysaccharide-stimulated Human MacrophagesPromoter via the Activation of Sp1 Transcription Factor in

The p38 Mitogen-activated Kinase Pathway Regulates the Human Interleukin-10

doi: 10.1074/jbc.M011157200 originally published online January 26, 20012001, 276:13664-13674.J. Biol. Chem. 

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