A Novel Role for IκB Kinase (IKK) α and IKK β in ERK-Dependent Up-Regulation of MUC5AC Mucin Transcription by Streptococcus pneumoniae This information is current as of September 29, 2021. Unhwan Ha, Jae Hyang Lim, Hirofumi Jono, Tomoaki Koga, Amit Srivastava, Richard Malley, Gilles Pagès, Jacques Pouysségur and Jian-Dong Li J Immunol 2007; 178:1736-1747; ; doi: 10.4049/jimmunol.178.3.1736 Downloaded from http://www.jimmunol.org/content/178/3/1736

References This article cites 63 articles, 33 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/178/3/1736.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Role for I␬B Kinase (IKK) ␣ and IKK␤ in ERK-Dependent Up-Regulation of MUC5AC Mucin Transcription by Streptococcus pneumoniae1

Unhwan Ha,2*† Jae Hyang Lim,2*† Hirofumi Jono,*† Tomoaki Koga,*† Amit Srivastava,‡ Richard Malley,‡ Gilles Page`s,§ Jacques Pouysse´gur,§ and Jian-Dong Li3*†

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading mi- crobes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in Downloaded from vitro and in mice in vivo. Interestingly, a “second wave” of ERK activation appears to be important in mediating MUC5AC induction. Moreover, I␬B kinase (IKK) ␣ and IKK␤ are distinctly involved in MUC5AC induction via an ERK1-dependent, but I␬B␣-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae. The Journal of Immunology, 2007, 178: 1736–1747.

n the innate immune system, epithelial cells represent the first S. pneumoniae, a Gram-positive bacterium, is an important hu- http://www.jimmunol.org/ line of host defense against invading microbes at mammalian man pathogen that colonizes the upper respiratory tract (9). It I mucosal surfaces (1). Epithelial cells are not passive barriers, causes potentially life-threatening diseases such as pneumonia, rather, they elaborate diverse molecules involved in efficient septicemia, and meningitis as well as otitis media, the most com- pathogen clearance. Mucins, the major constituent of mucus se- mon childhood bacterial infection and the leading cause of con- cretions, facilitate the mucociliary clearance by physically trapping ductive hearing loss (10). Using animal models of infection, nu- inhaled microbial pathogens in the upper respiratory tract (2, 3). merous S. pneumoniae virulence factors have been identified (11, Thus, up-regulation of mucins in infectious disease represents an 12). Among them, a 53-kDa cytoplasmic protein, pneumolysin, is important host innate defense response against invading microbes a key virulence factor produced by virtually all clinical isolates and (3). Excess mucin production, however, overwhelms the mucocili- released during respiratory infections in humans by bacterial au- by guest on September 29, 2021 ary escalator, contributing to airway obstruction. Similarly, pro- tolysis (13–15). Pneumolysin is classically defined as a cytolytic longed elevation of mucus production in the middle ear restricts toxin (16). At low sublytic concentrations, however, pneumolysin movement of the ossicles, contributing to hearing impairment dur- also serves as a potent inducer of host defense responses by, for ing otitis media (4–7). Indeed, mucin concentration in middle ear example, activation of host complement and induction of immune effusions correlates positively with hearing loss (8). Tight regula- cell-mediated inflammation (17, 18). The mechanisms by which S. tion of mucin production is thus critical for host mucosal defense, pneumoniae induces these responses and, in particular, the recep- and yet how mucin is up-regulated in upper respiratory infectious tor-mediated signaling pathways involved in mucin up-regulation, diseases, such as those involving Streptococcus pneumoniae, re- are unknown. main largely unknown. TLRs are integral to mediating effective host defense responses against invading pathogens (19, 20). To date, 11 members of the human TLR family have been identified. TLR-mediated MyD88- dependent signaling pathways activate NF-␬B and MAPK path- *Department of Microbiology and Immunology, University of Rochester Medical ways (20). A canonical NF-␬B pathway is commonly activated by † Center, Rochester, NY 14642; Gonda Department of Cell and Molecular Biology, proinflammatory cytokines as well as by pathogen-associated mo- House Ear Institute, University of Southern California, Los Angeles, CA 90057; ‡Children’s Hospital, Harvard Medical School, Boston, MA 02115; and §Institute of lecular patterns (21). These inducers interact with the TNFR and Signaling, Developmental Biology and Cancer Research, Nice, France TLR-IL-1R superfamilies, causing activation of the I␬B kinase Received for publication May 19, 2006. Accepted for publication November (IKK)4 complex. The most common form of this complex is com- 14, 2006. prised of IKK␣ and IKK␤ catalytic subunits plus the regulatory The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health Grants DC004562 and DC005843 (to J.-D.L.), AI067737 and AI057784 (to R.M.), Training Grants 4 Abbreviations used in this paper: IKK, I␬B kinase; IRAK1, IL-1R-associated kinase DC008703 (to U.H.) and AI07061 (to A.S.), and a grant from the Ligue Nationale 1; TRAF6, TNFR-associated factor 6; EGF, epidermal growth factor; WT, wild type; Contre le Cancer “Eqipes Labellise´es” (to G.P. and J.P.). Ply mt, pneumolysin-deficient mutant; LDH, lactate dehydrogenase; NHBE, primary 2 human bronchial epithelial; MEF, mouse embryonic fibroblast; DN, dominant nega- U.H. and J.H.L. contributed equally. tive; TK, thymidine kinase; siRNA, small-interfering RNA; Q-PCR, quantitative 3 Address correspondence and reprint requests to Dr. Jian-Dong Li, Department of PCR; NTHi, nontypeable Haemophilus influenzae; Tpl2, tumor progression locus 2; Microbiology and Immunology, University of Rochester Medical Center, Box 672, ELK, Ets-like transcription factor. 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: Jian-Dong_Li@ urmc.rochester.edu Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 1737 subunit IKK␥ (also known as NF-␬B essential modulator). Fol- ogies), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). HM3 (human lowing IKK␥ dissociation, IKK␤ drives the phosphorylation, colon epithelial) cells were maintained in DME H-21 (University of California polyubiquitination, and subsequent degradation of I␬B␣. Released Cell Culture Facility, San Francisco, CA), and mouse embryonic fibroblast (MEF) cells were maintained in DMEM (Invitrogen Life Technologies). WT, ␬ Ϫ Ϫ Ϫ Ϫ NF- B dimers (most commonly the p50–p65 heterodimer) trans- IKK␣ / , and IKK␤ / MEFs were provided by Dr. I. M. Verma (The Salk locate to the nucleus, bind DNA, and activate gene transcription. A Institute, La Jolla, CA) (26, 27). p65Ϫ/Ϫ MEF was provided by Dr. C.-Y. noncanonical NF-␬B pathway was recently discovered (21, 22), Wang (University of Michigan, Ann Arbor, MI) (28). Tlr4Lps-d and ERK1Ϫ/Ϫ Lps-d Ϫ/Ϫ typically induced by lymphotoxin ␤ receptor, B cell activating MEFs were isolated from Tlr4 mutant and ERK1 mice, respectively. HeLa (human cervix epithelial) cells were maintained in MEM (American factor of the TNF family, and CD40L. This alternative pathway is Type Culture Collection). HMEEC-1 (human middle ear epithelial) cells were strictly dependent on the formation of IKK␣ homodimers that in previously described (29) and maintained in bronchial epithelial basal medium turn induce posttranslational processing of p100 to the DNA-bind- (Cambrex). NHBE cells were purchased from Cambrex and maintained in ing subunit p52, ultimately leading to nuclear translocation of p52- bronchial epithelial growth medium (Cambrex) supplemented with defined RelB heterodimers and activating gene transcription. growth factors and retinoic acid as per Clonetics’ instructions. Only NHBE cells at passages 4 were used for experiments. All cells were cultured at 37°C In contrast to NF-␬B, MAPK pathways are quite diverse. in a humidified 5% CO2 water-jacketed incubator. For air-liquid interface cul- MAPKs are a superfamily of serine/threonine protein kinases ture, NHBE cells were cultured as previously described (30). Briefly, NHBE widely conserved among eukaryotes. They transduce a variety of cells were seeded at 2 ϫ 104 cells/cm2 onto 24-mm diameter, 0.4-␮m pore external signals, leading to an array of cellular responses that in- size, semipermeable membrane inserts (Transwell Permeable Supports; Corn- ing) in bronchial epithelial growth medium. The cultures were grown sub- clude growth, differentiation, apoptosis, and host defense response. merged for the first 7 days; an air-liquid interface was then created by remov- To date, three major MAPK pathways have been identified in ing media from the apical compartment of the cultures. Culture media were mammals: ERK, stress-activated protein kinase/JNK, and p38. changed every other day until creation of the air-liquid interface, then changed Downloaded from Growth factor-induced ERK activation is relatively well-elucidated, daily by replacing fresh media only to the basal compartment. NHBE cells but the signaling mechanisms underlying TLR-mediated activation of were grown in air-liquid interface for 2 wk before use in experiments. ERK remain to be determined. Plasmids, transfections, and luciferase assays In this study, we investigate the molecular mechanisms underlying The expression plasmids TLR2 dominant negative (DN), TLR4 DN, MyD88 S. pneumoniae-induced up-regulation of MUC5AC, a prominent mu- DN, IRAK1 DN, TRAF6 DN, I␬B␣ DN, ERK2 DN, IKK␣ DN, WT IKK␣, cin in respiratory secretions. We find that pneumolysin plays an im- IKK␤ DN, and WT IKK␤ have been previously described (31–37). The ex- http://www.jimmunol.org/ portant role in MUC5AC induction via TLR4-MyD88-IL-1R-associ- pression plasmid ERK1 DN was provided by Dr. Cobb (University of Texas ated kinase 1 (IRAK1)-TNFR-associated factor 6 (TRAF6)- Southwestern Medical Center, Dallas, TX). The construct containing the 5Ј- dependent activation of ERK in vitro and in vivo. Furthermore, flanking region of human MUC5AC gene was previously described (38). A deletion mutant of the promoter region was constructed by restriction enzyme delayed ERK activation appears to be crucial in mediating MUC5AC digestion and PCR amplification. Generated DNA fragments were ligated into induction. We also provide evidence for novel roles for IKK␣ and a luciferase reporter gene (pGL3-Basic vector; Promega). A MUC5AC thy- IKK␤ in mediating MUC5AC induction via an ERK1-dependent, but midine kinase (TK) luciferase construct (300TK) was obtained by subcloning Ϫ Ϫ I␬B␣-p65- and p100-p52-independent, mechanism. Our data thus re- the MUC5AC promoter (base pair 3752/ 3452) to upstream of the TK-32 promoter. An NF-␬B mutant TK construct was created by replacing the veal a novel IKK-mediated signaling pathway through which S. pneu- NF-␬B site (Ϫ3612/Ϫ3600) with an EcoRI site. All constructs were confirmed moniae stimulates production of MUC5AC. by DNA sequencing. Transient transfections were conducted using TransIT- by guest on September 29, 2021 LT1 reagent (Mirus) following the manufacturer’s instructions. In cotransfec- Materials and Methods tions, empty vector was used as a control. Transfected cells were pretreated Reagents with chemical inhibitors for 1 h, followed by S. pneumoniae or pneumolysin treatment for 7 h before lysis for luciferase assay. The luciferase assay was PD98059, SB203580, MG-132, and PMA were purchased from Calbio- conducted in triplicate and luciferase activity was normalized with respect to chem. Recombinant human epidermal growth factor (EGF) was purchased ␤-galactosidase activity. from R&D Systems. Polymyxin B was purchased from Sigma-Aldrich. Small-interfering RNA (siRNA) Bacterial strains and culture conditions RNA-mediated interference for down-regulating ERK1 and IKK␣ expres- Clinical isolates of S. pneumoniae wild-type (WT) strains D39, R6, 6B, 3, sions was done by the transfection of siRNA-ERK1 and siRNA-IKK␣ as 19F, and 23F were used in this study (23, 24). D39 isogenic pneumolysin- previously described (39). siRNA-ERK1 (human MAPK3), siRNA-IKK␣ deficient mutant (Ply mt) was developed through insertion-duplication mu- (human CHUK), and an siCONTROL Nontargeting siRNA Pool were pur- tagenesis as described (13). S. pneumoniae was grown on chocolate agar chased from Dharmacon. Cells, 40ϳ50% confluent HeLa, were transfected plates and in Todd-Hewitt broth supplemented with 0.5% yeast extract with a final concentration of 100 nM siRNA using Lipofectamine 2000

(THY) at 37°C in a humidified 5% CO2 water-jacketed incubator. The (Invitrogen Life Technologies). Forty hours after the start of transfection, whole bacterial cells cultured in THY (Autolysate) were harvested at cells were treated with S. pneumoniae for the indicated time before being 10,000 ϫ g for 20 min at 4°C to obtain supernatant and pellet after growth lysed for Western blot analysis and real-time quantitative PCR. to early stationary phase. The bacterial culture supernatant was filtered with 0.2-␮m pore size membrane to completely remove bacteria. The bacterial Western blot analysis pellet was suspended in PBS for the preparation of live bacteria. The bac- Abs were used to analyze total cell lysates as per the manufacturers’ in- terial cell suspension was sonicated on ice three times at 150 W for 3 min structions. Phospho-ERK (Thr202/Tyr204), ERK, phospho-Ets-like tran- at 5-min intervals. Residual intact cells were removed by centrifugation at scription factor (ELK) 1 (Ser383), phospho-IKK␣ (Ser180)/IKK␤ (Ser181), 12,000 ϫ g for 20 min at 4°C. The bacterial lysate was stored at Ϫ80°C ␣ ␤ ␮ IKK , IKK , and GST Abs were purchased from Cell Signaling. Mono- and 5 g/ml lysate was used in all experiments. clonal anti-␤-actin was purchased from Sigma-Aldrich. p100/p52 (N-ter- Purification of pneumolysin minal) Ab was provided by Dr. L.-F. Chen (University of Illinois at Ur- bana-Champaign, Urbana, IL). The 6 ϫ His-tag fused pneumolysin was expressed in and purified from an Escherichia coli strain and residual LPS was removed by passage over ELISA of MUC5AC protein End-X resin as previously described (25). The cytotoxicity of purified MUC5AC protein released from cells was measured with slight modifica- pneumolysin was quantified by lactate dehydrogenase (LDH) release assay tions to the ELISA described previously (40). Briefly, serial dilutions of using the CytoTox 96 Nonradioactive Cytotoxicity Assay kit (Promega). each sample were plated in duplicate on 96-well microtiter plates and dried Cell culture overnight at 40°C. After washing, plates were blocked with 2% BSA over- night at 4°C. To detect MUC5AC, Ag-coated wells were incubated with a All media described below except medium for primary human bronchial ep- 1/100 dilution of anti-mucin 5AC primary Ab (45M1; Neo Markers) for 1 h ithelial (NHBE) were supplemented with 10% FBS (Invitrogen Life Technol- at 37°C. After washing, the plates were incubated with a 1/10,000 dilution 1738 REGULATION OF MUC5AC BY IKK-ERK SIGNALING of HRP-conjugated anti-mouse IgG for 1 h. Plates were incubated with tetramethylbenzidine peroxidase substrate (Bio-Rad), and the reaction was stopped with1NH2SO4. Absorbance was measured at 450 nm. Bovine submaxillary gland mucin (type I; Sigma-Aldrich) was used as a standard. The data was expressed as a fold induction on the same experimental day due to various mucin productions with cell passage in HM3 cells. Real-time quantitative PCR (Q-PCR) analysis Total RNA was isolated using TRIzol reagent following the instruction of Invitrogen Life Technologies. Predeveloped TaqMan Assay Reagents (Ap- plied Biosystems) were used for Q-PCR. Synthesis of cDNA from total RNA was performed with MultiScribe Reverse Transcriptase. Primers and probes for human and mouse MUC5AC have been previously described (37) and synthesized by the Applied Biosystems Customer Oligo Synthesis Service (Applied Biosystems). Reactions were amplified and quantified using a 7500 Real-Time PCR System and the manufacturer’s software (Applied Biosystems). Relative quantities of MUC5AC mRNA were cal- culated using the comparative cycle threshold method and normalized by cyclophilin (human) and GAPDH (mouse) for the amount of RNA used in each reaction (Applied Biosystems). Assay for ERK MAPK activity Downloaded from Cells were harvested in cell lysis buffer following p44/42 MAPK Assay kit (Biolabs). Cell lysates were immunoprecipitated with immobilized phos- pho-ERK (Thr202/Tyr204) mAb (Cell Signaling), and total cell extracts were prepared with SDS-PAGE sample buffer. ERK activity was quantified in each immunoprecipitate using the Assay kit. Mice http://www.jimmunol.org/ ERK1Ϫ/Ϫ mice were previously described (41). Tlr4Lps-d mice (homozy- FIGURE 1. S. pneumoniae MUC5AC gous mice for the point mutation (Pro712 to His) in the Tlr4 gene), BALB/c acts as potent inducer for mucin and C57BL/6 mice were purchased from The Jackson Laboratory. Sex- and transcription in vitro and in vivo. A and B, Clinically isolated S. pneu- age-matched background mice were used as WT controls. All animal ex- moniae strains up-regulated MUC5AC expression at mRNA levels in HM3 periments were approved by the Institutional Animal Care and Use Com- cells (A) and at transcriptional levels in HeLa cells (B). C, HM3 cells were mittee at the House Ear Institute and the University of Rochester. treated with or without WT S. pneumoniae strain D39 (D39 wt). MUC5AC protein was measured by ELISA and expressed as a fold induction. D, D39 Animal experiments wt up-regulated MUC5AC expression at mRNA levels in human epithelial In our mouse model of upper respiratory (middle ear) infections, the middle cells including HeLa, HM3, HMEEC-1, and NHBE. E, S. pneumoniae D39 Ϫ/Ϫ Lps-d ear of anesthetized mice (WT, ERK1 , and Tlr4 ) were inoculated and 6B up-regulated Muc5ac expression at mRNA levels in the middle ear by guest on September 29, 2021 transtympanically with live S. pneumoniae (6.25 ϫ 105 CFU), lysate of BALB/c mice in vivo. F, D39 wt up-regulated Muc5ac expression at 5 (equivalent of 6.25 ϫ 10 CFU), or pneumolysin (200 ng). Saline was mRNA levels in the middle ear of both BALB/c and C57BL/6 mice in vivo. injected as a control. Bullae were dissected from mice 9 h after treatment, Data in A–D are expressed as mean Ϯ SD (n ϭ 3). Data in E and F are then subjected to MUC5AC mRNA expression analysis by Q-PCR. To p Ͻ 0.05 vs control group (A, C, and ,ء .(expressed as mean Ϯ SD (n ϭ 8 Ͻ ء examine the effect of systemic ERK inhibition on S. pneumoniae-induced MUC5AC mRNA expression, WT mice were pretreated with ERK inhib- D). , p 0.005 vs control group (B, E, and F). itor PD98059 (1 mg/kg, i.p.) or DMSO as vehicle control for 2 h before S. pneumoniae inoculation. Total RNA was extracted from whole murine bullae with TRIzol and Q-PCR was performed as described above. (Fig. 1B). These results suggest that the ability to induce mucin is well-conserved among clinical isolates. Consistent with MUC5AC Results mRNA induction, ELISA revealed increased MUC5AC protein S. pneumoniae acts as potent inducer for mucin MUC5AC production in response to S. pneumoniae (Fig. 1C). transcription in vitro and in vivo To evaluate the generalizability of these data, we assayed Up-regulated mucin production represents an important innate de- MUC5AC expression in a variety of epithelial cell lines including fense response against infectious agents such as S. pneumoniae. HeLa, HM3, and HMEEC-1 as well as primary bronchial epithelial Excess mucin production, however, may lead to detrimental air- NHBE cells. As shown in Fig. 1D, S. pneumoniae induced MUC5AC way obstruction or to conductive hearing loss in the middle ear. mRNA expression in all human epithelial cells tested, indicating that The molecular mechanism by which mucin is induced during S. MUC5AC induction by S. pneumoniae may be generalizable to most pneumoniae infection remains unknown. Because MUC5AC has human epithelial cells. To confirm whether MUC5AC is also induced been identified as a prominent mucin in respiratory secretions and in vivo, we next determined the effects of S. pneumoniae on Muc5ac in middle ear effusions, we focused on elucidating MUC5AC expression in mice. As shown in Fig. 1E, clinical isolates of S. pneu- up-regulation. To determine whether S. pneumoniae induces moniae (strains D39 and 6B) strongly induced Muc5ac mRNA ex- MUC5AC transcription, we first used Q-PCR to quantify MUC5AC pression in murine upper respiratory tracts. We confirmed again mRNA expression in human epithelial cells following incubation these results in two mouse strains, BALB/c and C57BL/6 (Fig. with S. pneumoniae strains. As shown in Fig. 1A, all clinical iso- 1F). Collectively, these data demonstrate that S. pneumoniae lates tested were capable of inducing MUC5AC expression. Tran- induces mucin MUC5AC transcription. scriptional regulation of MUC5AC was evaluated by transfecting epithelial cells with a MUC5AC promoter-driven luciferase re- Pneumolysin plays a major role in inducing MUC5AC mucin porter construct before treatment with S. pneumoniae. MUC5AC transcription in vitro and in vivo transcriptional activity was indeed increased upon exposure to all To identify the bacterial factors responsible for MUC5AC induc- clinically isolated S. pneumoniae strains tested, indicating that S. tion, we first compared the mucin-inducing activity of live bacte- pneumoniae induces MUC5AC expression at transcriptional levels ria, bacterial culture supernatant, autolysate, and bacterial lysate The Journal of Immunology 1739

FIGURE 2. Pneumolysin plays a major role in in- ducing MUC5AC mucin transcription in vitro and in vivo. A and B, D39 wt induced MUC5AC expression at transcriptional levels in HeLa cells (A) and at mRNA levels in the middle ear of BALB/c mice (B). Live, live bacteria; Sup, bacterial culture supernatant; Lysate, bac- terial lysate; Autolysate, bacterial autolysate. C, D39 wt and Ply mt were compared in their ability in inducing MUC5AC transcription in HM3 cells. D, Purified pneu- molysin (nanograms per milliliter) induced MUC5AC expression at transcriptional levels in HM3 cells in a dose-dependent manner. E, Cytotoxicity of purified pneumolysin (nanograms per milliliter) was assessed by Downloaded from LDH release assay in HM3, HeLa, and NHBE cells. F, Pneumolysin (200 ng/ml) was pretreated with poly- myxin B to determine the possible contamination of LPS. G, D39 wt, Ply mt, and purified pneumolysin (200 ng/ml) were compared in their ability in inducing MUC5AC mRNA expression in HM3 cells. H, D39 wt

and purified pneumolysin (200 ng) were compared in http://www.jimmunol.org/ their ability in inducing Muc5ac mRNA expression in the middle ear of BALB/c mice in vivo. Data in A and C–G are expressed as mean Ϯ SD (n ϭ 3). Data in B p Ͻ ,ء .(and H are expressed as mean Ϯ SD (n ϭ 6 p Ͼ 0.05 vs ,ء .(vs control group (A–D and G–H 0.005 p Ͼ 0.05 ,ءء .(mock in the presence of pneumolysin (F vs control group (C and G). by guest on September 29, 2021

containing soluble cytoplasmic components. As shown in Fig. 2A, MUC5AC expression at this dose. To eliminate the possibility that MUC5AC transcription was increased the most in response to S. the MUC5AC transcription was induced by LPS contaminated dur- pneumoniae lysate vs live bacteria and bacterial culture superna- ing purification from E. coli, we pretreated pneumolysin with poly- tant. Similar results were also observed in mice in vivo (Fig. 2B). myxin B, a well-characterized LPS inhibitor (26–28). As shown in Interestingly, autolysate induced MUC5AC expression to a degree Fig. 2F, polymyxin B pretreatment did not significantly reduce relatively comparable with lysate (Fig. 2A). Therefore, we con- pneumolysin-induced MUC5AC transcription, indicating that po- clude that S. pneumoniae cytoplasmic components are likely the tential LPS contamination was not involved. Finally, we compared primary mediators of MUC5AC induction. MUC5AC expression at mRNA levels induced by S. pneumonia S. pneumoniae produces numerous virulence factors contribut- and pneumolysin. As shown in Fig. 2G, D39 wt and purified pneu- ing to bacterial pathogenesis during infection (12). Of these, pneu- molysin induced MUC5AC mRNA to similar levels in epithelial molysin is a major cytoplasmic protein in inducing host immune cells. Similar results were observed in mice in vivo (Fig. 2H). responses (18, 42). To determine whether pneumolysin is respon- Taken together, these data demonstrate that S. pneumoniae pneu- sible for MUC5AC induction, WT S. pneumoniae strain D39 (D39 molysin plays a major role in inducing mucin MUC5AC. wt) and its isogenic Ply mt were compared in their MUC5AC- inducing activity. As shown in Fig. 2C, D39 wt potently induced MUC5AC transcription whereas Ply mt did not, indicating that TLR4-dependent MyD88-IRAK1-TRAF6 signaling is pneumolysin is important in MUC5AC induction. Indeed, purified required for MUC5AC induction by S. pneumoniae pneumolysin up-regulated MUC5AC transcription in a dose-de- in vitro and in vivo pendent manner (Fig. 2D). Because a 500 ng/ml dose of pneumo- Because of the important roles for TLR2 and TLR4 in cellular lysin induced MUC5AC transcription less strongly than 100ϳ200 responses to pneumococcal components (43–45), we investigated ng/ml, we examined pneumolysin cytotoxicity with an LDH re- whether TLR2 or TLR4 is involved in up-regulation of MUC5AC lease assay in HM3, HeLa, and NHBE epithelial cells. As shown by S. pneumoniae. As shown in Fig. 3A, overexpressing DN TLR4, in Fig. 2E, pneumolysin cytotoxicity was dose-dependent and but not TLR2, inhibited MUC5AC mRNA expression, suggesting peaked at 500 ng/ml, which may account for the decrease in TLR4-dependent MUC5AC induction by S. pneumoniae. The 1740 REGULATION OF MUC5AC BY IKK-ERK SIGNALING

TLR4-dependent ERK activation is required for MUC5AC induction in vitro and in vivo Having demonstrated that the TLR4-dependent signaling is re- quired in MUC5AC induction, still unclear is which downstream signaling pathway transduces signals to the nucleus to up-regulate MUC5AC transcription. Among the numerous host signaling path- ways downstream of TLR4, MAPKs have been shown to play important roles in mediating a variety of cellular responses (46). We have previously shown that p38 signaling is required for non- typeable Haemophilus influenzae (NTHi)-induced MUC5AC tran- scription (47). Thus, we initially investigated whether S. pneu- moniae-induced MUC5AC transcription is also mediated by MAPK signaling. As shown in Fig. 4A, pretreatment with SB203580, a specific chemical inhibitor for p38, did not signifi- cantly reduce MUC5AC transcription whereas pretreatment with PD98059, a specific chemical inhibitor for ERK, did. Similar re- sults were obtained using pneumolysin (Fig. 4B). Importantly, in air-liquid interface cultures of NHBE cells, PD98059 inhibited pneumolysin-induced MUC5AC expression at mRNA levels (Fig. Downloaded from 4C). In addition, D39 wt and purified pneumolysin potently in- duced phosphorylation of ERK as shown by Western blot analysis (Fig. 4D). Together, these data suggest that, in contrast to Gram- negative NTHi, Gram-positive S. pneumoniae uses ERK but not p38 to induce MUC5AC transcription. To confirm the requirement of ERK in S. pneumoniae-induced http://www.jimmunol.org/ MUC5AC transcription in vitro, we used more specific approaches to inhibit ERK signaling. As shown in Fig. 4, E and F, overex- FIGURE 3. TLR4-dependent MyD88-IRAK1-TRAF6 signaling is re- quired for MUC5AC induction by S. pneumoniae in vitro and in vivo. pressing DN ERK1 abolished MUC5AC expression at transcrip- A, D39 wt-induced MUC5AC expression at mRNA levels was inhibited tional and mRNA levels. In comparison, overexpressing DN by overexpression of DN TLR4 but not TLR2 in HM3 cells. B, Over- ERK2 reduced MUC5AC expression to a lesser extent, suggesting expression of DN MyD88, IRAK1, and TRAF6 inhibited D39 wt-in- that ERK1 may be more important in mediating MUC5AC expres- duced MUC5AC expression at mRNA levels in HM3 cells. C, Overex- sion by S. pneumoniae. Indeed, ERK1 knockdown using siRNA- pression of DN TLR4, MyD88, IRAK1, and TRAF6 also inhibited ERK1 also abolished MUC5AC expression (Fig. 4G, upper by guest on September 29, 2021 pneumolysin (200 ng/ml)-induced MUC5AC transcription in HM3 cells panel). The efficiency of siRNA-ERK1 in reducing endogenous as assessed by luciferase reporter assay. D, D39 wt induced MUC5AC ERK1 protein was confirmed by Western blot analysis (Fig. 4G, Lps-d transcription in WT but not in Tlr4 MEFs. E, D39 wt and pneu- lower panel). We then compared MUC5AC expression in WT molysin (200 ng) markedly induced Muc5ac at mRNA levels in BALB/c Ϫ Ϫ and ERK1 / MEFs. As shown in Fig. 4H, S. pneumoniae WT but not in Tlr4Lps-d mutant mice in vivo. Data in A–D are expressed as p Ͻ markedly induced MUC5AC transcription in WT but not in ,ء .(mean Ϯ SD (n ϭ 3). Data in E is expressed as mean Ϯ SD (n ϭ 6 Ϫ/Ϫ 0.01 vs mock groups in the presence of S. pneumoniae or pneumolysin ERK1 MEFs. p Ͻ 0.005 vs WT group (D and E) in the presence of S. pneu- The in vivo relevance of these data was confirmed by using ,ءء .(A–C) moniae or pneumolysin. PD98059 in mice. As shown in Fig. 4I, Muc5ac expression was abolished by pretreatment with PD98059 in two WT mouse strains (BALB/c and C57BL/6). Moreover, D39 wt and purified pneumo- lysin greatly induced Muc5ac expression at mRNA levels in known importance of MyD88-IRAK1-TRAF6 signaling down- BALB/c mice but not in ERK1Ϫ/Ϫ mice (Fig. 4J). Finally, we stream of TLR4 led us to examine the involvement of this pathway confirmed that ERK signaling is downstream of pneumolysin-ac- in MUC5AC induction. As shown in Fig. 3B, overexpressing DN tivated TLR4 by comparing WT and Tlr4Lps-d MEFs by perform- MyD88, IRAK1, or TRAF6 inhibited MUC5AC expression at ing Western blot analysis. As shown in Fig. 4K, phosphorylation of mRNA levels. Similarly, pneumolysin-induced MUC5AC tran- ERK by pneumolysin was reduced in Tlr4Lps-d MEF cells com- scription was also abolished by overexpressing DN TLR4, pared with WT MEF cells. Taken together, our data indicate that MyD88, IRAK1, or TRAF6 (Fig. 3C). These data implicate in- TLR4-dependent ERK activation is required for MUC5AC induc- volvement of the TLR4-MyD88-IRAK1-TRAF6 signaling path- tion by S. pneumoniae in vitro and in vivo. way in S. pneumoniae-induced MUC5AC up-regulation. To further confirm the requirement for TLR4, we next evaluated the effect of S. pneumoniae on MUC5AC transcription in WT and Tlr4Lps-d A “second wave” of ERK activation may be important in MEF cells. Fig. 3D shows that S. pneumoniae greatly up-regulated mediating MUC5AC induction by S. pneumoniae MUC5AC transcription in WT MEFs whereas MUC5AC induction Based on previous studies showing that the duration of ERK ac- was abolished in Tlr4Lps-d MEFs, indicating that TLR4 is indeed tivation can be relevant in directing the outcome of ERK activation required for MUC5AC induction by S. pneumoniae. Similarly, in- by various inducers (48), we evaluated the role of ERK activation duction of Muc5ac mRNA by both D39 wt and purified pneumo- in MUC5AC induction by S. pneumoniae. As shown in Fig. 5A, lysin was also abolished in Tlr4Lps-d mutant mice (Fig. 3E). We phosphorylation of ERK increased significantly by 10-min post- conclude from these data that TLR4-dependent signaling is re- treatment with S. pneumoniae, declined at 30 min, increased again quired for MUC5AC induction by S. pneumoniae in vitro and at 60 min, and declined thereafter, outlining a bimodal pattern in vivo. of ERK activation. Immunoprecipitation with anti-phospho-ERK The Journal of Immunology 1741

FIGURE 4. TLR4-dependent ERK activation is re- quired for MUC5AC induction in vitro and in vivo.A and B,10␮M PD98059 but not 10 ␮M SB203580 in- hibited both D39 wt- and pneumolysin (200 ng/ml)-in- duced MUC5AC transcription in HeLa cells. C,10␮M PD98059 also inhibited pneumolysin (200 ng/ml)-in- duced MUC5AC expression at mRNA levels in air-liq- uid interface cultures of NHBE cells. D, D39 wt and purified pneumolysin (200 ng/ml) but not Ply mt po- tently induced phosphorylation of ERK at 15 min of treatment as assessed by performing Western blot anal- ysis in HeLa cells. E and F, Overexpressing a DN ERK1 inhibited D39 wt-induced MUC5AC expression at both transcriptional (E) and mRNA (F) levels in HeLa cells. G, ERK1 knockdown by using siRNA-ERK1 (100 nM) Downloaded from inhibited MUC5AC expression at mRNA levels by D39 wt (upper panel) in HeLa cells. The efficiency of siRNA-ERK1 in reducing endogenous ERK1 protein was confirmed by Western blot analysis (lower panel). H, D39 wt markedly induced MUC5AC transcription in WT but not in ERK1Ϫ/Ϫ MEFs (upper panel). The ab- sence of ERK1 was confirmed by Western blot analysis http://www.jimmunol.org/ with ERK Ab (lower panel). I, Muc5ac induction by D39 wt was abolished in the lungs of two WT mouse strains, BALB/c and C57BL/6, by pretreatment with PD98059 (1 mg/kg, i.p.). J, D39 wt and purified pneu- molysin (200 ng) greatly induced Muc5ac expression at mRNA levels in the lungs of WT mice but not in ERK1Ϫ/Ϫ mice. K, Phosphorylation of ERK by pneu- molysin (200 ng/ml) was reduced in Tlr4Lps-d MEF cells

in comparison with that in WT. Data in A and B, E–H are by guest on September 29, 2021 expressed as mean Ϯ SD (n ϭ 3). Data in C are expressed as mean Ϯ SD (n ϭ 2). Data in I and J are expressed as mean Ϯ SD (n ϭ 6). Data in D and K are representative of p Ͻ 0.01 vs vehicle-treated ,ء .three separate experiments group in the presence of S. pneumoniae or pneumolysin (A p Ͼ 0.05 vs vehicle-treated group in the ,ءء .(and B, E–J presence of S. pneumoniae or pneumolysin (A and B). .p Ͻ 0.05 vs vehicle-treated group in the presence of S ,ءء pneumoniae or pneumolysin (C, E, and F).

Abs and in vitro kinase assays using ELK1-GST as ERK substrate TLR4-dependent activation of IKK␣ and IKK␤ play distinct revealed the same bimodal ERK activation. We next sought to roles in MUC5AC induction by S. pneumoniae determine which phase of ERK activation is responsible for max- Having shown that the TLR4-dependent ERK activation plays a imal MUC5AC induction. Because EGF induces transient activa- critical role in MUC5AC induction by S. pneumoniae, how ERK is tion of ERK (49), we confirmed transient activation by Western ␬ blot analysis using various concentrations of EGF (Fig. 5B). Then, activated remains unclear. We previously reported that the NF- B we compared MUC5AC induction by S. pneumoniae with that in- signaling cascade is involved in mucin induction by NTHi (36, ␬ duced by EGF at various concentrations (Fig. 5C). Interestingly, S. 37). In addition, the NF- B pathway represents one of the major pneumoniae potently induced MUC5AC transcription by ϳ6-fold signaling pathways downstream of TLR4 and involved in innate over the control, while 2.5 ng/ml EGF yielded only ϳ2-fold in- and acquired immune responses (50). We therefore investigated duction. Higher concentrations of EGF (10ϳ50 ng/ml) induced whether the NF-␬B signaling cascade is involved in S. pneu- even lower MUC5AC transcription compared with low-dose EGF moniae-induced MUC5AC transcription. First, we assessed the (2.5 ng/ml). Thus, the “second wave” of ERK phosphorylation effects on MUC5AC induction of inhibiting IKK␣ and IKK␤, may play an important role in S. pneumoniae-induced MUC5AC essential kinases upstream of NF-␬B. As shown in Fig. expression. 6A, overexpressing DN IKK␣ reduced MUC5AC induction by 1742 REGULATION OF MUC5AC BY IKK-ERK SIGNALING

FIGURE 5. A second wave of ERK activation may be important in mediating MUC5AC induction by S. pneumoniae. A, D39 wt induced phosphorylation and kinase activity of ERK in HeLa cells. Kinase activity of ERK immunoprecipitated with immobilized anti- phospho-ERK mAb was determined using ELK1 as a substrate. B, EGF induced phosphorylation of ERK in HeLa cells. C, D39 wt and EGF induced MUC5AC tran- scription in HeLa cells as assessed by luciferase reporter assay. Data in A and B are representative of three sep- arate experiments. Data in C is expressed as mean Ϯ SD p Ͻ 0.05 vs ,ءء .p Ͻ 0.005 vs control group ,ء .(n ϭ 3) control group. IP, immunoprecipitation; KA, kinase assay. Downloaded from

S. pneumoniae whereas, surprisingly, overexpressing DN IKK␤ enhanced it. IKK␣ therefore appears to be a positive regulator of MUC5AC induction, while IKK␤ acts as a negative regulator. Such distinct roles for IKK␣ and IKK␤ were confirmed at mRNA levels as shown in Fig. 6B. Consistent with these results, S. pneu- http://www.jimmunol.org/ moniae induced the phosphorylation of both IKK␣ and IKK␤ in a time-dependent manner (Fig. 6C). We next confirmed whether MUC5AC induction requires IKK␣ using a more specific siRNA approach. As shown in Fig. 6D (up- per panel), IKK␣ knockdown using siRNA-IKK␣ greatly reduced S. pneumoniae-induced MUC5AC mRNA expression. The effi- ciency of siRNA-IKK␣ in reducing endogenous IKK␣ protein was confirmed by Western blot analysis (Fig. 6D, lower panel). As

further confirmation of these results, we assessed MUC5AC up- by guest on September 29, 2021 regulation in WT, IKK␣Ϫ/Ϫ, and IKK␤Ϫ/Ϫ MEFs (Fig. 6E, upper panel). The absence of IKK␣ and IKK␤ proteins was again con- firmed by Western blot analysis (Fig. 6E, lower panel). In support of our findings to this point, MUC5AC induction was abolished in IKK␣Ϫ/Ϫ MEFs but markedly enhanced in IKK␤Ϫ/Ϫ MEFs. Re- sponsiveness to S. pneumoniae was restored by transfection of WT IKK␣ and IKK␤, respectively. Taken together, our data suggest that IKK␣ and IKK␤ play distinct roles in mediating MUC5AC induction by S. pneumoniae. We next sought to determine whether IKK␣ and IKK␤ act downstream of pneumolysin-activated TLR4. As shown in Fig. 6F, phosphorylation of IKK␣ and IKK␤ by pneumolysin was reduced in Tlr4Lps-d MEF cells compared with WT MEF cells. Thus, it is FIGURE 6. TLR4-dependent activation of IKK␣ and IKK␤ play dis- evident that IKK␣ and IKK␤ activation are TLR4-dependent and tinct roles in MUC5AC induction by S. pneumoniae. A and B, Overex- play distinct roles in mediating S. pneumoniae-induced MUC5AC pressing a DN IKK␣ reduced MUC5AC induction, whereas overexpressing transcription. aDNIKK␤ enhanced it, at transcriptional (A) and mRNA (B) levels in ␣ ␤ HeLa cells. C, D39 wt induced phosphorylation of IKK and IKK in S. pneumoniae-induced MUC5AC transcription involves neither HeLa cells. D, IKK␣ knockdown by siRNA-IKK␣ (100 nM) greatly re- the I␬B␣-p65-dependent canonical nor the p100-p52-dependent duced D39 wt-induced up-regulation of MUC5AC expression at mRNA ␬ levels in HeLa cells (upper panel). The efficiency of siRNA-IKK␣ in re- noncanonical NF- B pathway ducing endogenous IKK␣ protein was confirmed by Western blot analysis Because IKK␣ and IKK␤ are known to be distinctly involved in Ϫ Ϫ (lower panel). E, Up-regulation of MUC5AC was abolished in IKK␣ / MUC5AC induction, we next investigated whether the NF-␬B sig- Ϫ/Ϫ MEFs whereas the MUC5AC induction was enhanced in IKK␤ MEFs. naling cascade is involved in MUC5AC induction by S. pneu- Their distinct responsiveness to D39 wt was rescued by transfection of WT moniae. We first investigated whether an NF-␬B binding site in IKK␣ or IKK␤, respectively (upper panel). The absence of IKK␣ and the MUC5AC promoter is required for induction. Analysis of IKK␤ expression was confirmed by Western blot analysis (lower panel). F, Phosphorylation of IKK␣ and IKK␤ by pneumolysin (200 ng/ml) was MUC5AC promoter-driven luciferase activities revealed a S. pneu- Ϫ Ϫ reduced in Tlr4Lps-d MEF cells. Data in A and B, and D and E, are ex- moniae response element between base pairs 3752 and 3452 of pressed as mean Ϯ SD (n ϭ 3). Data in C and F are representative of three the MUC5AC promoter (Fig. 7A). This 300-bp region was sub- p Ͻ 0.005 vs mock group in the presence of S. cloned upstream of a TK-32 promoter (300TK); similar results ,ء .separate experiments pneumoniae (A and B, D and E). were observed with the full-length 3.7-kb and 300TK promoter The Journal of Immunology 1743

FIGURE 7. S. pneumoniae-induced MUC5AC tran- scription involves neither the I␬B␣-p65-dependent canonical nor the p100-p52-dependent noncanonical NF-␬B pathway. A, The S. pneumoniae response ele- ment resides in the base pair Ϫ3752/Ϫ3452 region of MUC5AC promoter, and the NF-␬B binding site located within this region (Ϫ3612/Ϫ3600) was not essential for induction of MUC5AC by D39 wt. B, D39 wt induced MUC5AC transcription in both WT and p65Ϫ/Ϫ MEFs (upper panel). The absence of p65 was confirmed by Western blot analysis (lower panel). C, D39 wt induced phosphorylation of ERK in WT and p65Ϫ/Ϫ MEFs at 60 min of treatment. D, D39 wt-induced ERK phosphory- lation in HeLa cells was not affected by 5 ␮M MG-132. E, Overexpressing a DN I␬B␣ did not alter MUC5AC transcription in HeLa cells as assessed by luciferase re- Downloaded from porter assay. F, D39 wt did not induce posttranslational processing of p100 to the DNA-binding subunit p52 in HeLa cells. Data in A, B, and E are expressed as mean Ϯ SD (n ϭ 3). Data in C, D, and F are representative of pϽ0.005 vs control group ,ء .three separate experiments pϾ0.05 vs WT (B) and mock group (E)inthe ,ء .(A) http://www.jimmunol.org/ presence of S. pneumoniae.

constructs. Sequence analysis revealed an NF-␬B binding site be- We next evaluated whether the noncanonical NF-␬B pathway is tween base pairs Ϫ3612 and Ϫ3600. Selective mutagenesis was per- involved in S. pneumoniae-induced MUC5AC transcription. As by guest on September 29, 2021 formed to replace the NF-␬B binding site with an EcoRI site. Sur- shown in Fig. 7F, S. pneumoniae did not induce posttranslational prisingly, as shown in Fig. 7A, 300TK (NF-␬B mt) retained the ability processing of p100 to the DNA-binding subunit p52, a mandatory to up-regulate MUC5AC promoter activation in response to S. pneu- event in noncanonical NF-␬B signaling. Thus, S. pneumoniae- moniae treatment. This result indicates that NF-␬B transcription induced MUC5AC transcription appears to be independent of factor may not be critically involved in mediating S. pneumoniae- both the I␬B␣-p65-dependent canonical and p100-p52-depen- induced MUC5AC expression. dent noncanonical NF-␬B pathways. IKKs therefore appear to IKK␣- and IKK␤-dependent activation of NF-␬B is mediated play a novel role in mediating induction of MUC5AC expression by by two distinct, canonical and noncanonical signaling pathways. In S. pneumoniae. the canonical pathway, IKK␤ is both necessary and sufficient for ␣ ␤ phosphorylation of I␬B␣, while the role of IKK␣ is unclear (22). IKK and IKK play distinct roles in mediating MUC5AC In contrast, the noncanonical pathway depends only on the IKK␣ induction by S. pneumoniae via an ERK1-dependent mechanism homodimer, which is essential in phosphorylating p100 before We have demonstrated that delayed ERK activation and IKK␣ ubiquitination and subsequent processing to p52 (22). To deter- signaling are positively involved in MUC5AC induction by S. mine whether the IKK␤-mediated canonical NF-␬B pathway is pneumoniae in an I␬B␣-p65- and p100-p52-independent manner. involved in MUC5AC induction, we compared S. pneumoniae-in- We further hypothesized that IKK␣ acts upstream of ERK in me- duced MUC5AC transcription in WT and p65Ϫ/Ϫ MEFs; p65 diating MUC5AC induction by S. pneumoniae. To test our hypoth- (RelA) is a key subunit of NF-␬B complex in the canonical path- esis, we assessed the effect of IKK␣ knockdown using IKK␣- way. As shown in Fig. 7B (upper panel), S. pneumoniae-induced siRNA on S. pneumoniae-induced ERK activation. Efficient MUC5AC transcription was almost equal in both WT and p65Ϫ/Ϫ reduction of endogenous IKK␣ protein was confirmed by Western MEFs, indicating that p65 is not required. The absence of p65 blot analysis (Fig. 8A). RNA-mediated interference for down-reg- protein was confirmed by Western blot analysis (Fig. 7B, lower ulating IKK␣ greatly reduced the second wave of ERK activation, panel). Consistent with this result, ERK phosphorylation is also especially ERK1, at 60 min, leaving transient activation of ERK at roughly equal in WT and p65Ϫ/Ϫ MEFs (Fig. 7C). Moreover, S. 10 min relatively unchanged, indicating that IKK␣ is involved in pneumoniae-induced ERK phosphorylation was not affected by delayed ERK activation by S. pneumoniae. We confirmed the in- pretreatment with MG-132, a chemical inhibitor known to prevent volvement of IKK␣ as well as IKK␤ in ERK activation by com- I␬B␣ degradation and subsequent inhibition of NF-␬B activation paring ERK phosphorylation in WT, IKK␣Ϫ/Ϫ, and IKK␤Ϫ/Ϫ (51) (Fig. 7D). Finally, overexpressing DN I␬B␣ did not alter MEFs. As shown in Fig. 8B, phosphorylation of ERK was mark- MUC5AC induction (Fig. 7E). Together, our data indicate that the edly reduced at 60 min in IKK␣Ϫ/Ϫ MEFs as compared with that I␬B␣-p65-dependent NF-␬B pathway is not required for S. pneu- at 10 min, whereas ERK activation was slightly enhanced in moniae-induced MUC5AC transcription. IKK␤Ϫ/Ϫ MEFs at both 10 and 60 min compared with that in WT 1744 REGULATION OF MUC5AC BY IKK-ERK SIGNALING

FIGURE 8. IKK␣ and IKK␤ play distinct roles in mediating MUC5AC induction by S. pneumoniae via an ERK1-dependent mechanism. A, IKK␣ knockdown us- ing siRNA-IKK␣ (100 nM) reduced second wave of activation of ERK1/2, especially ERK1, at 60 min in HeLa cells. B, Phosphorylation of ERK was markedly reduced at 60 min in IKK␣Ϫ/Ϫ MEFs, whereas ERK activation was slightly enhanced in IKK␤Ϫ/Ϫ MEFs at both 10 and 60 min compared with WT. C, S. pneu- moniae-induced kinase activity of ERK was greatly re- duced at 60 min in IKK␣Ϫ/Ϫ but not in IKK␤Ϫ/Ϫ MEFs. D, 0.1 ␮M PMA induced ERK activation in WT and IKK␣Ϫ/Ϫ MEFs. E, Overexpressing a DN IKK␤ or WT IKK␣ enhanced S. pneumoniae-induced MUC5AC tran- scription in WT MEFs pretreated with vehicle but not PD98059 (10 ␮M). Overexpressing a DN IKK␤ or Downloaded from WT IKK␣ did not enhance S. pneumoniae-induced MUC5AC transcription in ERK1Ϫ/Ϫ MEFs. Data in A–D are representative of three separate experiments. p Ͼ ,ء .(Data in E is expressed as mean Ϯ SD (n ϭ 3 0.05 vs control (E). IP, immunoprecipitation; KA, ki- nase assay. http://www.jimmunol.org/

MEFs, confirming that IKK␣ is indeed an upstream activator for delayed ERK activation; in contrast, IKK␤ may be somehow neg- atively involved in mediating ERK activation. Furthermore, im- by guest on September 29, 2021 munoprecipitation with anti-phospho-ERK Abs and in vitro kinase assay using ELK1-GST as the ERK substrate revealed a significant reduction in ERK kinase activity at 60 min in IKK␣Ϫ/Ϫ MEFs and increased kinase activity of ERK in IKK␤Ϫ/Ϫ MEFs (Fig. 8C). Using PMA, a specific inducer of MUC5AC via ERK in human epithelial cells, we next examined whether IKK␣ mediates delayed ERK activation generally for other ERK inducers or specifically for S. pneumoniae. As shown in Fig. 8D, PMA-induced ERK ac- tivation remained unaffected at both 10 and 60 min in WT and IKK␣Ϫ/Ϫ MEFs. We therefore conclude that S. pneumoniae in- duces delayed ERK activity via a unique pathway positively reg- ulated by IKK␣ and negatively regulated by IKK␤. We finally confirmed our conclusions by assessing ERK-medi- ated MUC5AC induction in the setting either of IKK␤ inhibition or IKK␣ activation in ERK1Ϫ/Ϫ and WT MEFs treated with the ERK inhibitor PD98059. If ERK1 signaling is already abrogated either by using PD98059 or using ERK1-deficient MEFs, altering IKK␤ or IKK␣ signaling should not further enhance MUC5AC induction by S. pneumoniae. Indeed, as shown in Fig. 8E, overexpressing DN IKK␤ or WT IKK␣ did not enhance S. pneumoniae-induced MUC5AC transcription in WT MEFs pretreated with PD98059 FIGURE 9. Schematic representation of the novel involvement of IKK␣ compared with WT MEFs not pretreated with PD98059. Likewise, and IKK␤ in up-regulation of MUC5AC mucin by S. pneumoniae via an overexpressing DN IKK␤ or WT IKK␣ did not further enhance S. ERK-dependent mechanism. As indicated, pneumolysin plays a crucial pneumoniae-induced MUC5AC transcription in ERK1Ϫ/Ϫ MEFs. role in up-regulating MUC5AC expression via TLR4-dependent activation Taken together, our data demonstrate distinct roles for IKK␣ and of ERK in vitro and in vivo. Interestingly, a second wave of ERK activa- ␤ tion appears to be important in mediating MUC5AC induction. Moreover, IKK in mediating S. pneumoniae-induced MUC5AC expression IKK␣ and IKK␤ are distinctly involved in MUC5AC induction via an by acting upstream of ERK1, thus revealing novel roles for IKKs ERK-dependent but I␬B␣-p65- and p100-p52-independent NF-␬B mech- in mediating up-regulation of MUC5AC mucin via an I␬B␣-p65- anism, thereby revealing novel roles for IKKs in regulating ERK-depen- and p100-p52-independent mechanism. dent MUC5AC mucin transcription by S. pneumoniae. The Journal of Immunology 1745

Discussion vation? The direct means by which IKK␣ induces ERK activation Here, we show that pneumolysin, a key cytoplasmic virulence pro- also remain to be determined. In addition, we cannot rule out the tein well-conserved among all clinical isolates of S. pneumoniae,is possibility that other pathways essential for MUC5AC expression, crucial for induction of MUC5AC, a prominent mucin in respira- in addition to the ERK pathway described here, are activated by tory secretions. We further found that S. pneumoniae induces pneumolysin and not by EGF. MUC5AC via TLR4-dependent activation of ERK both in vitro Pneumolysin is a multifunctional protein possessing both pore- and in vivo. Interestingly, delayed ERK activation appears to play forming cytolytic and immunomodulatory properties (58). Pneu- an important role in mediating MUC5AC induction by S. pneu- molysin is thought to be present at relatively low concentrations moniae. Our studies also provide evidence of distinct roles for early in infection, when it mediates both immunomodulatory ef- IKK␣ and IKK␤ via an ERK-dependent but I␬B␣-p65- and p100- fects and low sublytic activities (17, 18). Later in infection, pneu- p52-independent mechanism, thus revealing novel roles for IKKs molysin rises to lytic concentrations at which it mainly forms in mediating up-regulation of MUC5AC mucin by S. pneumoniae membrane pores that cause direct cellular and tissue damage, pro- (Fig. 9). moting widespread pneumococcal dissemination (16). In this ϳ The finding that pneumolysin induces MUC5AC via TLR4 is study, we found that 100 200 ng/ml doses of pneumolysin induce consistent with a previous report that TLR4 recognizes pneumo- high MUC5AC expression via TLR4-dependent activation of ERK, ϳ ϳ lysin to stimulate TNF-␣ and IL-6 production (43). However, re- though this concentration causes 5 25% cytotoxicity as mea- cent studies by van Rossum et al. (44) suggest that TLR2 is es- sured by LDH release assay. In contrast, 500 ng/ml pneumolysin induces pronounced cytotoxicity (60% by LDH assay) but signif- sential for clearance of S. pneumoniae. It is likely that TLR2 is the icantly lower MUC5AC expression. Recently, Ratner et al. (52) Downloaded from main pattern recognition receptor for live S. pneumoniae, while demonstrated that pneumolysin increases cell membrane perme- pneumolysin is predominantly recognized by TLR4. This model is ability by forming membrane pores, and the induced osmotic stress supported by the recent work of Schmeck et al. (45) who observed activates p38 independent of both TLR2 and TLR4. Implied that live S. pneumoniae-induced NF-␬B activity is exclusively de- therein is that activation of p38 is cytotoxicity-mediated and in- pendent on TLR2 whereas pneumolysin-induced NF-␬B activity is dependent of TLR signaling. We also observed p38 activation by exclusively dependent on TLR4. Their results are congruent with

200 ng/ml pneumolysin (data not shown), but p38 is apparently not http://www.jimmunol.org/ our finding that pneumolysin-induced activation of IKK-ERK sig- involved in MUC5AC induction as shown in Fig. 4B. MAPKs are naling is mainly TLR4-dependent. In contrast, van Rossum et al. important to both cell survival and apoptosis. Although p38 acti- (44) showed that pneumolysin promotes clearance of colonized S. vation is proapoptotic, activation of ERK can protect the cell. The pneumoniae in a TLR4-independent manner. The pore-forming ef- dynamic balance between these opposing pathways is crucial to fect of pneumolysin activates p38, contributing to the production determining whether a cell will survive or undergo apoptosis (59). of proinflammatory cytokines independent of both TLR2 and Therefore, it is likely that low sublytic concentrations of pneumo- TLR4 (52). It is logical that pneumolysin-activated p38 leads to lysin are sufficient to activate ERK, which predominates at these increased bacterial clearance by inducing proinflammatory re- concentrations for survival and MUC5AC induction via TLR4. At sponses independent of TLR. higher lytic concentrations, p38 activation predominates in pro- by guest on September 29, 2021 ␣ A novel finding in the present study is the distinct roles of IKK apoptotic responses, and interference with ERK activation by cell ␤ and IKK in mediating MUC5AC induction by S. pneumoniae.As damage may account for lower MUC5AC induction. described, these IKKs appear to act upstream of ERK, independent S. pneumoniae is capable of adhering to mucosal cells in the ␬ ␣ of both the I B -p65-mediated canonical and the p100-p52-me- upper respiratory tract, but pneumococcal carriage is initially ␬ diated noncanonical NF- B pathways. This unexpected finding asymptomatic. During the symptom-free period of colonization, ␣ ␤ seems to reveal novel functions for IKK and IKK in regulating pneumococci interact with host epithelial cells and activate intra- ERK-mediated biological responses during pneumococcal infec- cellular signaling pathways. In response, epithelial cells produce tion. It should be noted that no direct physical interaction was mucus, which facilitates bacterial clearance from airways via the ␣ ␤ observed between ERK and IKK or IKK when we performed mucociliary escalator. However, S. pneumoniae also develops dis- coimmunoprecipitation experiments (data not shown), implicating ease by provoking inflammatory responses, invading adjacent tis- the involvement of an as-yet unidentified signaling intermediate. sues and sometimes the bloodstream. Proinflammatory cytokine Recently, two separate studies have concluded that IKK␤ mediates release increases rapidly in response to bacterial cell wall compo- LPS- or TNF-␣-induced ERK activation via tumor progression nents released by pneumococcal autolysis (60), which may also locus 2 (Tpl2) (53, 54). However, by using Tpl2 siRNA knock- release relatively low concentrations of pneumolysin early in in- down, we found that Tpl2 is not involved in mediating MUC5AC fection. Released pneumolysin could interact with TLR4 to acti- induction or ERK activation by S. pneumoniae (data not shown). vate ERK signaling pathways and rapidly increase MUC5AC ex- Moreover, their studies described IKK␤ as a positive regulator of pression as shown in this study, both in human epithelial cells in ERK via Tpl2 whereas we found that IKK␤ acts as a negative vitro and in mice in vivo. Increased mucus production can lead to regulator for ERK activation by S. pneumoniae. Thus, our results airway obstruction which, in the setting of chronic inflammation, suggest novel roles for IKK␣ and IKK␤ in mediating ERK-depen- incapacitates the mucociliary escalator and impairs bacterial clear- dent MUC5AC mucin up-regulation by S. pneumoniae. ance. When bacterial load rises later in infection, increased bac- Differences in the duration and timing of ERK activation may terial autolysis releases lytic concentrations of pneumolysin, di- account for the very different outcomes observed with various rectly and significantly damaging the respiratory epithelium and ERK inducers (55). EGF transiently activates ERK (56, 57), but facilitating dissemination of pneumococci from alveoli into the induces MUC5AC transcription to a lesser extent compared with S. bloodstream (61–63). pneumoniae. To our surprise, S. pneumoniae induced bimodal ac- In the present study, pneumolysin was identified as a key viru- tivation of ERK. Delayed ERK activation is evidently important in lence factor in S. pneumoniae-induced MUC5AC expression via mediating MUC5AC induction, and IKK␣ mediates this second TLR4-dependent activation of ERK. These findings were consis- wave induced by S. pneumoniae. Why does treatment with S. tent in our in vitro and in vivo models, suggesting that the same pneumoniae and EGF lead to two different patterns of ERK acti- mechanisms likely act in vitro and in vivo. Furthermore, IKK␣ and 1746 REGULATION OF MUC5AC BY IKK-ERK SIGNALING

IKK␤ appear crucial in mediating MUC5AC induction via delayed 24. Briles, D. E., M. J. Crain, B. M. Gray, C. Forman, and J. Yother. 1992. Strong ERK activation. In the context of previous reports, our findings association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infect. Immun. 60: 111–116. outline a complex host signaling network responding to S. pneu- 25. Srivastava, A., P. Henneke, A. Visintin, S. C. Morse, V. Martin, C. Watkins, moniae infection. The signaling molecules that directly mediate J. C. Paton, M. R. Wessels, D. T. Golenbock, and R. Malley. 2005. The apoptotic ␣ ␤ response to pneumolysin is Toll-like receptor 4 dependent and protects against IKK and IKK regulation of ERK activation are yet to be de- pneumococcal disease. Infect. Immun. 73: 6479–6487. termined. Additional studies of how these pathways interact in 26. Li, Q., Q. Lu, J. Y. Hwang, D. Buscher, K. F. Lee, J. C. Izpisua-Belmonte, and vivo are also necessary to better characterize the integrated host I. M. Verma. 1999. IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev. 13: 1322–1328. response to S. pneumoniae infection. 27. Li, Q., G. Estepa, S. Memet, A. Israel, and I. M. Verma. 2000. Complete lack of NF-␬B activity in IKK1 and IKK2 double-deficient mice: additional defect in Acknowledgments neurulation. Genes Dev. 14: 1729–1733. 28. Yang, F., E. Tang, K. Guan, and C. Y. Wang. 2003. IKK ␤ plays an essential role We are grateful to Drs. D. Briles, J. R. Zhang, C. J. Kirschning, in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. M. H. Cobb, C. Y. Wang, L. F. Chen, and I. M. Verma for kindly providing J. Immunol. 170: 5630–5635. various reagents. We also appreciate the effort in professional editing of 29. Chun, Y. M., S. K. Moon, H. Y. Lee, P. Webster, D. E. Brackmann, J. S. Rhim, and D. J. Lim. 2002. 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