Interleukin-10 Induces Uteroglobin-related Protein (UGRP) 1 GeneExpression in Lung Epithelial Cells through HomeodomainTranscription Factor T/EBP/NKX2.1*□S

Received for publication, May 12, 2004, and in revised form, October 5, 2004Published, JBC Papers in Press, October 13, 2004, DOI 10.1074/jbc.M405331200

Achara Srisodsai‡§, Reiko Kurotani‡, Yoshihiko Chiba‡¶, Faruk Sheikh�, Howard A. Young**,Raymond P. Donnelly�, and Shioko Kimura‡ ‡‡

From the ‡Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,the �Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration,Bethesda, Maryland 20892, and the **Laboratory of Experimental Immunology, National Cancer Institute,Frederick, Maryland 21701

UGRP1 is a downstream target gene for homeodomaintranscription factor T/EBP/NKX2.1, which is predomi-nantly expressed in lung epithelial cells, and may playan anti-inflammatory role in lung inflammation. To un-derstand the role of UGRP1 in inflammation, its expres-sion was investigated in relation to cytokine signaling.In vivo experiments using mouse embryonic lung organculture and intranasal administration of interleukin(IL) 10 revealed that constitutive expression of Ugrp1mRNA is enhanced by IL-10. Increase of protein levelswas also demonstrated by immunohistochemistry usingembryonic lungs. This IL-10 induction of Ugrp1 geneexpression occurs at the transcriptional level when ex-amined using mouse embryonic lung primary cultures.In human lung NCI-H441 cells that in contrast to mouselung cells, do not exhibit constitutive expression of thegene, expression of the UGRP1 gene was induced in arapid and stable fashion. Two T/EBP, but not STAT3,binding sites located in the human UGRP1 gene pro-moter are responsible for IL-10 induction of the UGRP1gene as judged by transfection, gel shift, and chromatinimmunoprecipitation analyses. The IL-10 receptorchains, IL-10R1 and IL-10R2, are expressed in H441 cells,however, STAT3 was only weakly activated upon IL-10treatment. In contrast, STAT3 was strongly activatedwhen the cells were treated with other cytokines such asIL-22 and interferon-� but UGRP1 expression was notincreased. Together these results demonstrate thatIL-10 induces UGRP1 gene expression in lung epithelialcells through a T/EBP/NKX2.1-dependent pathway. Theresults further suggest that UGRP1 might be a target forIL-10 anti-inflammatory activities in the lung.

Uteroglobin-related protein 1 (UGRP1)1 was originally iden-tified as a downstream target gene for homeodomain transcrip-tion factor T/EBP (thyroid-specific enhancer-binding protein),also called thyroid transcription factor-1 (TTF-1) or NKX2.1(1). T/EBP regulates the expression of thyroid- and lung-spe-cific genes including thyroglobulin (2), thyroid peroxidase (3,4), thyrotropin receptor (5), and Na/I symporter (6) in thethyroid, and surfactant proteins A (7), B (8), and C (9), andClara cell secretory protein (10) in the lung. T/EBP is expressedin brain, thyroid, and lung epithelium during embryogenesisand is essential for the genesis of these organs (11). In adultlung, the expression of T/EBP is confined to both the conduct-ing airways and type II alveolar epithelial cells (12).

UGRP1, officially named Secretoglobin gene family 3A2 (13),is a novel gene encoding a homodimeric secretory protein of�10 kDa that is highly expressed in epithelial cells of thetrachea, bronchus, and bronchioles (1). Based on our previousresults, there is considerable evidence to suggest that UGRP1may function in the regulation of the local immune response inthe lung. First, the human UGRP1 gene is located on chromo-some 5q31-q32, a region where one of the asthma susceptibilitygenes was assigned and genes coding for several Th2-typecytokines such as interleukin (IL)-4, IL-5, and IL-13 are located(13). Second, the UGRP1 amino acid sequence exhibits similar-ity to the UG/Clara cell secretory protein, which is known tofunction as an anti-inflammatory agent via inhibition of phos-pholipase A2 (1). Third, a polymorphism (G/A) was identified inthe human UGRP1 gene promoter that is associated with anincreased risk of asthma in a Japanese population of adultasthmatic patients (14). Lastly, the mRNA level of Ugrp1 isdecreased in inflamed mouse lungs, and returns to basal levelsfollowing dexamethasone treatment (1).

IL-10 is a pleiotropic cytokine that was shown to mediateanti-inflammatory, immunosuppressive, and tissue protectivefunctions. The known IL-10 signaling is through binding tospecific receptors IL-10R1 and IL-10R2, which leads to activa-tion of the JAK-STAT (Janus kinase-signal transducers andactivators of transcription) signal transduction pathways (15).An anti-inflammatory role for IL-10 was demonstrated by lungcells and bronchoalveolar lavage fluid obtained from IL-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Fig. S1.

§ Supported by a partial grant from the National Cancer Instituteand by a Royal Golden Jubilee Ph.D. Scholarship from the ThailandResearch Fund. This work was carried out in partial fulfillment of therequirements for the Ph.D. degree from the Department of Pharmacol-ogy, Faculty of Science, Mahidol University, Bangkok, Thailand. Cur-rent address: Dept. of Pharmacology, Faculty of Science, Mahidol Uni-versity, Bangkok, 10400, Thailand.

¶ Current address: Dept. of Pharmacology, School of Pharmacy,Hoshi University, Tokyo 142-8501, Japan.

‡‡ To whom correspondence should be addressed: Bldg. 37, Rm.3112B, National Institutes of Health, 9000 Rockville Pike, Bethesda,MD 20892. Tel.: 301-496-0958; Fax: 301-496-8419; E-mail:shioko@helix.nih.gov.

1 The abbreviations used are: UGRP1, uteroglobin-related protein 1;T/EBP, thyroid-specific enhancer-binding protein; JAK-STAT, Januskinase-signal transducers and activators of transcription; EMSA, elec-trophoretic mobility shift analysis; ChIP, chromatin immunoprecipita-tion; MARCO, macrophage scavenger receptor with collagenous struc-ture; IL, interleukin; IFN, interferon; PBS, phosphate-buffered saline;RT, reverse transcriptase; Ab, antibody.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 52, Issue of December 24, pp. 54358–54368, 2004Printed in U.S.A.

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10(�/�) mice after repeated Aspergillus fumigatus challenge(16), which exhibited highly elevated levels of IL-4, IL-5, andIFN-� production, and exaggerated airway inflammation com-pared with wild-type controls. An anti-inflammatory role forIL-10 was also demonstrated by local delivery of IL-10 to theairway of mice using an adenovirus gene transfer system dur-ing ovalbumin challenge (17) or Pneumocystis carinii infection(18). Consistent with a role for IL-10 in allergic inflammation isthe observation that the lungs of asthmatic patients exhibitsignificantly lower levels of IL-10 when compared with normalcontrols (19). It is known that IL-10 inhibits: 1) the activationof a specific protein-tyrosine kinase involved in the bacteriallipopolysaccharide signaling pathway including the Ras signal-ing pathway (20) and 2) activation of the NF-�B pathway thatis involved in lipopolysaccharide-stimulated production of pro-inflammatory cytokines by human monocytes (21). However,the precise molecular mechanisms by which IL-10 exerts itsanti-inflammatory activities on different cell types have notbeen defined.

In this study, we demonstrate that IL-10 increased constitu-tive expression of the Ugrp1 gene in mouse lungs when exam-ined by mouse embryonic lung organ culture in the presence ofIL-10 and in vivo intranasal instillation of IL-10 to mice. IL-10also induced UGRP1 gene expression in human lung-derivedNCI-H441 cells that do not exhibit constitutive expression ofUGRP1. IL-10-modulated Ugrp1 gene expression was regu-lated at the transcriptional level when examined in primarymouse embryonic lung cells. The IL-10 responsiveness ofUGRP1 gene expression is mediated through the binding ofT/EBP, but not STAT3, to its specific binding sites in thepromoter region of the UGRP1 gene. This is the first demon-stration that the homeodomain transcription factor, T/EBP,plays an essential role in mediating the induction of an IL-10responsive gene in lung epithelial cells, and UGRP1 might be atarget for IL-10 anti-inflammatory activities in the lung.

EXPERIMENTAL PROCEDURES

Cell Culture, Embryonic Lung Organ Culture, and Materials—Hu-man pulmonary adenocarcinoma NCI-H441 cells were maintained inRPMI 1640 medium supplemented with 10% fetal bovine serum, glu-tamine, and penicillin/streptomycin. Mouse embryonic lungs obtainedfrom embryonic day (E) 15.5 pregnant C57BL/6 females were culturedby placing them on 0.4-�m Millipore filters (Millipore, Bedford, MA),supported by stainless steel grids in culture dishes containing Dulbec-co’s modified Eagle’s/F-12 medium supplemented with 10% fetal bovineserum, glutamine, and penicillin/streptomycin. Cells/lungs were cul-tured without serum for 24–48 h before addition of IL-10. ActinomycinD and cycloheximide were obtained from Sigma, and human and mouseIL-10 from R&D Systems (Minneapolis, MN).

Mouse Intranasal Instillation of IL-10—Six to 9-week-old 129Svmice (n � 5 in each group) were anesthetized with 2.5% Avertin andallowed to breathe spontaneously. Sterile PBS or various amounts ofmouse IL-10 were intranasally instilled into the trachea of each animal.After 24 h, total RNAs were prepared from whole lungs. All animalstudies were carried out in accordance with the Using Animals inIntramural Research Guidelines (NIH Animal Research Advisory Com-mittee, NIH, Bethesda, MD) after approval of the NCI Animal Care andUse Committee.

Immunohistochemistry—Cultured E15.5 embryonic lungs treatedwith 50 ng/ml IL-10 for 2 days were fixed with 4% paraformaldehyde at4 °C overnight and embedded in paraffin. Sections (4 �m) were incu-bated in 0.3% H2O2 in methanol for 30 min to inactivate endogenousperoxidases, followed by rinsing three times for 5 min each with PBS.Tissues for immunostaining for T/EBP were incubated in citrate buffer(pH 6.0) at 100 °C for 10 min. Tissues were blocked in 5% skim milk for30 min at room temperature, and then incubated overnight with rabbitanti-UGRP1 or anti-T/EBP antibodies produced in our laboratory (1:500 and 1:1000 dilution, respectively) at 4 °C in a humidified chamber.After washing three times for 5 min in PBS, tissues were processed bythe ABC method using a commercially available kit (Vector Laborato-ries, Burlingame, CA) according to the manufacturer’s instructions.Immunocomplexes were visualized with 3,3�-diaminobenzidine tetrahy-

drochloride (DAKO, Glostrup, Denmark).Northern Blotting—Total RNA, extracted from H441 cells or mouse

lungs by using TRIzol reagent (Invitrogen), was electrophoresed on a1% agarose gel containing 0.22 M formaldehyde and blotted onto Gene-Screen Plus nylon membranes (PerkinElmer Life Sciences). Hybridiza-tion was performed in PerfectHyb Plus hybridization buffer (Sigma) at65 °C overnight. The membranes were washed twice with 2� SSCcontaining 0.5% SDS at 65 °C for 20 min, followed by exposure to aStorm PhosphorImager screen and the signals were visualized withImageQuant software (Amersham Biosciences).

PCR-based Nuclear Run-on Experiments—Mouse embryonic lungprimary culture was used as a source for PCR nuclear run-on experi-ments. Briefly, E16.5 mouse embryo lungs were incubated in Dulbecco’smodified Eagle’s/F-12 media containing 10% fetal bovine serum, 20units/ml Dispase I (Roche Applied Science), and 10,000 units/ml colla-genase (Sigma) at 37 °C for 30 min with shaking. Dispersed cells werewashed three times in Dulbecco’s modified Eagle’s/F-12 media contain-ing 10% fetal bovine serum by centrifugation at 3,000 rpm at roomtemperature. Individual cells were plated out at the density of 1 � 106

cells per well in a 6-well dish and cultured in a CO2 incubator at 37 °Covernight. Cells were then treated with 100 ng/ml IL-10 or mock-treated with PBS for 2 h before harvest in PBS. Harvested cells werelysed by pipetting 10–15 times in 5 volumes of nucleus isolation bufferconsisting of 10 mM Tris, 10 mM NaCl, 3 mM MgCl2, and 0.5% IGEPALCA-630 (Sigma), and nuclei were collected by centrifugation at 14,000 �g for 5 min at 4 °C. Nuclei and the supernatant were separately sub-jected to RNA isolation using TRIzol to obtain nuclear and cytosolictotal RNAs.

Quantitative RT-PCR analysis was performed with ABI Prism 7900Sequence Detection System (Applied Biosystems, Foster City, CA) todetermine the level of Ugrp1 pre- and/or post-processed transcript inthe nuclei and/or cytosol. Total RNAs isolated from either nuclei orcytosol were treated with DNase I (Ambion, Austin, TX) to eliminatecontaminating genomic DNAs. Reverse transcription of isolated RNAswas carried out by using random hexamers and Superscript II reversetranscriptase (Invitrogen, Carlsbad, CA). To detect the Ugrp1 tran-script, two sets of primers were used (Fig. 2D); F1 (5�-AGTTCATTCAG-GCTTGGTCTTGAC-3�) and R1 (5�-GGAGTTTGTGTTTGCTGC-TATGC-3�) are specific to the sequences in intron 1 and amplify onlypre-spliced transcripts, whereas F2 (5�-GGTTATTCTGCCACTGCCCT-TCTC-3�) and R2 (5�-TACCAGGTGTGAAAGAGCCTCCAG-3�) primersequences are derived from the junctions of exon 1 and 2, and 2 and 3,respectively, and amplify only post-spliced transcripts. Reactions wereperformed using SYBR Green master mixture (Applied Biosystems,Foster City, CA) and the following conditions: 95 °C for 5 min, followedby 40 cycles of 95 °C for 30 s, 53 °C for pre-processed specific primers or62 °C for post-processed specific primers for 30 s, and at 72 °C for 60 s.Data were analyzed by the standard curve method, and normalized for18 S rRNA, which was measured by using the following primer pair:5�-CGGCTACCACATCCAAGGAA-3� (forward) and 5�-ATTGGAGCTG-GAATTACCGC-3�(reverse).

RT-PCR Analyses—The RT reactions were performed in a finalvolume of 20 �l containing DNase I-pretreated RNA (2.5 �g), 4 �l of 5�first strand synthesis buffer (Invitrogen, Carlsbad, CA), 1 �l of a mix-ture of four dNTPs (2.5 mM each), 2 �l of 0.1 M dithiothreitol, and 50 ngof oligo(dT) primers. After incubation at 42 °C for 2 min, 200 units ofSuperscript II reverse transcriptase (Invitrogen) was added, and theincubation was continued for 50 min. PCR was carried out using 0.5 �lof the reaction mixture, Advantage 2 polymerase (BD Biosciences, PaloAlto, CA), and the following oligonucleotide primers: human IL-10 R1(forward, 5�-GCTCCTGAGGTATGGAATAGAGTCC-3�; reverse, 5�-TA-TGTGTCATTTGCGGGGGC-3�), human IL-10 R2 (forward, 5�-TGGAT-GACACCATTATTGGACCC-3�; reverse, 5�-TTTGCTCACAGACAGGC-TCACTC-3�), human IL-22 R1 (forward, 5�-GCCACCAAGATGACTGA-CAGGTTC-3�; reverse, 5�-TTCTCTCTGCTTCCCTCCAAGG-3�), andhuman IL-28 R1 (forward, 5�-AAGTGGAAGAGTGTGCGGGAAC-3�; re-verse, 5�-TGGGGAGTGACTGGAAATAGGGTC-3�).

Luciferase Reporter Plasmid Construction and Transfection—Se-quence-specific primers were used to generate a series of reporter con-structs in pGL3 plasmid (Promega, Madison, WI) containing variouslengths of human UGRP1 gene promoter as follows: �31 hUGRP1,5�-GCGTGCTAGCATATAAATATGTGTGTGCAAAAGCAG-3�, �76hUGRP1, 5�-GCGTGCTAGCGGTGAGTCAAGTGGTAGGGAC-3�,�100 hUGRP1, 5�-GCGTGCTAGCTAACATTTATCCCTTTCTTTGGT-GG-3�, �148 hUGRP1, 5�-GCGTGCTAGCAACACACGGGGAAGTGG-AAAAC-3�, �178 hUGRP1, 5�-GCGTGCTAGCGCTGTACTGTAGAGC-TTTGTTTCTCA-3�, �322 hUGRP1, 5�-GCTGCTAGCTCTCCAAACTT-AAATGATTAGAAAACTG-3�, and 5�-AGATCTCGAGTCTGGGATATT-

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TTTC-3� (common reverse primer). The construct �209 hUGRP1 wasas previously described (1).

The following oligonucleotides were used to make pGL3-M1, M2,M1*2, and �76M3 mutant plasmids (mutated nucleotides boldfacedwith underline): M1, 5�-GCGTGCTAGCGCAAGGGTTTATGCCCGAG-G-3�, M2, 5�-GCGTGCTAGCGCAAGGGTTTATGCAAGAGGTACGGG-AGAAT-3�, M1*2, 5�-GCGTGCTAGCGCAAGGGTTTATGCCCGAGGT-ACGGGAGAAT-3�, M3, 5�-GCGTGCTAGCGGTGAGTCCCGTGGTAG-GGAC-3�, and 5�-AGATCTCGAGTCTGGGATATTTTTC-3� (commonreverse primer). The PCR fragments were digested with NheI (5� side)and XhoI, and inserted into the NheI and XhoI sites of the pGL3plasmid. For mutants M1*3, M2*3, and M1*2*3, the reverse primer5�-CTGAATTCTAGTCCCTACCACGGGACTCACCCCACC-3� wasused in combination with the above forward primers, and the resultantPCR fragment was digested with NheI and EcoRI (located downstreamof the 3rd T/EBP binding site), and swapped with the NheI-EcoRIfragment of the �209 hUGRP1 construct.

A series of UGRP1 luciferase reporter constructs and plasmidpRL-TK as a normalization control for transfection efficiency, andpCMV4-T/EBP (22) or pCMV4 were co-transfected using Opti-MEMmedia and Lipofectamine (Invitrogen) into H441 cells that were prein-cubated without serum for 24 h. IL-10 (25 ng/ml) was added 12 h aftertransfection, and 48 h later, cells were lysed, and luciferase activitieswere determined using the luciferase assay system (Promega).

EMSA—Nuclear extracts of NCI-H441 cells were prepared usingNE-PER (Pierce, Rockford, IL) according to the manufacturer’s in-struction. The following oligonucleotide sequences were used as probesor competitors: GRR (5�-ATGTATTTCCCAGAAA-3�) (23), PRL (5�-AG-ATTTCTAGGAATTCAAATCCAC-3�) (23), SIE (5�-GTCGACAGTTCC-CGTCAATC-3�) (23), T/EBP (5�-CACTGCCCAGTCAAGTGTTCTTGA-ACA-3�) (24), Cdx/A (5�-GGAACTGGTTTATCCTATTAGATTTGCCCT-3�) (25), C/EBP (5�-CAGTAAGGGAGTTTGCGCCACTATGCCC-3�) (26),HNF4 (5�-CTCAGCTTGTACTTTGGTACAACTA-3�; Santa Cruz Bio-technology, Santa Cruz, CA), and HTE-1 (5�-CACGATGACTCATCAC-TG-3�) (27).

Nuclear extract (3 �g) and unlabeled oligonucleotide competitorDNAs were preincubated in EMSA buffer (10 mM HEPES-KOH, pH 7.9,50 mM KCl, 0.6 mM EDTA, 5 mM MgCl2, 10% glycerol, 5 mM dithiothre-itol, 0.7 mM phenylmethylsulfonyl fluoride, 2 �g/�l pepstatin A, 2 �g/�lleupeptin, and 87 �g/�l poly(dI-dC) (Amersham Biosciences)) for 10 minon ice. An oligonucleotide probe (1 � 105 cpm) was added to the mixtureand incubated for 20 min on ice. For antibody supershift assay, 1 �l ofanti-TTF1 (T/EBP) monoclonal antibody (DAKO, Carpinteria, CA) oranti-STAT3 monoclonal antibody (Santa Cruz Biotechnology, SantaCruz, CA) was added to the mixture and the incubation continued for anadditional 30 min. Protein-DNA complexes were separated by 5% non-denaturing PAGE and analyzed with a Storm PhosphorImager (Amer-sham Biosciences).

ChIP—The ChIP assay was performed using a ChIP assay kit (Up-state Biotechnology, Lake Placid, NY) according to the manufacturer’sprotocol, except for the sonication procedure that was optimized forNCI-H441 cells. Briefly, pre-cultured H441 cells without serum for 48 hwere stimulated with 25 ng/ml IL-10 for 30 min. The DNA-proteinstructure was cross-linked with 1% formaldehyde at 37 °C for 10 minand cells were washed twice with cold PBS, harvested on ice, and spundown. The cell pellet was resuspended in 400 �l of SDS lysis buffer (1%SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1), and sonicated to shearDNAs to lengths between 600 and 1000 bp. The sonicate was centri-fuged and the supernatant was diluted 10-fold in dilution buffer (0.01%SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167mM NaCl). An aliquot (1% volume) of diluted cell supernatant wassaved to use as an input DNA. Salmon sperm DNA/protein A-agarose(Upstate Biotechnology) was added to the reminder of the supernatantto immunoprecipitate Ab-bound DNA complex, which was divided intothree fractions, each containing anti-TTF1 (T/EBP) (DAKO), mIgG(Santa Cruz Biotechnology), or no Ab as a negative control, and wasincubated overnight at 4 °C with rotation. After washing, the immuno-precipitated protein-DNA complex and the input were incubated at65 °C for 4 h to reverse protein/DNA cross-linking.

Primers used for PCR amplification are as follows: a fragment con-taining �179 to �209 T/EBP binding sites (forward, 5�-TCCAGGAGA-AGGATTCGTTGGG-3�; reverse, 5�-TTCCCCGTGTGTTCCCATGAG-3�), a fragment containing the �51 to �80 T/EBP binding site (forward,5�-TCTCATGGGAACACACGGGGAAG-3�; reverse, 5�-GTGTGATGGC-TGCTTTTGCACACAC-3�), or UGRP1 gene exon 3 as a nonspecific control(forward, 5�-GTGGCCAAACAGGACACTGG-3�; reverse, 5�-CGTCCT-TCTGAATAGAGTCC3�). PCR products were run on 2% agarose gel, andvisualized and quantitated with Eagle Eye (Stratagene, La Jolla, CA).

Phospho-STAT3 Western Blots—The levels of tyrosine-phospho-rylated STAT3 were measured by Western blotting as previouslydescribed (28). Briefly, H441 cells were treated with various cyto-kines (50 ng/ml each) for 30 min at 37 °C. At the end of this incuba-tion period, the cells were lysed, and STAT3 protein was immunopre-cipitated using a rabbit anti-STAT3 antiserum (Santa CruzBiotechnology). The levels of activated STAT3 were measured using amouse monoclonal anti-phospho-STAT3 (Tyr-705) antibody (CellSignaling Technology, Beverly, MA).

Anti-IL-10R1 Antibody Treatment—H441 cells were pretreated for 30min at 37 °C with or without a monoclonal antibody (3F9) (5 �g/ml) thatwas obtained in purified form from BD Pharmingen (San Diego, CA) andis known to specifically block the binding of IL-10 to the IL-10 receptor(29). IL-10 (50 ng/ml) was then added, and total RNA was prepared forNorthern blotting after an additional 6-h incubation period.

RESULTS

IL-10 Induces UGRP1 Gene Expression—Ugrp1 mRNA isconstitutively expressed in adult as well as embryonic mouselungs (Fig. 1) (1). The mRNA levels were found to be aug-mented by IL-10 when embryonic day (E) 15.5 lungs wereorgan-cultured in the presence of IL-10 and mice were intra-nasally administered IL-10. Thus, the organ-cultured embry-onic lungs exhibited an increase in Ugrp1 mRNA levels in astatistically significant time-dependent manner at 48 h afterthe initiation of IL-10 treatment (Fig. 1A), and intranasal IL-10administration demonstrated a dose-dependent increase ofUgrp1 mRNA levels after 24 h, with 600 ng being statisticallysignificant (Fig. 1B). When embryonic lungs were subjected toimmunohistochemistry, the increase of UGRP1 protein expres-sion after IL-10 treatment was clearly observed as comparedwith control, whereas no changes were seen for T/EBP expres-sion with or without IL-10 (Fig. 1C). These results demonstratethat IL-10 enhances Ugrp1 gene expression in mouse lungs.

To determine the mechanism for the IL-10 induced increasein UGRP1 mRNA expression, various lung cell lines were an-alyzed. Human pulmonary adenocarcinoma NCI-H441 cells,which do not demonstrate any constitutive expression ofUGRP1, were found to express UGRP1 mRNAs albeit at lowlevels, upon treatment with IL-10 at 25 and 50 ng/ml (Fig. 2A).The UGRP1 mRNA expression was first observed as early as2 h after the initiation of treatment, was drastically increasedby 4 h, and continued at elevated levels up to 24 h. Interest-ingly during this period, T/EBP mRNA levels did not signifi-cantly change (Fig. 2B). Next, H441 cells were pretreated witheither the RNA synthesis inhibitor, actinomycin D, or the pro-tein synthesis inhibitor, cycloheximide (Fig. 2C). The IL-10induction of UGRP1 mRNA expression was not observed in thepresence of actinomycin D, whereas cycloheximide did not haveany effect on IL-10-induced UGRP1 mRNA levels, demonstrat-ing that induction of UGRP1 gene expression by IL-10 in H441cells requires transcription but not a newly synthesized pro-tein(s). Also, of interest was that T/EBP mRNA expression wascompletely abolished by actinomycin D treatment.

PCR-based nuclear run-on experiments (30) were performedto confirm that the IL-10-induced increase in Ugrp1 mRNAoccurred at the transcriptional level. RNA was isolated fromnuclei and cytoplasm of primary mouse embryonic lung cellstreated with and without IL-10 for 2 h. Specific primers weredesigned that selectively recognize either pre-processed orpost-processed transcripts (Fig. 2D, illustration in upperpanel). In the nuclei, the levels of pre-spliced and post-splicedtranscripts were, respectively, 13- and 1.6-fold higher withIL-10 treatment than without, whereas in the cytosol, a 3-foldincrease of Ugrp1 mRNA levels was obtained as compared withno treatment (Fig. 2D, lower panels). Taken together, the re-sults indicate that IL-10 modulation of UGRP1 gene expressiontakes place at the transcriptional level.

Analysis of UGRP1 Gene Promoter—To determine the tran-

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scriptional control site(s) for IL-10 induction, the humanUGRP1 gene promoter activity was examined by transienttransfection analysis with reporter plasmids containing vari-ous lengths of the human UGRP1 gene promoter in the pres-ence or absence of IL-10 in H441 cells that have an endogenouslow level expression of T/EBP. Treatment with IL-10 increasedreporter gene activity driven by constructs containing se-quences between �179 and �209 bp of DNA upstream of thetranscription start site of the UGRP1 gene (Fig. 3A), suggestinga potential transcriptional control site for IL-10 signal trans-duction in this region.

In an attempt to identify possible transcription factors thatbind to DNA located between �179 and �209 bp that may beresponsible for IL-10 induction of UGRP1 gene expression,computer analysis was performed using the transcription fac-tor Search program (31). C/EBP (GGTTTATGCAAGAG), Cdx/A(GTTTATG), and two T/EBP (CTNNAG and CAAG) (1) bindingconsensus sequences were identified (Fig. 3B). Surprisingly,this region does not contain a typical STAT3 binding site.

IL-10 Activates DNA Binding of T/EBP—To identify tran-scription factors that might bind to the region between �179

and �209 bp that may be responsible for IL-10 induction ofUGRP1 gene expression, electrophoretic mobility shift analysis(EMSA) was performed using nuclear extracts prepared fromH441 cells treated with IL-10 and oligonucleotides containingsequence between �179 and �209 bp of the UGRP1 genepromoter, or the binding sites for GRR (STAT1, -3, and -5binding site), SIE (STAT-1 and -3 binding sites), PRL (STAT5binding site), C/EBP, Cdx/A, or T/EBP as probes or competi-tors. The 209-179 probe formed a strong specific complex withnuclear extracts prepared from cells treated with IL-10. Inter-estingly, the mobility of the complex was identical to thatobtained with the T/EBP consensus sequence as a probe (Fig.4A). The specific protein-DNA complex was only efficientlycompeted away by unlabeled oligonucleotide (209-179) orT/EBP-specific oligonucleotide, and not by other oligo probes(Fig. 4B). A competition assay was also performed usingHTE-1, an enhancer binding element, which IL-10-activatedIL-10E1 binds to and induces expression of the tissue inhibitorof matrix metalloproteinase 1 (Fig. 4B, right panel) (27). HTE-1did not compete with T/EBP for binding to the 209-179 probe,suggesting that IL-10 induction of UGRP1 gene expression is

FIG. 1. In vivo induction of Ugrp1 byIL-10 in mouse lung. A, upper panel,representative Northern blotting resultsusing RNAs isolated from E15.5 embry-onic mouse lungs cultured in the presenceof 50 ng/ml IL-10 for 24 and 48 h. Lowerpanel, relative UGRP1 mRNA levels(mUgrp1) plotted against time. Valuesare the mean of four independent experi-ments � S.E., each comprising 5–6 em-bryos at each time point (n � 20). Notethat a significant difference (p � 0.05)was observed between 0 and 48 h. B, up-per panel, representative Northern blot-ting results using lung RNAs isolatedfrom mice that received intranasal instil-lation of 0, 20, 60, and 600 ng of IL-10 andwere euthanized 24 h later. Lower panel,relative Ugrp1 mRNA levels plotted(mUgrp1) against the amount of IL-10 ad-ministered. Values are the mean of thoseobtained from 5 mice � S.E. Note that asignificant difference (p � 0.05) was ob-served between 0 and 600 ng IL-10. C, exvivo cultured E15.5 embryonic mouselungs were treated with 50 ng/ml IL-10for 48 h, followed by immunohistochemi-cal analysis using anti-UGRP1 andT/EBP antibodies.

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not mediated by IL-10E1. The antibody against T/EBP pro-duced a faint but distinct supershifted band, whereas anti-STAT3 antibody had no effect (Fig. 4B), confirming that uponIL-10 activation T/EBP binds to its binding site(s) between�179 and �209 bp of the human UGRP1 gene promoter. Theformation of the IL-10-induced specific DNA-T/EBP complexwas observed as early as 2 min and remained high for up to24 h, suggesting that the IL-10 signal transduction is rapid andstable (Fig. 4C).

Two T/EBP Binding Sites Are Involved in IL-10 Induction ofUGRP1 Gene Expression—To confirm the involvement ofT/EBP in the IL-10 induction of UGRP1 gene expression, mu-tations were introduced to the 1st (Mut 1; �193 to �196 bp)and 2nd (Mut 2; �182 to �187 bp) T/EBP binding sites within�179 to �209 bp (distal) of the UGRP1 gene promoter (Figs. 3B5B). An additional 3rd T/EBP binding site was found at �65 to�68 bp (proximal) (Fig. 3B), at which a mutation was alsointroduced (Mut 3; Fig. 5B). Co-transfection assays were per-formed with either pCMV4 vector or pCMV4-T/EBP expressionplasmid in the presence or absence of IL-10 (Fig. 5A).

The construct �76 showed an increased promoter activity inthe presence of the T/EBP expression plasmid, suggesting that

the 3rd T/EBP binding site is capable of activating gene tran-scription when T/EBP is present, whereas IL-10 did not en-hance promoter activity. Introduction of a mutation into the3rd T/EBP binding site (�76M3) abolished the T/EBP-stimu-lated promoter activity as expected. The activity of the �209construct increased to similar levels by IL-10 treatment or byco-transfection of T/EBP. Both IL-10 and T/EBP overexpres-sion together further increased the promoter activity. Interest-ingly, promoter activity of the construct �209M3, which has amutation at the 3rd (proximal) T/EBP binding site, completelyabolished the IL-10 effects, suggesting involvement of the 3rdT/EBP binding site in IL-10 activation of the UGRP1 gene.

Co-transfection analysis was further carried out using vari-ous mutant constructs: M1 (1st T/EBP binding site mutated),M2 (2nd site mutated), M1*2 (both 1st and 2nd sites mutated),M1*3 (both 1st and 3rd sites mutated), M2*3 (both 2nd and 3rdsites mutated), and M1*2*3 (all three sites mutated). Amongthree mutants (M1, M2, and M1*2) that have one or two of thedistal (�179 to �209 bp) T/EBP binding sites mutated buthaving an intact proximal 3rd T/EBP binding site, only mutantM1 exhibited IL-10 induction of the promoter activity in theabsence of the co-transfected T/EBP. The M1 construct in the

FIG. 2. IL-10 induces UGRP1 mRNA levels. A, dose effects of IL-10. H441 cells were treated with various amounts of IL-10 (6.25, 12.5. 25,and 50 ng/ml) for 12 h. The representative results from three independent experiments are shown. B, time responses for IL-10 induction. H441 cellswere treated with 25 ng/ml IL-10 and were harvested at various time points (2, 4, 8, 12, and 24 h). The representative results from threeindependent experiments are shown. Lower panel, relative mRNA expression of UGRP1 (hUGRP1) or T/EBP versus glyceraldehyde-3-phosphatedehydrogenase (GAPDH) shown in the upper panel was plotted versus time. C, effect of actinomycin D or cycloheximide. H441 cells were added witheither 5 �g/ml actinomycin D (Act D) or 10 �g/ml cycloheximide (CHX) 2 h before the addition of 25 ng/ml IL-10. Total RNAs were isolated 5 or10 h later. IL-10 treatment only (IL-10) is shown as a positive control. The representative results from two independent experiments are shown.All Northern blots were hybridized serially with UGRP1, T/EBP, and GAPDH probe as a loading control. D, PCR-based nuclear run-onexperiments. Mouse embryonic lung primary cultures were treated with (�) or without (�) 100 ng/ml IL-10 for 2 h. Total RNAs were separatelyisolated from nuclei (Nuc) or cytosol (Cyt) fractions, and were subjected to real-time PCR using pre- (F1/R1) and post-transcript (F2/R2) specificprimer pairs (illustrated in the upper panel, exon size is not in scale). PCR products are normalized to 18 S rRNA, and the relative expression levelsare shown based on the value obtained without IL-10 treatment as set to 1 in each experimental group. The representative results from threeindependent experiments are shown.

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presence of T/EBP, or the M2 and M1*2 constructs with orwithout T/EBP did not exhibit any increase upon IL-10 treat-ment. These results suggest that the 2nd T/EBP binding site isessential for IL-10 induction of UGRP1 gene expression,whereas the 1st site is dispensable, but required for maximalIL-10 induction of UGRP1 gene expression. When the proximal3rd T/EBP binding site was mutated in addition to the distalsites (M1*3, M2*3, and M1*2*3), IL-10 induction was not ob-served with any of the constructs, further suggesting a criticalrole for the 3rd binding site for IL-10 induction of the gene.Co-transfection of the T/EBP expression plasmid increased pro-moter activities albeit to different degrees in all �209 mutantshaving at least one intact T/EBP binding site. The M1*2*3 thathas all T/EBP binding sites mutated has almost completely lostboth T/EBP and IL-10 induction of promoter activity. Thesedata indicate that both the 2nd and 3rd T/EBP binding sitesare essential for IL-10 induction of UGRP1 gene expression,however, all three sites are required for maximal IL-10 induc-tion of promoter activity.

When human and mouse UGRP1 gene promoter sequencesare compared, the areas containing the 2nd and 3rd T/EBPbinding sites in human UGRP1 gene promoter aligned verywell with T/EBP binding regions II and IV in the mouse Ugrp1gene promoter, previously identified by DNase I footprintinganalysis (1) (Fig. 5C). This suggests that IL-10 induction ofmouse Ugrp1 gene expression is likely to occur through asimilar, if not the same, T/EBP-mediated mechanism.

The requirement of the 2nd and 3rd T/EBP binding sites forIL-10 signal transduction was further confirmed by EMSAusing probes 209-179, 80-51, and oligonucleotides having mu-tations at the 1st (Mut 1), 2nd (Mut 2), both 1st and 2nd (Mut1*2), and 3rd (Mut 3) T/EBP binding sites as probes and/orcompetitors (Fig. 5B). As expected, only the Mut 1 oligonucleo-tide competed for the formation of a specific DNA-proteincomplex (Fig. 5D).

To determine whether IL-10-induced binding of T/EBP to theUGRP1 gene promoter actually takes place in situ, a ChIPassay was performed. Three sets of primers were used to spe-cifically amplify either distal (1st and 2nd together) or proximal(3rd) T/EBP binding sites or exon 3 of the UGRP1 gene as anonspecific control (Fig. 6A). Both distal and proximal T/EBPbinding sites were specifically amplified only in the IL-10treated group after precipitating with anti-T/EBP antibody(Fig. 6, B and C). These results are consistent with co-trans-fection and EMSA, indicating that T/EBP binds in situ uponIL-10 stimulation to the UGRP1 gene promoter at the distaland proximal binding sites.

IL-10 Induces UGRP1 Gene Expression through the IL-10Receptor, but Does Not Require JAK-STAT Signaling—To un-derstand the mechanism of IL-10 induction of UGRP1 geneexpression, RT-PCR was performed to first examine whetherthe IL-10 receptor chains, IL-10R1 and IL-10R2, are expressedby H441 cells. RT-PCR yielded products of 291 bp for IL-10R1and 300 bp for IL-10R2 from both H441 cells and human

FIG. 3. Determination of IL-10 re-sponsive region. A, UGRP1 gene pro-moter deletion analysis in the presence(closed box) or absence (open box) ofIL-10 treatment. Constructs are illus-trated on the left with possible transcrip-tion factor binding sites depicted in B.The relative luciferase activity of cellstransiently transfected with the indi-cated constructs is shown based on theactivity obtained with the basic pGL3vector that was arbitrarily set as 1. Val-ues are the means of three independentexperiments � S.E., each done in tripli-cate. B, human UGRP1 gene promotersequence. Relative positions of the 5�end of each construct are shown by ar-rowheads. The IL-10 responsive region(�179-209 bp) contains binding sites fortranscription factors C/EBP (under-lined), Cdx/A (overlined), and T/EBP(boxes 1 and 2). The proximal T/EBPbinding site (box 3) is also shown, andthe TATA box and translation initiationcodon ATG are indicated in boldface.The polymorphic nucleotide previouslyreported (14) is marked with an asterisk(*).

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monocytes (positive control), demonstrating that H441 cellsexpress both receptors (Fig. 7A). The presence of IL-10R1 andIL-10R2 subunits was also confirmed in mouse embryonic andadult lungs by RT-PCR (data not shown).

A family of IL-10-related cytokines, including IL-10, IL-22,IL-26, and IL-28, share a common receptor subunit, IL-10R2but have distinct receptor 1 subunits (28, 32, 33). The presenceof IL-22R1 and IL-28R1 was demonstrated by RT-PCR in H441cells but not in human monocytes. Low level expression wasobserved in liver and NCI-A549 cells that were used as positivecontrols (Fig. 7B). IL-22R1 and IL-28R1 are known to be ex-pressed by epithelial cell lines such as H441 and A549, but arenot expressed on hematopoietic cells such as monocytes.

To determine whether IL-10 and other IL-10 family membersactivate the JAK-STAT pathway in H441 cells, cells weretreated with IL-10, IL-19, IL-20, IL-22, or IFN-�, and the levelsof phosphorylated STAT3, the major STAT protein activated byall five cytokines, were examined. It is known that IL-10 stim-ulation does not result in serine phosphorylation of STAT3 (34).Furthermore, it is interesting to note that H441 cells haveconstitutive serine phosphorylation of STAT3.2 When the tyro-sine phosphorylation of STAT3 was examined, it was predom-inantly observed in IL-22- and IFN-�-treated cells, whereasonly low levels of activation were detected in IL-10-treated cells(Fig. 7C), indicating that the STAT3 signal transduction path-way is active in H441 cells. Of particular interest is the factthat the induction of UGRP1 mRNA expression was observedonly in IL-10-treated cells despite the low level of STAT3 acti-vation compared with IL-22 or IFN-�-treated cells (Fig. 7D).These results indicate that IL-10 induction of UGRP1 geneexpression is STAT3-independent.

Finally, to confirm that the induction of UGRP1 gene expres-sion by IL-10 is mediated by signaling through IL-10 receptors,the effect of anti-IL-10R1 antibody on IL-10 induction ofUGRP1 mRNA expression was examined (Fig. 7E). This anti-body has been shown to specifically block the binding of IL-10to the IL-10 receptor (29). Northern blotting analysis clearlydemonstrated that IL-10 induces UGRP1 mRNA expression bysignaling through the IL-10 receptor.

DISCUSSION

We report here a novel IL-10 signal transduction pathway forUGRP1 gene expression in lung epithelial cells that is medi-ated through the IL-10 receptor and the homeodomain tran-scription factor T/EBP. UGRP1 gene expression was enhancedby IL-10 in in vivo and ex vivo lungs that have constitutiveexpression of Ugrp1, whereas the expression was induced byIL-10 in H441 cells where no constitutive expression wasfound. It is not surprising that we found a difference in expres-sion patterns between in vivo/ex vivo tissues and a tumor cellline, the latter of which may not express factor(s) that arenecessary for constitutive expression of UGRP1. We furtherfound that the IL-10 modulation of UGRP1 gene expression istranscriptional, and T/EBP binding sites, present in the pro-moter region of the UGRP1 gene, are essential for induction ofthis gene by IL-10. Our results demonstrate for the first timethat T/EBP plays a role in a cytokine signaling pathway inaddition to the established roles for this transcription factor inregulating thyroid (2–6) and lung-specific gene expression (7–

2 A. Srisodsai, R. Kurotani, Y. Chiba, F. Sheikh, H. A. Young,R. P. Donnelly, and S. Kimura, unpublished observation.

asterisk. Right panel, competition assays using the HTE-1 oligonucleo-tide as a competitor. C, EMSA time course. H441 cells nuclear extractswere prepared at various time points after the addition of 25 ng/mlIL-10. All EMSA experiments were repeated at least 3–4 times.

FIG. 4. EMSA. A, various oligonucleotides were used as probes, in-cluding a sequence between �179 and �209 bp of the UGRP1 genepromoter (probe 209-179), GRR, SIE, PRL, T/EBP, Cdx/A, and C/EBP.H441 cells were incubated with or without 25 ng/ml IL-10 for 12 hbefore harvest for nuclear extract (NE) preparation. B, EMSA compe-tition or antibody supershift (Ab) assay using the labeled 209-179oligonucleotide as a probe and 50-fold excess of various unlabeled spe-cific oligonucleotides as competitors or antibodies as indicated. A su-pershifted band obtained with antibody against T/EBP is shown by an

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10) and in the genesis of the thyroid, lung, and ventral fore-brain during embryogenesis (11).

Our results further demonstrate the constitutive expressionof IL-10R1 by H441 cells. The IL-10 receptor complex is com-posed of two receptor chains, IL-10R1 and IL-10R2 (15, 35). TheIL-10R1 subunit is primarily expressed by hematopoietic cellssuch as B cells, T cells, NK cells, monocytes, and macrophages,whereas IL-10R2 is constitutively expressed by most cells andtissues (15, 35). The presence of IL-10R1 in H441 cells andmouse lungs is an addition to a short list of non-hematopoieticcells that express IL-10R1 that includes fibroblasts, epidermalcells, and keratinocytes (35). The constitutive IL-10R1 expres-sion has been described in placental cytotrophoblasts (36) andcolonic epithelium (37). The level of IL-10R1 expression inH441 cells may be low because IL-10 treatment yielded weakSTAT3 activation as compared with IL-22 or IFN-�. Under lowIL-10R1 expression, no tyrosine phosphorylation of STAT3 andSTAT1, nor induction of the IL-10 target gene suppressor of

cytokine signaling (SOCS) 3 were observed in human neutro-phils (38). In our case, the expression of UGRP1 mRNA washighly induced by IL-10 despite low levels of IL-10R1 andSTAT3 activation. It is interesting to note that SOCS3 wasconstitutively expressed in H441 cells.2 We attempted smallinterfering RNA experiments to suppress STAT3 activity tofurther demonstrate that STAT3 is not involved in the novelIL-10 signaling pathway, however, the experiments did notwork. Further work is required before any conclusions can bereached regarding this point. Furthermore, although ourEMSA results did not suggest the involvement of STAT1 orSTAT5 in the current novel IL-10 signaling pathway, this pos-sibility cannot be excluded and further experiments are re-quired to address this question.

Currently, the known IL-10 signal transduction pathwayinvolves IL-10R, the receptor-associated Janus tyrosine ki-nases, JAK1 and TYK2, and the latent transcription factorSTAT3 (15, 35). STAT3-independent IL-10 signaling pathways

FIG. 5. Analysis of mutant T/EBP binding sites. A, co-transfection analysis of constructs �76 and �209, and their mutants in the presenceor absence of T/EBP and/or IL-10. Mutant constructs are illustrated on the left. The relative luciferase activity of cells transiently transfected withthe indicated constructs is shown based on the activity obtained with the basic pGL3 vector that was arbitrarily set as 1. Values are the meansof three independent experiments � S.E., each done in triplicate. B, sequences of mutants used in transfection analysis and EMSA. The putativeT/EBP consensus binding site and mutated sequences are shown in boldface. C, alignment of mouse and human UGRP1 gene promoter sequences.T/EBP binding regions or sites for mouse Ugrp1 (II, III, and IV) and human UGRP1 (1, 2, and 3) gene promoters are overlined and underlined,respectively. D, EMSA competition assay using probe 209-179 or 80-51 and 50-fold excesses of unlabeled wild-type or specific mutated oligonu-cleotides. A T/EBP antibody supershifted band is shown by an asterisk.

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have been described for the macrophage de-activation re-sponses to IL-10 in the J774 mouse macrophage cell line (39)and the IL-10 induction of tissue inhibitor of matrix metallo-proteinase 1 expression in human prostate cells (27). Themechanism for IL-10 de-activation of macrophage has not yetbeen fully defined, although phosphorylation of certain cyto-plasmic tyrosine residues is required for this process to occur.In the case of tissue inhibitor of matrix metalloproteinase 1,phosphorylated tyrosine residues further phosphorylate theIL-10E1 protein (27), which triggers rapid translocation of IL-10E1 to the nucleus where it binds to a specific enhancerelement, termed HTE-1, located in intron 1, upstream of the5�-ATG translation start site of the tissue inhibitor of matrixmetalloproteinase 1 gene. Because the HTE-1 oligonucleotidedid not compete for binding to the IL-10 responsive region ofthe UGRP1 gene in the EMSA binding reaction, it is unlikelythat IL-10E1 is involved in the IL-10 signal transduction path-way of UGRP1 induction in H441 cells. On the other hand,T/EBP clearly bound to this region although the T/EBP-specificantibody produced a very faint supershifted band. The findingthat only a small component was supershifted upon antibodyaddition was previously observed when recombinant T/EBPwas used in EMSA with mouse Ugrp1 gene promoter bindingsequences as probes (1). One of these T/EBP binding sitescorresponds very well to the 2nd T/EBP binding site describedin the current study. We have no explanation for the weaksupershift in this study, but it might be because of conforma-

tional differences in the binding of the protein to this specificsite, thus reducing the ability of the antibody to recognize theprotein or possibly the interaction of other proteins withT/EBP, thus masking the epitope.

The novel IL-10 signaling in H441 cells occurs very rapidlyand is stable because the specific DNA-T/EBP complex isformed as early as 2 min, and remains elevated even after 24 hof IL-10 treatment. Whereas the mechanism responsible forIL-10 signaling observed in our study is unknown, T/EBP couldbe phosphorylated by kinases such as JAK1 and TYK2, andthen translocated to the nucleus where it binds to its specificbinding sites. In fact, several studies on phosphorylation ofT/EBP and its relation to DNA binding and transcriptionalactivity have been reported. The results vary from phosphoryl-ation by protein kinase C without any changes in bindingaffinity (40), to phosphorylation by protein kinase A and in-crease of DNA binding activity and transcription (41, 42).Mammalian sterile 20-like 2 kinase (43) and extracellular sig-nal-regulated kinase (44) were also shown to phosphorylateT/EBP. In all cases, phosphorylation occurs at serine residues.To determine whether T/EBP is phosphorylated upon IL-10treatment, we performed the following experiments: 1) additionof the general kinase inhibitor staurosporin (1 �M) in culturemedia to examine its effect on IL-10 induced UGRP1 mRNAlevels; 2) in vivo phosphorylation assays; and 3) Western blot-ting to demonstrate the effect of IL-10 on the phosphorylationstatus of T/EBP (Supplemental Materials Fig. 1A). These re-

FIG. 6. ChIP analysis of T/EBP bind-ing to the UGRP1 gene promoter. A,schematic representation of T/EBP bind-ing sites (oval 1, 2, and 3) within �209 bpupstream of the UGRP1 gene promoter. Apair of primers F1/R1, F2/R2, and Ex3-F/Ex3-R used to amplify T/EBP bindingsites and UGRP1 exon 3 sequences as anonspecific control, are shown by arrows.B, representative ethidium bromidestaining of amplified products after ChIPwith mouse IgG (mIgG), no antibody (noAb), or T/EBP antibody (with Ab). Theinput DNAs prior to immunoprecipitationand purified human DNA (Clontech) wereused as a positive control. The negativecontrol contains no DNA. C, the resultsobtained in B were quantitated with anEagel Eye photodocumentation systemand plotted. The ChIP assay was repeatedtwice with the same result. Open bar, un-treated; closed bar, IL-10.

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sults, although limited, did not indicate an involvement ofT/EBP phosphorylation in the novel IL-10 signaling pathway.Furthermore, T/EBP is a nuclear protein and is located in thenucleus regardless of IL-10 treatment (Supplemental Materi-als Fig. 1, B and C), suggesting that T/EBP may not be aprotein that translocates to the nucleus upon IL-10 treatment.Alternatively, there is a possibility that an unknown proteinmay be phosphorylated upon IL-10 signaling, which thentranslocates to the nucleus and phosphorylates or binds di-rectly to T/EBP or T/EBP interacting protein(s) such as co-activators, resulting in enhancement of binding affinity ofT/EBP to its binding site and a subsequent increase in targetgene transcriptional activity. To understand the mechanism ofIL-10 induction of UGRP1 gene expression, additional experi-ments must be conducted as to determine: 1) whether phospho-rylation of T/EBP is indeed involved, and if so, what are thetype and site of phosphorylation and kinases involved; 2)whether other factors might be interacting with T/EBP totransduce IL-10 signals; and 3) what roles JAKs might play inthis novel signaling pathway.

Lastly, our findings may provide a rationale to further ex-ploring the possibility that the anti-inflammatory activity ofIL-10 is mediated through UGRP1 expression in lung tissues.Our current results clearly demonstrate that UGRP1 expres-sion is induced in lung epithelial cells by IL-10, a cytokineknown to have potent anti-inflammatory and immunoregula-

tory activities. We previously hypothesized that UGRP1 maybe involved in regulating lung inflammation (1, 14). In supportof our hypothesis, intranasally administered IL-10 reducedproduction of Th-2 cytokines such as IL-4 and IL-5, and eosi-nophilia in bronchoalveolar lavage fluid of ragweed-inducedallergic mice (45). Similarly, IL-10 abrogated ovalbumin-induced airway inflammation and tumor necrosis factor-�production (46). Interestingly, IL-10 induction of UGRP1gene expression is independent of the polymorphism in theUGRP1 gene promoter that was demonstrated to be associ-ated with an increased risk of asthma (14). Recently, macro-phage scavenger receptor with collagenous structure(MARCO) expressed by alveolar macrophages in the lung wasidentified as a receptor for UGRP1 (47). Lipopolysaccharide,a previously identified MARCO ligand, competes withUGRP1 for binding to MARCO and bacteria, thus suggestingthat the UGRP1-MARCO ligand-receptor pair is likely in-volved in inflammation and pathogen clearance in the lung.Whether UGRP1 is a mediator of anti-inflammatory activityof IL-10 awaits further experiments.

In conclusion, a novel IL-10 induction of UGRP1 gene ex-pression was found in lung epithelial cells that is mediated bya homeodomain transcription factor T/EBP. This signalingpathway might play a role in the anti-inflammatory activitiesof IL-10 in the lung.

FIG. 7. Analysis of IL-10 family signaling pathway. A, RT-PCR analysis for the presence of IL-10R1 and IL-10R2 in human monocytes andH441 cells. The size of PCR products are 291 and 300 bp for IL-10R1 and IL-10R2, respectively. The negative control is shown as H2O and withoutRT (�RT). B, RT-PCR analysis for the presence of IL-22R1 and IL-28R1 in human monocytes, H441 cells, human liver, and NCI-A549 cells.RT-PCR generated PCR products of 240 and 325 bp for IL-22R1 and IL-28R1, respectively. The negative control is shown as H2O and without RT(�RT). C, Western blot analysis of tyrosine phospho-STAT 3 (pYSTAT3) levels in H441 cells treated with IL-10, IL-19, IL-20, IL-22, and IFN-� for30 min. D, Northern blot analysis of UGRP1 mRNA levels in H441 cells treated with IFN-�, IL-10, IL-22, and IFN-�/IL-28 for 30 min. Human lungRNA was used as a positive control and �-actin for a loading control. E, anti-IL-10R1 antibody treatment. H441 cells were pretreated with orwithout a monoclonal antibody (3F9) before incubation with IL-10 (50 ng/ml) for 6 h. Human lung RNA was used as a positive control andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as a loading control in Northern blotting analysis.

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Acknowledgments—We thank Drs. Frank J. Gonzalez, PorntipSupavilai, and Amnuay Thithapandha for continuous support duringthe course of this work, Dr. Kyung S. Lee for help on the phosphoryla-tion assays, Drs. Atsushi Yamada, Takashi Kusakabe, Akihide Kamiya,and Kimihiko Matsusue for helpful advise and discussions, and Dr.Francisco DeMayo (Baylor College of Medicine) for advice on mouseembryo organ cultures.

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T/EBP/NKX2.1-dependent IL-10 Induction of UGRP1 Gene Expression54368

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Raymond P. Donnelly and Shioko KimuraAchara Srisodsai, Reiko Kurotani, Yoshihiko Chiba, Faruk Sheikh, Howard A. Young,

Lung Epithelial Cells through Homeodomain Transcription Factor T/EBP/NKX2.1Interleukin-10 Induces Uteroglobin-related Protein (UGRP) 1 Gene Expression in

doi: 10.1074/jbc.M405331200 originally published online October 13, 20042004, 279:54358-54368.J. Biol. Chem. 

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