Transforming Growth Factor-�-Smad Signaling PathwayCooperates with NF-�B to Mediate Nontypeable Haemophilusinfluenzae-induced MUC2 Mucin Transcription*

Received for publication, July 10, 2002, and in revised form, September 4, 2002Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M206883200

Hirofumi Jono‡, Tsuyoshi Shuto‡§, Haidong Xu‡, Hirofumi Kai§, David J. Lim‡,James R. Gum, Jr.¶, Young S. Kim¶, Shoji Yamaoka�, Xin-Hua Feng**, and Jian-Dong Li‡ ‡‡

From the ‡Gonda Department of Cell and Molecular Biology, House Ear Institute, and Department of Otolaryngology,University of Southern California, Los Angeles, California 90057, the §Department of Molecular Medicine,Kumamoto University, Kumamoto 862-0973, Japan, the ¶Gastrointestinal Research Laboratory, Veterans Affairs MedicalCenter and Department of Medicine, University of California, San Francisco, California 94143, the �Department ofMolecular Virology, Tokyo Medical and Dental University, Tokyo 113-8519 Japan, and the **Michael E. DeBakeyDepartment of Surgery and Department of Molecular and Cellular Biology, Baylor College of Medicine,Houston, Texas 77030

Transforming growth factor-� (TGF-�) and relatedfactors are multifunctional cytokines that regulatediverse cellular processes, including proliferation, dif-ferentiation, apoptosis, and immune response. The in-volvement of TGF-� receptor-mediated signaling in bac-teria-induced up-regulation of mucin, a primary innatedefensive response for mammalian airways, however,still remains unknown. Here, we report that the bacte-rium nontypeable Haemophilus influenzae (NTHi), animportant human respiratory pathogen, utilizes theTGF-�-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKK�/�-I�B� pathway to mediateNF-�B-dependent MUC2 mucin transcription. The NTHi-induced TGF-� receptor Type II phosphorylationoccurred at as early as 5 min. Pretreatment of NTHi withTGF-� neutralization antibody reduced up-regulation ofMUC2 transcription. Moreover, functional cooperationof NF-�B p65/p50 with Smad3/4 appears to positivelymediate NF-�B-dependent MUC2 transcription. Thesedata are the first to demonstrate the involvement ofTGF-� receptor-mediated signaling in bacteria-inducedup-regulation of mucin transcription, bring insightsinto the novel role of TGF-� signaling in bacterial patho-genesis, and may lead to new therapeutic interventionof NTHi infections.

The Gram-negative bacterium nontypeable Haemophilus in-fluenzae (NTHi)1 is an important human respiratory pathogen

in children and adults (1). In children, it causes otitis media(OM), the most common childhood infection and the leadingcause of conductive hearing loss (2, 3) whereas in adults, itexacerbates chronic obstructive pulmonary diseases (COPD),the fourth leading cause of death in the United States (4, 5). Ahallmark of both OM and COPD is mucus overproduction thatmainly results from up-regulation of mucin, a primary innatedefensive response for mammalian airways (6, 7). Mucins, themajor component of mucus secretions, are high molecularweight and heavily glycosylated proteins synthesized by themucosal epithelial cells lining the middle ear, trachea, diges-tive, and reproductive tracts (8). They protect the epithelialsurface by binding and trapping inhaled infectious particles,including bacteria and viruses, for mucociliary clearance, atleast in part because of the extraordinary diversity of theircarbohydrate side chains (6, 9). However, in patients with OMwith effusion and COPD whose mucociliary clearance mecha-nisms have become defective, excessive production of mucinwill lead to airway obstruction in COPD and conductive hear-ing loss in OM with effusion (8, 10, 11). To date, 14 mucin geneshave been identified (9, 10, 12, 13). Among these, at leastMUC2, MUC5AC, and MUC5B have been shown to play animportant role in the pathogenesis of respiratory infectiousdiseases. Understanding the signaling mechanisms underlyingup-regulation of mucin may open up novel therapeutic targetsfor these diseases.

In contrast to the relatively well known mechanism by whichMUC5AC mucin is up-regulated by NTHi (14), the signalingmechanism underlying NTHi-induced MUC2 mucin transcrip-tion remains totally unknown. Based on our previous studiesshowing that MUC2 is up-regulated by Gram-negative bacte-rium Pseudomonas aeruginosa in cystic fibrosis via activationof NF-�B (15, 16) and that NTHi, also a Gram-negative bacte-rium, strongly activates NF-�B (17), it is plausible that activa-tion of NF-�B might be also required for NTHi-induced MUC2up-regulation via specific signaling pathways.

In addition to the NF-�B pathway, the TGF-�-Smad path-way represents another important signaling pathway partici-pating in regulation of diverse biological processes, includingcell proliferation, differentiation, death, inflammatory, and

* This work was supported in part by grants from the NationalInstitutes of Health (RO1-DC04562) (to J. D. L.), CA24321 (to Y. S. K.and J. G.) and GM63773 (to X.-H. F.), the Department of VeteransAffairs Medical Research Service (to J. G. and Y. S. K.), American Can-cer Society Research Project Grant RPG-00214-01-CCG (to X.-H. F.),and the Henry L. Guenther Foundation (to D. J. L. and J. D. L.). 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.

‡‡ To whom correspondence should be addressed: the Gonda Dept. ofCell and Molecular Biology, House Ear Inst., University of SouthernCalifornia, 2100 W. 3rd St., Los Angeles, CA 90057. Tel.: 213-273-8083;Fax: 213-273-8088; E-mail: jdli@hei.org.

1 The abbreviations used are: NTHi, nontypeable Haemophilus influ-enzae; ELISA, enzyme-linked immunosorbent assay; SBE, Smad-binding element; TGF-�, transforming growth factor-�; COPD, chronicobstructive pulmonary diseases; OM, otitis media; R-Smad, receptor-activated Smad; T�R, TGF-� receptor; CAPE, caffeic acid phenethyl

ester; HM3, human colon epithelial cell line; HMEEC-1, human middleear epithelial cell line; NHBE, primary human bronchial epithelial cell;PAI-1, plasminogen activator inhibitor-1; MUC, mucin; TLR, Toll-likereceptor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 45547–45557, 2002Printed in U.S.A.

This paper is available on line at http://www.jbc.org 45547

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

immune responses (18–24). The TGF-� superfamily is a largegroup of secreted growth factors of which three subgroups havebeen defined: the TGF-�s, activins, and bone morphonegeticproteins (BMPs) (21, 23). TGF-� initiates signaling through theligand-dependent activation of a heteromeric complex of type IIand type I receptors. The type II receptor kinase then phospho-rylates the type I receptor in a conserved glycine-serine domain(GS domain), resulting in activation of the type I receptor. Theactivated type I receptor subsequently recognizes and phospho-rylates the Smad subgroup known as receptor-activated Smads(R-Smad), including Smad2 and Smad3. This causes dissocia-tion of R-Smad from the receptor, stimulates the assembly of aheteromeric complex between the phosphorylated R-Smad andthe Co-Smad, Smad4, and then induces the translocation of theSmad complex to the nucleus, where the Smad complex regu-lates the expression of target genes (19, 21). In addition to itsdirect interaction with Smad DNA-binding element (SBE),growing evidence suggests that Smads also regulate gene tran-scription by direct interaction and functional cooperation withother transcription factors, such as NF-�B (25–27). Despite itsimportant role in regulation of diverse biological processes, it isstill unclear if activation of the TGF-�-Smad signaling pathwayalso mediates up-regulation of mucin, a primary host innatedefensive response to bacteria.

Because of the important role of NF-�B and TGF-� signalingin mediating diverse cellular responses as well as the reportedfunctional cooperation between NF-�B and TGF-�-Smad, wehypothesized that the TGF-�-Smad signaling pathway cooper-ates with NF-�B to mediate up-regulation of MUC2 mucintranscription in response to NTHi infections in human epithe-lial cells. Here, we show that activation of TGF-� receptor-Smad3/4 signaling, together with TLR2-MyD88-TAK1-NIK-IKK�/�-I�B�-dependent activation of NF-�B, mediates NTHi-induced MUC2 mucin transcription. These findings provideddirect evidence, for the first time, that the bacterium NTHiuses the TGF-�-Smad pathway for transducing signals intonucleus, at least in part, via an autocrine-independent mecha-nism and that the functional cooperation between NF-�B andTGF-� receptor-Smad is required for host defensive response toNTHi. These studies may bring insights into the novel role ofTGF-�-Smad signaling in bacterial pathogenesis and may leadto novel therapeutic intervention for OM and COPD.

MATERIALS AND METHODS

Reagents—Caffeic acid phenethyl ester (CAPE) and MG-132 werepurchased from Calbiochem (La Jolla, CA). Recombinant humanTGF-�1 and TGF-� neutralization antibody were purchased from R&DSystems.

Bacterial Strains and Culture Conditions—NTHi strain 12, a clinicalisolate, was used in this study (14, 17, 28). Bacteria were grown onchocolate agar at 37 °C in an atmosphere of 5% CO2. For making NTHicrude extract, NTHi were harvested from a plate of chocolate agar afterovernight incubation and incubated in 30 ml of brain heart infusion(BHI) broth supplemented with NAD (3.5 �g/ml). After overnight incu-bation, NTHi were centrifuged at 10,000 � g for 10 min, and thesupernatant was discarded. The resulting pellet of NTHi was sus-pended in 10 ml of phosphate-buffered saline and sonicated. Subse-quently, the lysate was collected and stored at �70 °C. NTHi lysates (5�g/ml) were used in all the experiments. We chose to use NTHi lysatesbecause of the following reasons: First, NTHi has been shown to behighly fragile and has the tendency to autolyse. Its autolysis can betriggered in vivo under various conditions including antibiotic treat-ment (14, 17, 28). Therefore, using lysates of NTHi represents a com-mon clinical condition in vivo, especially after antibiotic treatment.

Cell Culture—Human colon epithelial cell line HM3 was maintainedin Dulbecco’s modified Eagle’s medium supplemented with 10% fetalbovine serum (Invitrogen) (14, 16). Human cervix epithelial cell lineHeLa and human middle ear epithelial cell line HMEEC-1 were main-tained as described (17, 28). Primary human bronchial epithelial cells(NHBE) were purchased from Clonetics (San Diego, CA). NHBE cellswere maintained in Clonetics’ recommended bronchial epithelial

growth media (BEGM), which includes supplements of bovine pituitaryextract, hydrocortisone, human recombinant epidermal growth factor,epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, gen-tamicin, and amphotericin B (Clonetics), to a confluence of 60–80%(37 °C, 5% CO2). Media were replaced every other day. Only cells atpassages 4 were used for experiments. Wild-type mink Mv1Lu cells andtwo cell lines, DR26 and R1B that are derived from Mv1Lu and lackfunctional T�RII and T�RI, respectively, were kindly provided by Dr.Joan Massague (Memorial Sloan-Kettering Cancer Center, New York),and were maintained as described previously (29). Wild-type rat Rat-1cells and a cell line R5 that is derived from Rat-1 and lacks functionalIKK� were maintained as described previously (30). All media receivedadditions of 100 units/ml penicillin and 0.1 mg/ml streptomycin.

Real-time Quantitative RT-PCR Analysis—Total RNA was isolatedfrom human epithelial cells using TRIzol® Reagent (Invitrogen) follow-ing the manufacturer’s instruction. For the real-time quantitative RT-PCR, predeveloped TaqMan assay reagents (Applied Biosystems) wereused. Synthesis of cDNA from total RNA samples was performed withMultiScribeTM reverse transcriptase. To normalize MUC2 expressionrelative to cDNA, we used primers and a TaqMan probe correspondingto cyclophilin. Expression of MUC2 was measured relative to cyclophi-lin. Primers and the TaqMan probe for MUC2 and cyclophilin weredesigned by using Primer Express software (Applied Biosystems) andsynthesized by Applied Biosystem Customer Oligo Synthesis Service(Applied Biosystems). TaqMan probes were labeled with FAM on the5�-end and TAMARA on the 3�-end. The primers and probes for MUC2were: forward primer, 5�-TCCATCCTGCTGACCATCAA-3� and reverseprimer, 5�-GTAGGCATCGCTCTTCTCAATGA-3�, and TaqMan probe,5�-FAM-TGACACCATCTACCTCACCCGCCATAMRA-3�. Reactionswere amplified and quantified using an ABI 7700 sequence detector andmanufacturer’s software (Applied Biosystems). Relative quantity ofMUC2 mRNA was obtained using Comparative CT Method (for details,see user Bulletin 2 for the ABI PRISM 7700 Sequence Detection Systemunder www.appliedbiosystems.com/support/tutorials) and was normal-ized using predeveloped TaqMan assay reagent human cyclophilin asan endogenous control (Applied Biosystems).

Plasmids, Transfections, and Luciferase Assays—The expressionplasmids I�B�(S32A/S36A), IKK�(K44M), IKK�(K49A), IKK�, NIK(K429A/K430A), TAK1 DN, MyD88 DN, hTLR2 DN, �GF�RIIDN andwild-type, �GF�RI DN and wild-type, Smad2 DN, Smad3DN and wild-type, Smad4 DN and wild-type were previously described (17, 27, 28,31–33). The reporter constructs, 5�-flanking region of the human MUC2gene, NF-�B-luc, SBE-Luc, and plasminogen activator inhibitor-1 (PAI-1)-Luc, were also previously described (16, 18, 27). All transient trans-fections were carried out in HM3 cells in triplicate using TransIT-LT1reagent (Mirus, Medison, WI) following the manufacturer’s instruction,unless otherwise indicated. In all co-transfections with either a wild-type or a dominant-negative mutant of signaling molecules, an emptyvector was used as a control. Transfected cells were pretreated with orwithout chemical inhibitors including CAPE and MG-132 for 2 h. NTHior recombinant human TGF-�1 was then added to the transfected cells42 h after transfection. After 5 h, the cells were harvested for luciferaseassay. In experiments using neutralization TGF-� antibody, NTHilysates were pretreated with either TGF-� neutralization antibody orcontrol antibody for 1 h before being added to the transfected cells for5 h.

Immunofluorescent Staining—Cells were cultured on 4-chamber mi-croscope slides. After NTHi treatment, the cells were fixed in paraform-aldehyde solution (4%), incubated with mouse anti-p65 NF-�B or mouseanti-Smad4 monoclonal antibodies for 1 h (Santa Cruz Biotechnology,Inc., Santa Cruz, CA). Primary antibody was detected with fluoresceinisothiocyanate (FITC)-conjugated goat anti-mouse IgG (Santa Cruz Bio-technology, Inc.). Samples were viewed and photographed using a ZeissAxiophot microscope.

Western Blot Analysis and ELISA Assays—Antibodies against phos-pho-I�B�(Ser-32), I�B� were purchased from Cell Signaling (Beverly,MA). Antibodies against phospho-T�RII (Tyr-336) and T�RII were pur-chased from Santa Cruz Biotechnology. Phosphorylation of I�B� andT�RII were detected as described and following the manufacturer’sinstructions (17). TGF-�1, 2, and 3 released from the cells were ana-lyzed by standard sandwich ELISA assays using ELISA kits as de-scribed and following the manufacturer’s instructions. TGF-�1 ELISAkit was purchased from BioSource Europe S. A. (Nivelles, Belgium).TGF-�2 and TGF-�3 ELISA kits were purchased from the R&D system.

Regulation of MUC2 by Nontypeable H. influenzae45548

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

RESULTS

NTHi Up-regulates MUC2 Mucin Transcription in HumanEpithelial Cells—We first examined whether NTHi up-regu-lates MUC2 mucin expression in human epithelial cells byperforming real-time quantitative PCR analysis. NTHistrongly up-regulates MUC2 expression at mRNA level in hu-man epithelial HM3 cells (Fig. 1A). To investigate whethertranscriptional regulation is involved in MUC2 induction, wenext transfected HM3 cells with an expression vector contain-ing 2.8 kb of the human MUC2 5�-flanking region fused to aluciferase reporter gene. When we exposed the transfected cellswith NTHi, the luciferase activity driven by the MUC2 pro-moter increased in a time- and dose-dependent manner, sug-gesting the involvement of transcriptional regulation (Fig. 1Band data not shown). Because we were interested in the poten-tial generality of NTHi-induced MUC2 up-regulation, we as-sayed a variety of human epithelial MUC2-expressing cell linesas well as primary cells. Results from HeLa, HM3, HMEEC-1,and NHBE cells are shown in Fig. 1, C and D. Interestingly,NTHi-induced up-regulation of MUC2 expression at both tran-scriptional and mRNA levels is well conserved among allhuman epithelial cell lines and primary epithelial cells that wetested. Thus, these findings indicate that NTHi up-regulatesMUC2 mucin transcription in human epithelial cells.

Activation of NF-�B Via a TLR2-MyD88-dependent TAK1-NIK-IKK�/�-I�B� Pathway Is Required for NTHi-inducedMUC2 Up-regulation—We next performed experiments to de-fine the NTHi-response element in the 5�-flanking region of thehuman MUC2 mucin gene and the related transcription factorsin the HM3 cell, the human epithelial cell line yielding thestrongest NTHi response. Analysis of luciferase activity from apanel of deletion mutants of the MUC2 promoter-luciferasereporter gene revealed a NTHi response element between basepairs �1528 and �1430 (Fig. 2A and data not shown). Subse-quent sequence analysis showed that this region contains aNF-�B binding site. Based on our recent report showing thatNTHi strongly activates NF-�B (17), we next explored the

possibility that activation of NF-�B is required for NTHi-in-duced MUC2 transcription by performing selective mutagene-sis of the NF-�B binding site. As shown in Fig. 2A, mutantconstructs M1, M2, and M3, in which the NF-�B site is mu-tated, markedly reduced the responsiveness of MUC2 promoterconstruct, whereas mutant construct M4 in which NF-�B siteremains intact did not reduce MUC2 induction. These resultssuggested that NF-�B activation is required for NTHi-inducedMUC2 transcription. Because nuclear translocation is a keystep for NF-�B to exert its transcriptional activity, we nextsought to determine whether NF-�B nuclear translocation isalso required for MUC2 induction. As shown in Fig. 2B (upperpanel), p65, a key subunit of NF-�B complex, was translocatedinto the nucleus upon exposure to NTHi as we reported previ-ously in HeLa cells. The NTHi-induced NF-�B translocationwas blocked by a chemical inhibitor CAPE, which is known tospecifically block the translocation of p65 without affectingI�B� degradation (Fig. 2B, upper panel) (34). Moreover, CAPEabrogated NTHi-induced MUC2 transcription (Fig. 2B, lowerpanel), confirming that nuclear translocation and activationof NF-�B is indeed required for NTHi-induced MUC2transcription.

Based on our recent report that TAK1-NIK-IKK�-dependentI�B� phosphorylation and degradation is required for NTHi-induced NF-�B activation, we next sought to determine theinvolvement of I�B� phosphorylation and degradation inMUC2 induction (17). We first investigated whether NTHiinduces I�B� phosphorylation and degradation in HM3 cells.As shown in Fig. 2C (upper panel), phosphorylation and deg-radation of I�B� was observed in HM3 cells treated with NTHi.The NTHi-induced I�B� degradation was blocked by MG132, aproteasome inhibitor that prevents the degradation of I�B�(35), whereas phosphorylated I�B� was no longer degraded inthe presence of MG-132 and thus persists in the cytoplasm. Wenext sought to determine the requirement of I�B� degradationby assessing the effect of MG132 on NF-�B nuclear transloca-tion and MUC2 induction. As expected, MG132 completely

FIG. 1. NTHi up-regulates humanMUC2 mucin transcription in humanepithelial cells. A, NTHi up-regulatesMUC2 expression at mRNA level in HM3cells as assessed by real-time quantitativeRT-PCR. B, NTHi up-regulates MUC2transcription in HM3 cells in a time-dependent manner. HM3 cells were tran-siently transfected with human MUC22.8-kb promoter luciferase reporter con-struct (pMUC2-2864luc) and stimulatedwith NTHi for various times as indicated.Luciferase activity was then assessed inNTHi-treated and -untreated cells. C,NTHi-induced up-regulation of MUC2transcription was observed in a variety ofhuman epithelial cell lines includingHM3, HeLa, and HMEEC-1 as well asNHBE. D, NTHi-induced MUC2 up-regulation was also observed at mRNAlevel in HM3, HeLa, and HMEEC-1 aswell as NHBE cells as assessed by real-time quantitative RT-PCR. Values are themeans � S.D. (n � 3).

Regulation of MUC2 by Nontypeable H. influenzae 45549

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 2. Activation of NF-�B via a TLR2-MyD88-dependent TAK1-NIK-IKK�/�-I�B� pathway is required for NTHi-induced MUC2up-regulation. A, human MUC2 regulatory regions (base pairs: �1528 to �1430) containing the wild-type (WT) or various mutated sites withinthe region �1458/�1430 as indicated were subcloned upstream of the TK-32 promoter in a luciferase vector (designated M1 to M4) and transfectedinto HM3 cells. B, CAPE (20 �g/ml), a chemical inhibitor that is known to specifically block the translocation of p65 without affecting I�B�degradation, inhibits NTHi-induced NF-�B nuclear translocation and MUC2 transcription. C, MG-132 (1 �M), a specific proteosome inhibitor,blocks NTHi-induced I�B� degradation, NF-�B nuclear translocation, and up-regulation of MUC2 transcription. In addition, overexpression of atransdominant mutant of I�B� (S32/36A) also inhibits NTHi-induced MUC2 up-regulation. D, co-expression of dominant-negative mutants ofIKK� (K44A), NIK (K429A/K430A), and TAK1, but not IKK�, inhibits NTHi-induced MUC2 transcription. E, NTHi-induced MUC2 transcription

Regulation of MUC2 by Nontypeable H. influenzae45550

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

blocked NTHi-induced NF-�B translocation in HM3 cells (Fig.2C, middle panel). Concomitantly, MUC2 induction was alsoinhibited by MG132 (Fig. 2C, lower panel). Moreover, overex-pression of a transdominant mutant of I�B� (S32A/S36A)greatly inhibited MUC2 induction, further confirming theinvolvement of I�B� degradation in MUC2 induction (36).Finally, overexpression of a dominant-negative mutant form ofeither IKK� or NI� or TAK1, but not IKK�, markedly inhibitedMUC2 induction (Fig. 2D). Consistent with these results, R5, acell line that is derived from Rat-1 cells and lacks functionalIKK�, did not show NTHi-induced transcriptional activationfrom both the NF-�B-driven promoter and MUC2 promoter(30). Their responsiveness to NTHi could be rescued by co-transfection of wild-type IKK� in R5 cells, similarly to theresponse of wild-type Rat-1 cells (Fig. 2E). Taken together,these findings demonstrate that NTHi induces MUC2 up-reg-ulation via a TAK1-NIK-IKK�/�-dependent I�B� phosphoryla-tion and degradation, which, in turn, leads to NF-�B nucleartranslocation and activation.

Having identified TAK1-NIK-IKK�/�-I�B�-dependent acti-vation of NF-�B involved in NTHi-induced MUC2 transcrip-tion, still unknown is which cell surface receptor(s) is involvedin transmitting signals from cell surface to the cytoplasm inMUC2 induction. Because of the important role of Toll-likereceptor 2 (TLR2) in mediating bacteria-induced NF-�B acti-vation (17), we sought to first determine the involvement ofTLR2 in NTHi-induced MUC2 transcription. As shown in Fig.2F, overexpression of a dominant-negative mutant of humanTLR2 inhibited MUC2 induction. Similarly, co-transfectionwith a dominant-negative mutant MyD88, a key adaptor pro-tein downstream of TLR2, also inhibited MUC2 induction (37).These data indicate that the TLR2-MyD88 signaling pathwayis also involved in MUC2 induction.

Because NTHi is a major bacterial pathogen causing middleear and airway infections, we next determined whether thissignaling pathway also mediates NTHi-induced MUC2 tran-scription in HMEEC-1 and NHBE. As shown in Fig. 2G, over-expressing dominant-negative mutants of I�B�, IKK�, TAK1,MyD88, and TLR2 inhibits NTHi-induced MUC2 transcriptionin both HMEEC-1 (left panel) and NHBE cells (right panel).Thus, it is clear that NTHi-induced MUC2 up-regulation viathe TLR2-MyD88-TAK1-NIK-IKK-I�B�-NF-�B signaling path-way is also well conserved in the relevant human middle earand bronchial epithelial cells.

To further confirm whether the endogenous MUC2 gene andMUC2 promoter-driven luciferase reporter gene are similarlyup-regulated, we evaluated the effects of overexpressing dom-inant-negative mutants of several key signaling moleculesidentified above, including I�B�, TAK1, and MyD88. All thesetreatments inhibited NTHi-induced up-regulation of MUC2mRNA, confirming that the TLR2-MyD88-TAK1-NIK-IKK-I�B�-NF-�B signaling pathway revealed by luciferase reportergene assays are indeed responsible for induction of endogenousMUC2 gene expression by NTHi (Fig. 2H).

Activation of TGF-� Receptor I/II-Smad3/4 Signaling Path-way Is Also Required for NTHi-induced MUC2 Transcription—Because of the important role of TGB-� signaling in regulationof diverse cellular responses, we were interested in determin-ing whether TGF-� receptor signaling is also required for

NTHi-induced MUC2 transcription. We first examined if NTHiactivates TGF-�-Smad signaling by evaluating NTHi-inducednuclear translocation of Smad4, a key step for Smad3/4 com-plex to exert its transcriptional activity (18), As seen in Fig. 3A,it is evident that NTHi potently induces nuclear translocationof Smad4. As expected, Smad4 translocation was also inducedby TGF-�1. To further confirm whether NTHi activates TGF-�-Smad-dependent transcriptional activity, we assessed theeffect of NTHi on SBE-dependent promoter activity by usingSBE luciferase reporter (38) and TGF-�-responsive promoteractivity of a PAI-1-Luc in HM3 cells (39). When we exposed thetransfected cells to NTHi or TGF-�1, SBE-driven luciferaseactivity greatly increased in cells treated with NTHi or TGF-�1(Fig. 3A), confirming that NTHi indeed activates the TGF-�-Smad signaling pathway. Next, we investigated the require-ment of TGF-� signaling in NTHi-induced MUC2 transcriptionby overexpressing dominant-negative mutant of T�RII (27, 32,33). Interestingly, co-transfection with dominant-negativeT�RII greatly inhibited NTHi-induced MUC2 transcription(Fig. 3B). Concomitantly, overexpression of a dominant-nega-tive mutant of T�RI also blocked MUC2 induction (Fig. 3B). Inaccordance with these results, no NTHi-induced MUC2 tran-scription was shown in either R1B or DR26 cells, two cell linesthat are derived from Mv1Lu cells and lack functional T�RIand T�RII, respectively (29), whereas the wild-type Mv1Lucells still showed potent MUC2 induction by NTHi (Fig. 3C).Moreover, co-transfecting R1B and DR26 cells with wild-typeT�RI and T�RII expression plasmids rescued the responsive-ness to NTHi (Fig. 3C). Thus, these data suggest the require-ment of TGF-� receptor type I/II signaling in NTHi-inducedMUC2 transcription.

To determine the involvement of Smad2/3-Smad4 in MUC2induction, dominant-negative mutants of Smad2, 3, and 4 wereco-transfected into HM3 cells with MUC2 luciferase reporterconstruct (27). As shown in Fig. 3D, overexpression of a domi-nant-negative mutant of Smad3 or Smad4, but not Smad2,greatly inhibited NTHi-induced MUC2 transcription, suggest-ing the involvement of Smad3/4 in MUC2 induction.

To address whether the TGF-�-Smad signaling pathway alsomediates NTHi-induced MUC2 transcription in HMEEC-1 andNHBE, we next assessed the effects of overexpressing domi-nant-negative mutants of T�RII and Smad4 on NTHi-inducedMUC2 transcription. Interestingly, all these treatments inhib-ited NTHi-induced up-regulation of MUC2 in both HMEEC-1(Fig. 3E, left panel) and NHBE cells (right panel), confirmingthat this signaling pathway is indeed well conserved in therelevant human middle ear and bronchial epithelial cells.

To further confirm whether the endogenous MUC2 gene andMUC2 promoter-driven luciferase reporter gene are up-regu-lated similarly by the TGF-�-Smad signaling pathway identi-fied above, we evaluated the effects of overexpressing domi-nant-negative mutants of T�RII and Smad4 on NTHi-inducedMUC2 induction. All these treatments inhibited NTHi-inducedup-regulation of MUC2 mRNA, confirming that the T�RII-Smad4 signaling pathway revealed by luciferase reporter geneassays is indeed responsible for induction of endogenous MUC2gene expression by NTHi (Fig. 3F).

NTHi Activates TGF-�-Smad Signaling Likely Via an Auto-crine-independent Mechanism—Although we have demon-

requires IKK�. An NF-�B-regulated luciferase reporter plasmid or a MUC2 �1.5/�1.3 TK-luciferase reporter vector was transfected into wild-typeRat-1 cells expressing IKK�, or the derivative R5 cells lacking functional IKK�, respectively. Expression plasmid of wild-type IKK� wasco-transfected, as marked. F, co-expression of a dominant-negative mutant of TLR2 or MyD88 inhibits NTHi-induced MUC2 transcription. G,co-expression of dominant-negative mutants of I�B�, IKK�, TAK1, MyD88, and TLR2 also inhibits NTHi-induced MUC2 transcription inHMEEC-1 (left panel) and NHBE cells (right panel). H, co-expression of dominant-negative mutants of I�B�, TAK1, and MyD88 attenuateNTHi-induced MUC2 up-regulation at mRNA level. In all the experiments shown above, transfections were carried out in triplicate. NTHi wasadded to the transfected cells for 5 h before being lysed for luciferase assay. Values are the means � S.D. (n � 3).

Regulation of MUC2 by Nontypeable H. influenzae 45551

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 3. Activation of the TGF-� receptor I/II-Smad3/4 signaling pathway is also required for NTHi-induced MUC2 transcription. A,NTHi and TGF-�1 induce nuclear translocation of Smad4, Smad-regulated promoter activity of SBE-Luc and TGF-�-responsive promoter activity

Regulation of MUC2 by Nontypeable H. influenzae45552

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

strated that activation of the TGF-�-Smad signaling pathwayis required for NTHi-induced MUC2 transcription, it is stillunclear whether TGF-� signaling is activated directly byNTHi-derived TGF-�-like factor or indirectly by NTHi-inducedTGF-� autocrine signaling. We first evaluated the time courseof NTHi-induced phosphorylation of T�RII by using an anti-body against phosphorylated tyrosine at position 336 of T�RII.Fig. 4A shows phosphorylation of T�RII in cells treated withNTHi for various times. The NTHi-induced T�RII phosphoryl-ation became evident at as early as 5 min. Given such an earlyphosphorylation of T�RII, it is likely that the early phospho-rylation of T�RII occurred as a result of direct activation of T�Rsignaling by NTHi, rather than NTHi-induced TGF-� autocrinesignaling. To determine whether NTHi-derived TGF-�-likefactor is responsible for the activation of TGF-� signaling, weassessed the effect of NTHi lysates pretreated with TGF-�neutralization antibody or control antibody on the transcrip-tional activity of MUC2 promoter. As shown in Fig. 4B, TGF-�1-induced PAI-1 promoter activity was attenuated by TGF-�neutralization antibody treatment, thus validating the effi-ciency of the TGF-� neutralization antibody in blocking TGF-�signaling. Pretreatment of NTHi lysates with the same TGF-�antibody reduced its ability to induce MUC2 transcription,thereby suggesting that a direct activation of T�R-mediatedsignaling by NTHi mediates MUC2 transcription. To furtherdetermine whether TGF-� signaling is also activated indirectlyby NTHi-induced TGF-� autocrine signaling, we next deter-mined whether NTHi induces any increase in three majorTGF-� family members, TGF-�1, 2, and 3, in the conditionedmedia of HM3 cells using TGF-�1, 2, and 3 ELISA kits. Nota-bly, NTHi did not induce any detectable increase in TGF-�1, 2,and 3 (Fig. 4C). Together, these data suggest that NTHi-induced MUC2 transcription is mediated by the TGF-�-Smadsignaling pathway, at least in part, via a mechanism independ-ent of TGF-�1, 2, and 3 autocrine signaling, although our datado not preclude the involvement of the latent TGF-�s stored inthe extracellular matrix that might be activated by NTHi andthen cross-talk with T�RI and T�RII. In addition, it is stillunclear whether other TGF-� family members may be involvedin mediating NTHi-induced MUC2 transcription in an auto-crine manner.

Functional Cooperation of Smad3/4 with NF-�B p65/p50Appears to Mediate NF-�B-dependent MUC2 Transcription—Because our data (Fig. 2) demonstrated that activation ofNF-�B is required for NTHi-induced MUC2 transcription andno functional SBE was found within the functional promoterregion of MUC2, we next determined whether the TGF-�-Smadsignaling pathway mediates MUC2 transcription via its func-tional interaction with NF-�B (26). We first assessed the effectof overexpressing dominant-negative mutants of TGF-� recep-tor type II and Smad4 on NTHi-induced NF-�B-driven pro-moter activity. As seen in Fig. 5A, NTHi-induced NF-�B acti-vation was abrogated by both treatments, indicating the directinvolvement of TGF-�-Smad signaling in NTHi-induced NF-�Bactivation. We next investigated whether activation of the

TGF-� signaling pathway contributes to NTHi-induced pro-moter activity of both MUC2-luc and NF-�B-luc in a similarway. As shown in Fig. 5B, activation of TGF-� signaling byTGF-�1 or co-expression of wild-type Smad3 and Smad4greatly enhanced NTHi-induced NF-�B activation as well asMUC2 transcription. These data indicate that the TGF-�-Smadsignaling pathway mediates MUC2 transcription likely viacross-talk with NF-�B, rather than with other DNA responseelements in MUC2 promoter. However, it is still unclear whichNF-�B subunits are involved in mediating its interaction withSmad3 and Smad4.

Based on the importance of NF-�B subunits p65 and p50 inmediating a variety of cellular responses, we first overex-pressed Smad3 and Smad4, both alone and in concert withNF-�B subunits p65 and p50 and then assessed their NF-�Btransactivation potential. As expected, overexpression of eitherp65 alone, or together with p50, induced potent NF-�B reportergene activation (lanes 3 and 4), whereas overexpression ofeither p50, or Smad3 or Smad4 alone only induced weak NF-�Breporter gene activation (Fig. 5C, lanes 2, 5, and 6). Interest-ingly, co-expression of p65 but not p50 with Smad3, or Smad4induced relatively potent NF-�B-dependent promoter activity(lanes 8–11). Moreover, co-expression of Smad3 and Smad4also induced potent NF-�B activation (lane 7), which was fur-ther greatly enhanced by co-transfection with p65 but not withp50 (lanes 14 and 15). It should be noted that, although over-expression of p50 alone did not appear to activate NF-�Bpotently, it greatly enhanced NF-�B reporter gene activationinduced by co-expression of either p65 and Smad3 or Smad4(lanes 12 and 13) or p65, Smad3, and Smad4 (lane 16). Takentogether, these results indicate that, at least p65, p50, Smad3,and Smad4 are involved in the functional cooperation of NF-�Bwith the TGF-� signaling pathway.

DISCUSSION

Activation of TGF-�-Smad Signaling by Bacterium NTHiAppears to be Required for Up-regulation of MUC2 Mucin inHuman Epithelial Cells—TGF-� and related factors are multi-functional cytokines that regulate diverse cellular processes,including proliferation, differentiation, apoptosis, and immuneresponse (19–24). However, the activation of TGF-� receptor-mediated signaling in bacteria-induced up-regulation of mucin,a primary innate defensive response for mammalian airways,has not been reported. Here, we provided clear evidence thatactivation of the TGF-� receptor-Smad3/4 signaling pathwayby Gram-negative bacterium NTHi is required for induction ofMUC2 mucin transcription in human epithelial cells. Severallines of evidence strongly support this notion. First, overex-pression of dominant-negative mutants of T�RI, T�RII,Smad3, and Smad4 greatly inhibited NTHi-induced MUC2transcription (Fig. 3, B and D–F). In accordance with theseresults, no NTHi-induced MUC2 transcription was shown ineither R1B or DR26 cells, mutant Mv1Lu cells that lack func-tional T�RI and T�RII, respectively (29), whereas the wild-typeMv1Lu cells still showed potent MUC2 induction by NTHi (Fig.

of PAI-1-Luc in HM3, a human epithelial cell line that has been shown to express both TGF-� receptor types I and II (data not shown).Representative fields of Smad4 fluorescence (upper panel) are shown in HM3 cells that are treated with NTHi or TGF-�1 (1 ng/ml) for 45 min,respectively. B, co-expression of dominant-negative mutants of TGF-� receptor I/II inhibits NTHi-induced MUC2 up-regulation. Human MUC2�1.5/�1.3 TK-luciferase reporter vector was co-transfected into HM3 cells with either an empty vector or dominant-negative mutants of TGF-�receptors I and II as indicated. The cells were then stimulated with NTHi for 5 h before being harvested for measurement of luciferase activity.C, NTHi-induced MUC2 transcription requires TGF-� receptors I and II. A MUC2 �1.5/�1.3TK-luciferase reporter vector was transfected intowild-type Mv1Lu cells expressing T�RI and T�RII or the derivative R1B and DR26 cells lacking functional T�RI and T�RII, respectively.Expression plasmid of wild-type T�RI or T�RII was co-transfected, as marked. D, co-expression of dominant-negative mutants of Smad3 andSmad4, but not Smad2, inhibits NTHi-induced MUC2 up-regulation. E, co-expression of dominant-negative mutants of T�RII and Smad4 alsoinhibits NTHi-induced MUC2 transcription in HMEEC-1 (left panel) and NHBE cells (right panel). F, co-expression of dominant-negative mutantsof T�RII and Smad4 attenuate NTHi-induced MUC2 up-regulation at mRNA level. In all the experiments shown above, transfections were carriedout in triplicate. NTHi was added to the transfected cells for 5 h before being lysed for luciferase assay. Values are the means � S.D. (n � 3).

Regulation of MUC2 by Nontypeable H. influenzae 45553

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

3C). Moreover, co-transfecting R1B and DR26 cells with wild-type T�RI and T�RII expression plasmids rescued the respon-siveness to NTHi. These observations implicated the require-ment of TGF-� receptor-Smad signaling in NTHi-inducedMUC2 up-regulation. Second, NTHi potently induced phospho-rylation of T�RII, nuclear translocation of Smad4 and TGF-�responsive PAI-1 promoter activity as well as SBE-driven pro-moter activity, demonstrating the ability of NTHi in activatingthe TGF-� signaling pathway (Figs. 3A and 4A). Finally, theNTHi-induced T�RII phosphorylation occurred at as early as 5min, suggesting that NTHi may activate T�R-mediated signal-

ing via a TGF-� autocrine-independent mechanism. AlthoughT�RI and T�RII are known as serine/threonine kinases, thereis also evidence that T�RII can function as a dual specificitykinase and tyrosine phosphorylation may have an importantrole in T�R signaling (40). Thus, NTHi-induced tyrosine phos-phorylation at 5 min may be at least interpreted as a T�R-mediated early response to NTHi independent of TGF-� auto-crine. Additionally, pretreatment of NTHi lysates with theneutralization TGF-� antibody reduced its ability to induce thetranscriptional activity of MUC2 promoter (Fig. 4B). Moreover,as evidenced by our ELISA experiments (Fig. 4C), NTHi did

FIG. 4. NTHi activates TGF-�-Smad signaling likely via an autocrine-independent mechanism. A, NTHi induces phosphorylation ofT�RII in HM3 cells in a time-dependent manner as assessed using an antibody against phosphorylated T�RII (Tyr-336). B, pretreatment of TGF-�1(1 ng/ml) or NTHi lysates with the neutralization TGF-� antibody reduced its ability to induce the transcriptional activity of the PAI-1 promoterand MUC2 promoter in HM3 cells. C, NTHi did not induce any detectable increase in TGF-�1, 2, and 3 in the conditioned media of HM3 cells asassessed using TGF-�1, 2, and 3 ELISA kits.

Regulation of MUC2 by Nontypeable H. influenzae45554

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 5. Functional cooperation ofSmad3/4 with NF-�B p65/p50 appearsto mediate NF-�B-dependent MUC2transcription. A, co-expression of a dom-inant-negative mutant of TGF-� receptorII or Smad4 blocks NTHi-induced NF-�Bactivation. NF-�B luciferase reporter vec-tor was co-transfected into HM3 cellswith either an empty vector or a domi-nant-negative mutant of TGF-� receptorII or Smad4. B, activation of TGF-� sig-naling by TGF-�1 (1 ng/ml) or co-expres-sion of wild-type Smad3 and Smad4greatly enhanced NTHi-induced NF-�Bactivation as well as MUC2 transcription.C, effects of overexpression of Smad3 andSmad4 on p65 and/or p50-induced NF-�Bactivation. In these experiments, lucifer-ase reporter vector was co-transfectedinto HM3 cells with either an empty vectoror vectors containing wild-type Smad3,Smad4, p50, and p65 as indicated. Valuesare the means � S.D. (n � 3).

Regulation of MUC2 by Nontypeable H. influenzae 45555

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

not induce any detectable increase in TGF-�1, 2 and 3 in theconditioned media of the epithelial cells. Collectively, thesedata suggest that NTHi-induced MUC2 transcription is medi-ated, at least in part, by direct activation of the TGF-�-Smadsignaling pathway, independent of TGF-�1, 2, and 3 autocrinesignaling, although our data do not preclude the involvement ofthe latent TGF-�s stored in the extracellular matrix that mightbe activated by NTHi and then cross-talk with T�RI and T�RII(Fig. 6). In addition, it is still unclear whether other TGF-�family members may be involved in mediating NTHi-inducedMUC2 transcription in an autocrine manner. Previously, therehas been a report for the involvement of parasite Trypanosomacruzi-derived TGF-�-like factors in T. cruzi invasion (41).Given the fact that bacterial lysate of NTHi was used in ourstudies, it is possible that a bacterial-derived TGF-�-like prod-uct may be responsible for directly inducing TGF-� receptorsignaling. It should be noted that lipooligosaccharide fromNTHi did not significantly up-regulate MUC2 transcription.Therefore, the molecular identity of NTHi-derived TGF-�-likefactors should be further investigated. Additionally, we willalso determine whether NTHi also activates the latent TGF-�stored in the extracellular matrix that, in turn, leads to theactivation of TGF-� signaling.

Functional Cooperation of Smad with NF-�B Is Positively

Involved in Mediating NTHi-induced Up-Regulation of MUC2Transcription—In addition to the activation of the TGF-� sig-naling pathway by NTHi for MUC2 induction, another inter-esting finding in this study is the functional cooperation of theTGF-�-Smad signaling pathway with NF-�B to positivelymediate MUC2 up-regulation by NTHi. In contrast to the roleof TGF-� signaling in suppressing the killing activity of macro-phage and enhancing intracellular proliferation of infectiouspathogen such as Leishmania, activation of TGF-� signalingactually cooperates with NF-�B to positively mediate hostdefensive responses to bacterium NTHi. Recently, growing ev-idence suggests that the TGF-�-Smad signaling pathway reg-ulates gene transcription either by functional cooperation withtranscription factors bound to adjacent transcription factors ordirectly interacting with transcription factors bound to DNAresponse element (21–22). For instance, functional cooperationbetween Smad and NF-�B that are bound to Smad- and TNF�-response elements, respectively, activates the expression of anextracellular matrix-related gene, COL7A1 (42). In contrast,Smad3 has been shown to stimulate transcription from theHIV-1 LTR promoter via an interaction with NF-�B bound to a�B site (43). Similarly, a �B site has also been identified as aTGF-�-responsive region in the 3�-downstream junB promoterregion (26). Concurrent with the later cases, the TGF-�-Smadsignaling pathway mediates NTHi-induced MUC2 transcrip-tion also via a �B site in the 5�-upstream MUC2 promoterregion, providing the first identification of a functional cooper-ation between Smad and NF-�B to positively mediate bacterial-induced transcription of a host defense gene.

We provided evidence for the involvement of NF-�B p65, p50,Smad3, and Smad4 in the functional interaction betweenNF-�B and TGF-� signaling pathways. It should be noted that,in contrast to the potent activation of NF-�B induced by over-expression of p65 and Smad3/4, both alone and in concert withone another, the NF-�B transactivation potential of p50 israther weak. In addition, NF-�B activation induced by co-expression of p65 with Smad4 is more potent than that inducedby co-transfection of p65 with Smad3. Our data are consistentwith the recent report by Lopez-Rovira et al. (26) showing thatoverexpression of Smad3 and Smad4 enhances transactivationof NF-�B and co-expression of Smad3 or Smad4 together withthe NF-�B subunit p65 further increases those responses. Ad-ditionally, they showed that co-expression of Smad3 or Smad4with NF-�B p52 also further greatly enhances NF-�B activa-tion. Thus, it seems necessary to further explore the involve-ment of NF-�B p52 in the functional cooperation betweenNF-�B and TGF-� signaling involved in NTHi-induced MUC2transcription.

In conclusion, our studies demonstrate that bacterium NTHiutilizes the TGF-� receptor type I/II-Smad3/4 signaling path-way together with the TLR2-MyD88-TAK1-NIK-IKK�/�-I�B�

pathway to induce NF-�B-dependent MUC2 mucin transcrip-tion, a primary innate defensive response for mammalian air-ways. Functional cooperation of NF-�B p65/p50 with TGF-�-Smad3/4 is likely involved in mediating NTHi-induced MUC2transcription. These studies provide evidence for the first timethat demonstrate the activation of TGF-� receptor-mediatedsignaling in bacteria-induced up-regulation of mucin transcrip-tion, bring insights into the novel role of TGF-� signaling inbacterial pathogenesis, and may lead to new therapeutic inter-vention of NTHi infections.

Acknowledgments—We thank A. S. Baldwin for p65 and p50 expres-sion plasmids and J. Massague for mutant mink cell lines DR26 andR1B.

FIG. 6. Schematic representation of NTHi-induced signalingpathways involved in MUC2 up-regulation in human epithelialcells. NTHi, an important human respiratory pathogen, utilizes theTGF-� receptor type I/II-Smad3/4 signaling pathway together with theTLR2-MyD88-TAK1-NIK-IKK�/�-I�B� pathway to mediate NF-�B-de-pendent MUC2 mucin transcription. NTHi-induced MUC2 transcrip-tion appears to be mediated, at least in part, by direct activation of theTGF-�-Smad signaling pathway, independent of TGF-�1, 2, and 3 au-tocrine signaling. Moreover, functional cooperation of NF-�B p65/p50with Smad3/4 appears to positively mediate NF-�B-dependent MUC2transcription. Up-regulation of mucin production, known as a primaryinnate defensive response for mammalian airways, contributes signifi-cantly to mechanical clearance of inhaled infectious particles. However,in patients with OM with effusion and COPD whose mucociliary clear-ance mechanisms have become defective, excessive production of mucinwill lead to airway obstruction in COPD and conductive hearing loss inOM with effusion.

Regulation of MUC2 by Nontypeable H. influenzae45556

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

REFERENCES

1. Foxwell, A. R., Kyd, J. M., and Cripps, A. W. (1998) Microbiol. Mol. Biol. Rev.62, 294–308

2. Rao, V. K., Krasan, G. P., Hendrixson, D. R., Dawid, S., and St Geme, J. W., III(1999) FEMS Microbiol. Rev. 23, 99–129

3. Sethi, S., and Murphy, T. F. (2000) N. Engl. J. Med. 343, 1969–19704. ATS Statement (1995) Am. J. Respir. Crit. Care Med. 152, 78S-121S.5. Murphy, T. F. (2000) Semin. Respir. Infect. 15, 41–516. Knowles, M. R., and Boucher, R. C. (2002) J. Clin. Invest. 109, 571–5777. Lemjabbar, H., and Basbaum, C. (2002) Nat. Med. 8, 41–468. Kim, W. D. (1997) Eur. Respir. J. 10, 1914–19179. Rose, M. C., Nickola, T. J., and Voynow, J. A. (2001) Am. J. Respir. Cell Mol.

Biol. 25, 533–53710. Basbaum, C., Lemjabbar, H., Longphre, M., Li, D., Gensch, E., and

McNamara, N. (1999) Am. J. Respir. Crit. Care. Med. 160, S44–4811. Carrie, S., Hutton, D. A., Birchall, J. P., Green, G. G., and Pearson, J. P. (1992)

Acta Otolaryngol. 112, 504–51112. Kim, Y. S., and Gum, J. R., Jr. (1995) Gastroenterology 109, 999–100113. Gendler, S. J., and Spicer, A. P. (1995) Annu. Rev. Physiol. 57, 607–63414. Wang, B., Lim, D. J., Han, J., Kim, Y. S., Basbaum, C. B., and Li, J. D. (2002)

J. Biol. Chem. 277, 949–95715. Li, J. D., Dohrman, A., Gallup, M., Miyata, S., Gum, J., Kim, Y., Nadel, J.,

Prince, A., and Basbaum, C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,967–972

16. Li, J. D., Feng, W. J., Gallup, M. G., Gum, J., Kim, Y., and Basbaum, C. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 5718–5723

17. Shuto, T., Xu H., Wang, B, Han, J., Kai, H., Gu, X. X., Murphy, T., Lim, D. J.,and Li, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 8774–8779

18. Derynck, R., and Feng, X.-H. (1997) Biochim. Biophys. Acta 1333, F105-15019. Derynck, R, Zhang, Y., and Feng, X.-H. (1998) Cell 95, 737–74020. Piek, E., Heldin, C. H., and Ten Dijke, P. (1999) FASEB J. 13, 2105–212421. Massague, J., and Wotton, D. (2000) EMBO J. 19, 1745–175422. Massague, J. (2000) Nat. Rev. Mol. Cell. Biol. 1, 169–17823. Hill, C. S. (2001) Curr. Opin. Genet. Dev. 11, 533–54024. Roberts, A. B. (2002) Cytokine Growth Factor Rev. 13, 3–5

25. Zhang, Y., Feng, X.-H., and Derynck, R. (1998) Nature 394, 909–91326. Lopez-Rovira, T., Chalaux, E., Rosa, J. L., Bartrons, R., and Ventura, F. (2000)

J. Biol. Chem. 275, 28937–2894627. Feng, X.-H., Lin, X., and Derynck, R. (2000) EMBO J. 19, 5178–519328. Shuto, T., Imasato, A., Jono, H., Sakai, A, Xu, H., Watanabe, T., Rixter, D. D.,

Kai, H., Andalibi, A., Linthicum, F., Guan Y. L., Han, J., Cato, A. C., Lim,D. J., Akira, S., and Li, J. D. (2002) J. Biol. Chem. 277, 17263–17270

29. Laiho, M., Weis, M. B., and Massague, J. (1990) J. Biol. Chem. 265,18518–18524

30. Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T, Weil, R., Agou, F., Kirk,H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 1231–1240

31. Beg, A. A., Ruben, S. M., Scheinman, R. I., Haskill, S., Rosen, C. A., andBaldwin, A. S., Jr. (1992) Genes Dev. 6, 1899–1913

32. Feng, X.-H., Filvaroff, E. H., and Derynck, R. (1995) J. Biol. Chem. 270,24237–24245

33. Feng, X.-H., and Derynck, R. (1997) EMBO J. 16, 3912–392334. Natarajan, K., Singh, S., Burke, T. R., Jr., Grunberger, D., and Aggarwal, B. B.

(1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9090–909535. Neish, A. S., Gewirtz, A. T., Zeng, H., Young, A. N., Hobert, M. E., Karmali, V.,

Rao, A. S., and Madara, J. L. (2000) Science 289, 1560–156336. Beauparlant, P., Kwon, H., Clarke, M., Lin, R., Sonenberg, N., Wainberg, M.,

and Hiscott, J. (1996) J. Virol. 70, 5777–578537. Horng, T., and Medzhitov R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,

12654–1265838. Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague, J., and Pavletich,

N. P. (1998) Cell 94, 585–59439. Keeton, M. R., Curriden, S. A., van Zonneveld, A. J., and Loskutoff, D. J. (1991)

J. Biol. Chem. 266, 23048–2305240. Lawler, S, Feng, X. H., Chen, R. H., Maruoka, E. M., Turck, C. W., Griswold-

Prenner, I., and Derynck, R. (1997) J. Biol. Chem. 272, 14850–1485941. Ming, M., Ewen, M. E., and Pereira, M. E. (1995) Cell 82, 287–29642. Kon, A., Vindevoghel, L., Kouba, D. J., Fujimura, Y., Uitto, J., and Mauviel, A.

(1999) Oncogene 18, 1837–184443. Li, J. M., Shen, X., Hu, P. P., and Wang, X. F. (1998) Mol. Cell. Biol. 18,

110–121

Regulation of MUC2 by Nontypeable H. influenzae 45557

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Gum, Jr., Young S. Kim, Shoji Yamaoka, Xin-Hua Feng and Jian-Dong LiHirofumi Jono, Tsuyoshi Shuto, Haidong Xu, Hirofumi Kai, David J. Lim, James R.

Mucin Transcription MUC2-induced Haemophilus influenzaeMediate NontypeableB toκ-Smad Signaling Pathway Cooperates with NF-βTransforming Growth Factor-

doi: 10.1074/jbc.M206883200 originally published online September 16, 20022002, 277:45547-45557.J. Biol. Chem. 

  10.1074/jbc.M206883200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/277/47/45547.full.html#ref-list-1

This article cites 43 references, 20 of which can be accessed free at

by guest on May 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from