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J. Biol. Chem., Vol. 277, Issue 47, 45547-45557, November 22, 2002

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Transforming Growth Factor--Smad Signaling Pathway Cooperates with NF-B to Mediate Nontypeable Haemophilus influenzae-induced MUC2 Mucin Transcription^*

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 Medical Center and Department of Medicine, University of California, San Francisco, California 94143, the  Department of Molecular Virology, Tokyo Medical and Dental University, Tokyo 113-8519 Japan, and the ^** Michael E. DeBakey Department of Surgery and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, July 10, 2002, and in revised form, September 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transforming growth factor- (TGF-) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF- receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF--Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKK/-IB pathway to mediate NF-B-dependent MUC2 mucin transcription. The NTHi-induced TGF- receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF- neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-B p65/p50 with Smad3/4 appears to positively mediate NF-B-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF- receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF- signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Gram-negative bacterium nontypeable Haemophilus influenzae (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 leading cause of conductive hearing loss (2, 3) whereas in adults, it exacerbates chronic obstructive pulmonary diseases (COPD), the fourth leading cause of death in the United States (4, 5). A hallmark of both OM and COPD is mucus overproduction that mainly results from up-regulation of mucin, a primary innate defensive response for mammalian airways (6, 7). Mucins, the major component of mucus secretions, are high molecular weight and heavily glycosylated proteins synthesized by the mucosal epithelial cells lining the middle ear, trachea, digestive, and reproductive tracts (8). They protect the epithelial surface by binding and trapping inhaled infectious particles, including bacteria and viruses, for mucociliary clearance, at least in part because of the extraordinary diversity of their carbohydrate side chains (6, 9). However, in patients with OM with effusion and COPD whose mucociliary clearance mechanisms have become defective, excessive production of mucin will lead to airway obstruction in COPD and conductive hearing loss in OM with effusion (8, 10, 11). To date, 14 mucin genes have been identified (9, 10, 12, 13). Among these, at least MUC2, MUC5AC, and MUC5B have been shown to play an important role in the pathogenesis of respiratory infectious diseases. Understanding the signaling mechanisms underlying up-regulation of mucin may open up novel therapeutic targets for these diseases.

In contrast to the relatively well known mechanism by which MUC5AC mucin is up-regulated by NTHi (14), the signaling mechanism underlying NTHi-induced MUC2 mucin transcription remains totally unknown. Based on our previous studies showing that MUC2 is up-regulated by Gram-negative bacterium Pseudomonas aeruginosa in cystic fibrosis via activation of NF-B (15, 16) and that NTHi, also a Gram-negative bacterium, strongly activates NF-B (17), it is plausible that activation of NF-B might be also required for NTHi-induced MUC2 up-regulation via specific signaling pathways.

In addition to the NF-B pathway, the TGF--Smad pathway represents another important signaling pathway participating in regulation of diverse biological processes, including cell proliferation, differentiation, death, inflammatory, and immune responses (18-24). The TGF- superfamily is a large group of secreted growth factors of which three subgroups have been defined: the TGF-s, activins, and bone morphonegetic proteins (BMPs) (21, 23). TGF- initiates signaling through the ligand-dependent activation of a heteromeric complex of type II and type I receptors. The type II receptor kinase then phosphorylates the type I receptor in a conserved glycine-serine domain (GS domain), resulting in activation of the type I receptor. The activated type I receptor subsequently recognizes and phosphorylates the Smad subgroup known as receptor-activated Smads (R-Smad), including Smad2 and Smad3. This causes dissociation of R-Smad from the receptor, stimulates the assembly of a heteromeric complex between the phosphorylated R-Smad and the Co-Smad, Smad4, and then induces the translocation of the Smad complex to the nucleus, where the Smad complex regulates the expression of target genes (19, 21). In addition to its direct interaction with Smad DNA-binding element (SBE), growing evidence suggests that Smads also regulate gene transcription by direct interaction and functional cooperation with other transcription factors, such as NF-B (25-27). Despite its important role in regulation of diverse biological processes, it is still unclear if activation of the TGF--Smad signaling pathway also mediates up-regulation of mucin, a primary host innate defensive response to bacteria.

Because of the important role of NF-B and TGF- signaling in mediating diverse cellular responses as well as the reported functional cooperation between NF-B and TGF--Smad, we hypothesized that the TGF--Smad signaling pathway cooperates with NF-B to mediate up-regulation of MUC2 mucin transcription in response to NTHi infections in human epithelial cells. Here, we show that activation of TGF- receptor-Smad3/4 signaling, together with TLR2-MyD88-TAK1-NIK-IKK/-IB-dependent activation of NF-B, mediates NTHi-induced MUC2 mucin transcription. These findings provided direct evidence, for the first time, that the bacterium NTHi uses the TGF--Smad pathway for transducing signals into nucleus, at least in part, via an autocrine-independent mechanism and that the functional cooperation between NF-B and TGF- receptor-Smad is required for host defensive response to NTHi. These studies may bring insights into the novel role of TGF--Smad signaling in bacterial pathogenesis and may lead to novel therapeutic intervention for OM and COPD.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Caffeic acid phenethyl ester (CAPE) and MG-132 were purchased from Calbiochem (La Jolla, CA). Recombinant human TGF-1 and TGF- neutralization antibody were purchased from R&D Systems.

Bacterial Strains and Culture Conditions-- NTHi strain 12, a clinical isolate, was used in this study (14, 17, 28). Bacteria were grown on chocolate agar at 37 °C in an atmosphere of 5% CO₂. For making NTHi crude extract, NTHi were harvested from a plate of chocolate agar after overnight incubation and incubated in 30 ml of brain heart infusion (BHI) broth supplemented with NAD (3.5 µg/ml). After overnight incubation, NTHi were centrifuged at 10,000 × g for 10 min, and the supernatant was discarded. The resulting pellet of NTHi was suspended in 10 ml of phosphate-buffered saline and sonicated. Subsequently, the lysate was collected and stored at 70 °C. NTHi lysates (5 µg/ml) were used in all the experiments. We chose to use NTHi lysates because of the following reasons: First, NTHi has been shown to be highly fragile and has the tendency to autolyse. Its autolysis can be triggered in vivo under various conditions including antibiotic treatment (14, 17, 28). Therefore, using lysates of NTHi represents a common clinical condition in vivo, especially after antibiotic treatment.

Cell Culture-- Human colon epithelial cell line HM3 was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) (14, 16). Human cervix epithelial cell line HeLa and human middle ear epithelial cell line HMEEC-1 were maintained as described (17, 28). Primary human bronchial epithelial cells (NHBE) were purchased from Clonetics (San Diego, CA). NHBE cells were maintained in Clonetics' recommended bronchial epithelial growth media (BEGM), which includes supplements of bovine pituitary extract, hydrocortisone, human recombinant epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, gentamicin, and amphotericin B (Clonetics), to a confluence of 60-80% (37 °C, 5% CO₂). Media were replaced every other day. Only cells at passages 4 were used for experiments. Wild-type mink Mv1Lu cells and two cell lines, DR26 and R1B that are derived from Mv1Lu and lack functional TRII and TRI, 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-1 cells and a cell line R5 that is derived from Rat-1 and lacks functional IKK were maintained as described previously (30). All media received additions of 100 units/ml penicillin and 0.1 mg/ml streptomycin.

Real-time Quantitative RT-PCR Analysis-- Total RNA was isolated from human epithelial cells using TRIzol^® Reagent (Invitrogen) following the manufacturer's instruction. For the real-time quantitative RT-PCR, predeveloped TaqMan assay reagents (Applied Biosystems) were used. Synthesis of cDNA from total RNA samples was performed with MultiScribe^TM reverse transcriptase. To normalize MUC2 expression relative to cDNA, we used primers and a TaqMan probe corresponding to cyclophilin. Expression of MUC2 was measured relative to cyclophilin. Primers and the TaqMan probe for MUC2 and cyclophilin were designed by using Primer Express software (Applied Biosystems) and synthesized by Applied Biosystem Customer Oligo Synthesis Service (Applied Biosystems). TaqMan probes were labeled with FAM on the 5'-end and TAMARA on the 3'-end. The primers and probes for MUC2 were: forward primer, 5'-TCCATCCTGCTGACCATCAA-3' and reverse primer, 5'-GTAGGCATCGCTCTTCTCAATGA-3', and TaqMan probe, 5'-FAM-TGACACCATCTACCTCACCCGCCATAMRA-3'. Reactions were amplified and quantified using an ABI 7700 sequence detector and manufacturer's software (Applied Biosystems). Relative quantity of MUC2 mRNA was obtained using Comparative CT Method (for details, see user Bulletin 2 for the ABI PRISM 7700 Sequence Detection System under www.appliedbiosystems.com/support/tutorials) and was normalized using predeveloped TaqMan assay reagent human cyclophilin as an endogenous control (Applied Biosystems).

Plasmids, Transfections, and Luciferase Assays-- The expression plasmids IB(S32A/S36A), IKK(K44M), IKK(K49A), IKK, NIK (K429A/K430A), TAK1 DN, MyD88 DN, hTLR2 DN, TGFRIIDN and wild-type, TGFRI 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 MUC2 gene, NF-B-luc, SBE-Luc, and plasminogen activator inhibitor-1 (PAI-1)-Luc, were also previously described (16, 18, 27). All transient transfections were carried out in HM3 cells in triplicate using TransIT-LT1 reagent (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 empty vector was used as a control. Transfected cells were pretreated with or without chemical inhibitors including CAPE and MG-132 for 2 h. NTHi or recombinant human TGF-1 was then added to the transfected cells 42 h after transfection. After 5 h, the cells were harvested for luciferase assay. In experiments using neutralization TGF- antibody, NTHi lysates were pretreated with either TGF- neutralization antibody or control antibody for 1 h before being added to the transfected cells for 5 h.

Immunofluorescent Staining-- Cells were cultured on 4-chamber microscope slides. After NTHi treatment, the cells were fixed in paraformaldehyde solution (4%), incubated with mouse anti-p65 NF-B or mouse anti-Smad4 monoclonal antibodies for 1 h (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Primary antibody was detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.). Samples were viewed and photographed using a Zeiss Axiophot microscope.

Western Blot Analysis and ELISA Assays-- Antibodies against phospho-IB(Ser-32), IB were purchased from Cell Signaling (Beverly, MA). Antibodies against phospho-TRII (Tyr-336) and TRII were purchased from Santa Cruz Biotechnology. Phosphorylation of IB and TRII were detected as described and following the manufacturer's instructions (17). TGF-1, 2, and 3 released from the cells were analyzed by standard sandwich ELISA assays using ELISA kits as described and following the manufacturer's instructions. TGF-1 ELISA kit was purchased from BioSource Europe S. A. (Nivelles, Belgium). TGF-2 and TGF-3 ELISA kits were purchased from the R&D system.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NTHi Up-regulates MUC2 Mucin Transcription in Human Epithelial Cells-- We first examined whether NTHi up-regulates MUC2 mucin expression in human epithelial cells by performing real-time quantitative PCR analysis. NTHi strongly up-regulates MUC2 expression at mRNA level in human epithelial HM3 cells (Fig. 1A). To investigate whether transcriptional regulation is involved in MUC2 induction, we next transfected HM3 cells with an expression vector containing 2.8 kb of the human MUC2 5'-flanking region fused to a luciferase reporter gene. When we exposed the transfected cells with NTHi, the luciferase activity driven by the MUC2 promoter increased in a time- and dose-dependent manner, suggesting the involvement of transcriptional regulation (Fig. 1B and data not shown). Because we were interested in the potential generality of NTHi-induced MUC2 up-regulation, we assayed a variety of human epithelial MUC2-expressing cell lines as 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 transcriptional and mRNA levels is well conserved among all human epithelial cell lines and primary epithelial cells that we tested. Thus, these findings indicate that NTHi up-regulates MUC2 mucin transcription in human epithelial cells.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   NTHi up-regulates human MUC2 mucin transcription in human epithelial cells. A, NTHi up-regulates MUC2 expression at mRNA level in HM3 cells as assessed by real-time quantitative RT-PCR. B, NTHi up-regulates MUC2 transcription in HM3 cells in a time-dependent manner. HM3 cells were transiently transfected with human MUC2 2.8-kb promoter luciferase reporter construct (pMUC2-2864luc) and stimulated with NTHi for various times as indicated. Luciferase activity was then assessed in NTHi-treated and -untreated cells. C, NTHi-induced up-regulation of MUC2 transcription was observed in a variety of human epithelial cell lines including HM3, HeLa, and HMEEC-1 as well as NHBE. D, NTHi-induced MUC2 up-regulation was also observed at mRNA level in HM3, HeLa, and HMEEC-1 as well as NHBE cells as assessed by real-time quantitative RT-PCR. Values are the means ± S.D. (n = 3).

Activation of NF-B Via a TLR2-MyD88-dependent TAK1-NIK-IKK/-IB Pathway Is Required for NTHi-induced MUC2 Up-regulation-- We next performed experiments to define the NTHi-response element in the 5'-flanking region of the human MUC2 mucin gene and the related transcription factors in the HM3 cell, the human epithelial cell line yielding the strongest NTHi response. Analysis of luciferase activity from a panel of deletion mutants of the MUC2 promoter-luciferase reporter gene revealed a NTHi response element between base pairs 1528 and 1430 (Fig. 2A and data not shown). Subsequent sequence analysis showed that this region contains a NF-B binding site. Based on our recent report showing that NTHi strongly activates NF-B (17), we next explored the possibility that activation of NF-B is required for NTHi-induced MUC2 transcription by performing selective mutagenesis of the NF-B binding site. As shown in Fig. 2A, mutant constructs M1, M2, and M3, in which the NF-B site is mutated, markedly reduced the responsiveness of MUC2 promoter construct, whereas mutant construct M4 in which NF-B site remains intact did not reduce MUC2 induction. These results suggested that NF-B activation is required for NTHi-induced MUC2 transcription. Because nuclear translocation is a key step for NF-B to exert its transcriptional activity, we next sought to determine whether NF-B nuclear translocation is also required for MUC2 induction. As shown in Fig. 2B (upper panel), p65, a key subunit of NF-B complex, was translocated into the nucleus upon exposure to NTHi as we reported previously in HeLa cells. The NTHi-induced NF-B translocation was blocked by a chemical inhibitor CAPE, which is known to specifically block the translocation of p65 without affecting IB degradation (Fig. 2B, upper panel) (34). Moreover, CAPE abrogated NTHi-induced MUC2 transcription (Fig. 2B, lower panel), confirming that nuclear translocation and activation of NF-B is indeed required for NTHi-induced MUC2 transcription.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Activation of NF-B via a TLR2-MyD88-dependent TAK1-NIK-IKK/-IB pathway is required for NTHi-induced MUC2 up-regulation. A, human MUC2 regulatory regions (base pairs: 1528 to 1430) containing the wild-type (WT) or various mutated sites within the region 1458/1430 as indicated were subcloned upstream of the TK-32 promoter in a luciferase vector (designated M1 to M4) and transfected into HM3 cells. B, CAPE (20 µg/ml), a chemical inhibitor that is known to specifically block the translocation of p65 without affecting IB degradation, inhibits NTHi-induced NF-B nuclear translocation and MUC2 transcription. C, MG-132 (1 µM), a specific proteosome inhibitor, blocks NTHi-induced IB degradation, NF-B nuclear translocation, and up-regulation of MUC2 transcription. In addition, overexpression of a transdominant mutant of IB (S32/36A) also inhibits NTHi-induced MUC2 up-regulation. D, co-expression of dominant-negative mutants of IKK (K44A), NIK (K429A/K430A), and TAK1, but not IKK, inhibits NTHi-induced MUC2 transcription. E, NTHi-induced MUC2 transcription requires IKK. An NF-B-regulated luciferase reporter plasmid or a MUC2 1.5/1.3 TK-luciferase reporter vector was transfected into wild-type Rat-1 cells expressing IKK, or the derivative R5 cells lacking functional IKK, respectively. Expression plasmid of wild-type IKK was co-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 IB, IKK, TAK1, MyD88, and TLR2 also inhibits NTHi-induced MUC2 transcription in HMEEC-1 (left panel) and NHBE cells (right panel). H, co-expression of dominant-negative mutants of IB, TAK1, and MyD88 attenuate NTHi-induced MUC2 up-regulation at mRNA level. In all the experiments shown above, transfections were carried out 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).

Based on our recent report that TAK1-NIK-IKK-dependent IB phosphorylation and degradation is required for NTHi-induced NF-B activation, we next sought to determine the involvement of IB phosphorylation and degradation in MUC2 induction (17). We first investigated whether NTHi induces IB phosphorylation and degradation in HM3 cells. As shown in Fig. 2C (upper panel), phosphorylation and degradation of IB was observed in HM3 cells treated with NTHi. The NTHi-induced IB degradation was blocked by MG132, a proteasome inhibitor that prevents the degradation of IB (35), whereas phosphorylated IB was no longer degraded in the presence of MG-132 and thus persists in the cytoplasm. We next sought to determine the requirement of IB degradation by assessing the effect of MG132 on NF-B nuclear translocation and MUC2 induction. As expected, MG132 completely blocked NTHi-induced NF-B translocation in HM3 cells (Fig. 2C, middle panel). Concomitantly, MUC2 induction was also inhibited by MG132 (Fig. 2C, lower panel). Moreover, overexpression of a transdominant mutant of IB (S32A/S36A) greatly inhibited MUC2 induction, further confirming the involvement of IB degradation in MUC2 induction (36). Finally, overexpression of a dominant-negative mutant form of either IKK or NIK or TAK1, but not IKK, markedly inhibited MUC2 induction (Fig. 2D). Consistent with these results, R5, a cell line that is derived from Rat-1 cells and lacks functional IKK, did not show NTHi-induced transcriptional activation from 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 the response of wild-type Rat-1 cells (Fig. 2E). Taken together, these findings demonstrate that NTHi induces MUC2 up-regulation via a TAK1-NIK-IKK/-dependent IB phosphorylation and degradation, which, in turn, leads to NF-B nuclear translocation and activation.

Having identified TAK1-NIK-IKK/-IB-dependent activation of NF-B involved in NTHi-induced MUC2 transcription, still unknown is which cell surface receptor(s) is involved in transmitting signals from cell surface to the cytoplasm in MUC2 induction. Because of the important role of Toll-like receptor 2 (TLR2) in mediating bacteria-induced NF-B activation (17), we sought to first determine the involvement of TLR2 in NTHi-induced MUC2 transcription. As shown in Fig. 2F, overexpression of a dominant-negative mutant of human TLR2 inhibited MUC2 induction. Similarly, co-transfection with a dominant-negative mutant MyD88, a key adaptor protein downstream of TLR2, also inhibited MUC2 induction (37). These data indicate that the TLR2-MyD88 signaling pathway is also involved in MUC2 induction.

Because NTHi is a major bacterial pathogen causing middle ear and airway infections, we next determined whether this signaling pathway also mediates NTHi-induced MUC2 transcription in HMEEC-1 and NHBE. As shown in Fig. 2G, overexpressing dominant-negative mutants of IB, IKK, TAK1, MyD88, and TLR2 inhibits NTHi-induced MUC2 transcription in both HMEEC-1 (left panel) and NHBE cells (right panel). Thus, it is clear that NTHi-induced MUC2 up-regulation via the TLR2-MyD88-TAK1-NIK-IKK-IB-NF-B signaling pathway is also well conserved in the relevant human middle ear and bronchial epithelial cells.

To further confirm whether the endogenous MUC2 gene and MUC2 promoter-driven luciferase reporter gene are similarly up-regulated, we evaluated the effects of overexpressing dominant-negative mutants of several key signaling molecules identified above, including IB, TAK1, and MyD88. All these treatments inhibited NTHi-induced up-regulation of MUC2 mRNA, confirming that the TLR2-MyD88-TAK1-NIK-IKK-IB-NF-B signaling pathway revealed by luciferase reporter gene assays are indeed responsible for induction of endogenous MUC2 gene expression by NTHi (Fig. 2H).

Activation of TGF- Receptor I/II-Smad3/4 Signaling Pathway Is Also Required for NTHi-induced MUC2 Transcription-- Because of the important role of TGB- signaling in regulation of diverse cellular responses, we were interested in determining whether TGF- receptor signaling is also required for NTHi-induced MUC2 transcription. We first examined if NTHi activates TGF--Smad signaling by evaluating NTHi-induced nuclear translocation of Smad4, a key step for Smad3/4 complex to exert its transcriptional activity (18), As seen in Fig. 3A, it is evident that NTHi potently induces nuclear translocation of Smad4. As expected, Smad4 translocation was also induced by TGF-1. To further confirm whether NTHi activates TGF--Smad-dependent transcriptional activity, we assessed the effect of NTHi on SBE-dependent promoter activity by using SBE luciferase reporter (38) and TGF--responsive promoter activity of a PAI-1-Luc in HM3 cells (39). When we exposed the transfected cells to NTHi or TGF-1, SBE-driven luciferase activity 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 requirement of TGF- signaling in NTHi-induced MUC2 transcription by overexpressing dominant-negative mutant of TRII (27, 32, 33). Interestingly, co-transfection with dominant-negative TRII greatly inhibited NTHi-induced MUC2 transcription (Fig. 3B). Concomitantly, overexpression of a dominant-negative mutant of TRI also blocked MUC2 induction (Fig. 3B). In accordance with these results, no NTHi-induced MUC2 transcription was shown in either R1B or DR26 cells, two cell lines that are derived from Mv1Lu cells and lack functional TRI and TRII, respectively (29), whereas the wild-type Mv1Lu cells still showed potent MUC2 induction by NTHi (Fig. 3C). Moreover, co-transfecting R1B and DR26 cells with wild-type TRI and TRII expression plasmids rescued the responsiveness to NTHi (Fig. 3C). Thus, these data suggest the requirement of TGF- receptor type I/II signaling in NTHi-induced MUC2 transcription.

View larger version (19K): [in this window] [in a new window]   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 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 into wild-type Mv1Lu cells expressing TRI and TRII or the derivative R1B and DR26 cells lacking functional TRI and TRII, respectively. Expression plasmid of wild-type TRI or TRII was co-transfected, as marked. D, co-expression of dominant-negative mutants of Smad3 and Smad4, but not Smad2, inhibits NTHi-induced MUC2 up-regulation. E, co-expression of dominant-negative mutants of TRII and Smad4 also inhibits NTHi-induced MUC2 transcription in HMEEC-1 (left panel) and NHBE cells (right panel). F, co-expression of dominant-negative mutants of TRII and Smad4 attenuate NTHi-induced MUC2 up-regulation at mRNA level. In all the experiments shown above, transfections were carried out 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).

To determine the involvement of Smad2/3-Smad4 in MUC2 induction, dominant-negative mutants of Smad2, 3, and 4 were co-transfected into HM3 cells with MUC2 luciferase reporter construct (27). As shown in Fig. 3D, overexpression of a dominant-negative mutant of Smad3 or Smad4, but not Smad2, greatly inhibited NTHi-induced MUC2 transcription, suggesting the involvement of Smad3/4 in MUC2 induction.

To address whether the TGF--Smad signaling pathway also mediates NTHi-induced MUC2 transcription in HMEEC-1 and NHBE, we next assessed the effects of overexpressing dominant-negative mutants of TRII and Smad4 on NTHi-induced MUC2 transcription. Interestingly, all these treatments inhibited NTHi-induced up-regulation of MUC2 in both HMEEC-1 (Fig. 3E, left panel) and NHBE cells (right panel), confirming that this signaling pathway is indeed well conserved in the relevant human middle ear and bronchial epithelial cells.

To further confirm whether the endogenous MUC2 gene and MUC2 promoter-driven luciferase reporter gene are up-regulated similarly by the TGF--Smad signaling pathway identified above, we evaluated the effects of overexpressing dominant-negative mutants of TRII and Smad4 on NTHi-induced MUC2 induction. All these treatments inhibited NTHi-induced up-regulation of MUC2 mRNA, confirming that the TRII-Smad4 signaling pathway revealed by luciferase reporter gene assays is indeed responsible for induction of endogenous MUC2 gene expression by NTHi (Fig. 3F).

NTHi Activates TGF--Smad Signaling Likely Via an Autocrine-independent Mechanism-- Although we have demonstrated that activation of the TGF--Smad signaling pathway is required for NTHi-induced MUC2 transcription, it is still unclear whether TGF- signaling is activated directly by NTHi-derived TGF--like factor or indirectly by NTHi-induced TGF- autocrine signaling. We first evaluated the time course of NTHi-induced phosphorylation of TRII by using an antibody against phosphorylated tyrosine at position 336 of TRII. Fig. 4A shows phosphorylation of TRII in cells treated with NTHi for various times. The NTHi-induced TRII phosphorylation became evident at as early as 5 min. Given such an early phosphorylation of TRII, it is likely that the early phosphorylation of TRII occurred as a result of direct activation of TR signaling by NTHi, rather than NTHi-induced TGF- autocrine signaling. To determine whether NTHi-derived TGF--like factor is responsible for the activation of TGF- signaling, we assessed the effect of NTHi lysates pretreated with TGF- neutralization antibody or control antibody on the transcriptional 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 efficiency 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 TR-mediated signaling by NTHi mediates MUC2 transcription. To further determine whether TGF- signaling is also activated indirectly by NTHi-induced TGF- autocrine signaling, we next determined whether NTHi induces any increase in three major TGF- family members, TGF-1, 2, and 3, in the conditioned media of HM3 cells using TGF-1, 2, and 3 ELISA kits. Notably, 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--Smad signaling pathway, at least in part, via a mechanism independent of TGF-1, 2, and 3 autocrine signaling, although our data do not preclude the involvement of the latent TGF-s stored in the extracellular matrix that might be activated by NTHi and then cross-talk with TRI and TRII. In addition, it is still unclear whether other TGF- family members may be involved in mediating NTHi-induced MUC2 transcription in an autocrine manner.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   NTHi activates TGF--Smad signaling likely via an autocrine-independent mechanism. A, NTHi induces phosphorylation of TRII in HM3 cells in a time-dependent manner as assessed using an antibody against phosphorylated TRII (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 promoter and 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 as assessed using TGF-1, 2, and 3 ELISA kits.

Functional Cooperation of Smad3/4 with NF-B p65/p50 Appears to Mediate NF-B-dependent MUC2 Transcription-- Because our data (Fig. 2) demonstrated that activation of NF-B is required for NTHi-induced MUC2 transcription and no functional SBE was found within the functional promoter region of MUC2, we next determined whether the TGF--Smad signaling pathway mediates MUC2 transcription via its functional interaction with NF-B (26). We first assessed the effect of overexpressing dominant-negative mutants of TGF- receptor type II and Smad4 on NTHi-induced NF-B-driven promoter activity. As seen in Fig. 5A, NTHi-induced NF-B activation was abrogated by both treatments, indicating the direct involvement of TGF--Smad signaling in NTHi-induced NF-B activation. We next investigated whether activation of the TGF- signaling pathway contributes to NTHi-induced promoter activity of both MUC2-luc and NF-B-luc in a similar way. As shown in Fig. 5B, activation of TGF- signaling by TGF-1 or co-expression of wild-type Smad3 and Smad4 greatly enhanced NTHi-induced NF-B activation as well as MUC2 transcription. These data indicate that the TGF--Smad signaling pathway mediates MUC2 transcription likely via cross-talk with NF-B, rather than with other DNA response elements in MUC2 promoter. However, it is still unclear which NF-B subunits are involved in mediating its interaction with Smad3 and Smad4.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Functional cooperation of Smad3/4 with NF-B p65/p50 appears to mediate NF-B-dependent MUC2 transcription. A, co-expression of a dominant-negative mutant of TGF- receptor II or Smad4 blocks NTHi-induced NF-B activation. NF-B luciferase reporter vector was co-transfected into HM3 cells with either an empty vector or a dominant-negative mutant of TGF- receptor II or Smad4. B, activation of TGF- signaling by TGF-1 (1 ng/ml) or co-expression of wild-type Smad3 and Smad4 greatly enhanced NTHi-induced NF-B activation as well as MUC2 transcription. C, effects of overexpression of Smad3 and Smad4 on p65 and/or p50-induced NF-B activation. In these experiments, luciferase reporter vector was co-transfected into HM3 cells with either an empty vector or vectors containing wild-type Smad3, Smad4, p50, and p65 as indicated. Values are the means ± S.D. (n = 3).

Based on the importance of NF-B subunits p65 and p50 in mediating a variety of cellular responses, we first overexpressed Smad3 and Smad4, both alone and in concert with NF-B subunits p65 and p50 and then assessed their NF-B transactivation potential. As expected, overexpression of either p65 alone, or together with p50, induced potent NF-B reporter gene activation (lanes 3 and 4), whereas overexpression of either p50, or Smad3 or Smad4 alone only induced weak NF-B reporter gene activation (Fig. 5C, lanes 2, 5, and 6). Interestingly, co-expression of p65 but not p50 with Smad3, or Smad4 induced relatively potent NF-B-dependent promoter activity (lanes 8-11). Moreover, co-expression of Smad3 and Smad4 also induced potent NF-B activation (lane 7), which was further greatly enhanced by co-transfection with p65 but not with p50 (lanes 14 and 15). It should be noted that, although overexpression of p50 alone did not appear to activate NF-B potently, it greatly enhanced NF-B reporter gene activation induced by co-expression of either p65 and Smad3 or Smad4 (lanes 12 and 13) or p65, Smad3, and Smad4 (lane 16). Taken together, these results indicate that, at least p65, p50, Smad3, and Smad4 are involved in the functional cooperation of NF-B with the TGF- signaling pathway.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activation of TGF--Smad Signaling by Bacterium NTHi Appears to be Required for Up-regulation of MUC2 Mucin in Human Epithelial Cells-- TGF- and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response (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 that activation of the TGF- receptor-Smad3/4 signaling pathway by Gram-negative bacterium NTHi is required for induction of MUC2 mucin transcription in human epithelial cells. Several lines of evidence strongly support this notion. First, overexpression of dominant-negative mutants of TRI, TRII, Smad3, and Smad4 greatly inhibited NTHi-induced MUC2 transcription (Fig. 3, B and D-F). In accordance with these results, no NTHi-induced MUC2 transcription was shown in either R1B or DR26 cells, mutant Mv1Lu cells that lack functional TRI and TRII, respectively (29), whereas the wild-type Mv1Lu cells still showed potent MUC2 induction by NTHi (Fig. 3C). Moreover, co-transfecting R1B and DR26 cells with wild-type TRI and TRII expression plasmids rescued the responsiveness to NTHi. These observations implicated the requirement of TGF- receptor-Smad signaling in NTHi-induced MUC2 up-regulation. Second, NTHi potently induced phosphorylation of TRII, nuclear translocation of Smad4 and TGF- responsive PAI-1 promoter activity as well as SBE-driven promoter activity, demonstrating the ability of NTHi in activating the TGF- signaling pathway (Figs. 3A and 4A). Finally, the NTHi-induced TRII phosphorylation occurred at as early as 5 min, suggesting that NTHi may activate TR-mediated signaling via a TGF- autocrine-independent mechanism. Although TRI and TRII are known as serine/threonine kinases, there is also evidence that TRII can function as a dual specificity kinase and tyrosine phosphorylation may have an important role in TR signaling (40). Thus, NTHi-induced tyrosine phosphorylation at 5 min may be at least interpreted as a TR-mediated early response to NTHi independent of TGF- autocrine. Additionally, pretreatment of NTHi lysates with the neutralization TGF- antibody reduced its ability to induce the transcriptional activity of MUC2 promoter (Fig. 4B). Moreover, as evidenced by our ELISA experiments (Fig. 4C), NTHi did not induce any detectable increase in TGF-1, 2 and 3 in the conditioned media of the epithelial cells. Collectively, these data suggest that NTHi-induced MUC2 transcription is mediated, at least in part, by direct activation of the TGF--Smad signaling pathway, independent of TGF-1, 2, and 3 autocrine signaling, although our data do not preclude the involvement of the latent TGF-s stored in the extracellular matrix that might be activated by NTHi and then cross-talk with TRI and TRII (Fig. 6). In addition, it is still unclear whether other TGF- family members may be involved in mediating NTHi-induced MUC2 transcription in an autocrine manner. Previously, there has been a report for the involvement of parasite Trypanosoma cruzi-derived TGF--like factors in T. cruzi invasion (41). Given the fact that bacterial lysate of NTHi was used in our studies, it is possible that a bacterial-derived TGF--like product may be responsible for directly inducing TGF- receptor signaling. It should be noted that lipooligosaccharide from NTHi did not significantly up-regulate MUC2 transcription. Therefore, the molecular identity of NTHi-derived TGF--like factors should be further investigated. Additionally, we will also determine whether NTHi also activates the latent TGF- stored in the extracellular matrix that, in turn, leads to the activation of TGF- signaling.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Schematic representation of NTHi-induced signaling pathways involved in MUC2 up-regulation in human epithelial cells. NTHi, an important human respiratory pathogen, utilizes the TGF- receptor type I/II-Smad3/4 signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKK/-IB pathway to mediate NF-B-dependent MUC2 mucin transcription. NTHi-induced MUC2 transcription appears to be mediated, at least in part, by direct activation of the TGF--Smad signaling pathway, independent of TGF-1, 2, and 3 autocrine signaling. Moreover, functional cooperation of NF-B p65/p50 with Smad3/4 appears to positively mediate NF-B-dependent MUC2 transcription. Up-regulation of mucin production, known as a primary innate defensive response for mammalian airways, contributes significantly to mechanical clearance of inhaled infectious particles. However, in patients with OM with effusion and COPD whose mucociliary clearance mechanisms have become defective, excessive production of mucin will lead to airway obstruction in COPD and conductive hearing loss in OM with effusion.

Functional Cooperation of Smad with NF-B Is Positively Involved in Mediating NTHi-induced Up-Regulation of MUC2 Transcription-- In addition to the activation of the TGF- signaling pathway by NTHi for MUC2 induction, another interesting finding in this study is the functional cooperation of the TGF--Smad signaling pathway with NF-B to positively mediate MUC2 up-regulation by NTHi. In contrast to the role of TGF- signaling in suppressing the killing activity of macrophage and enhancing intracellular proliferation of infectious pathogen such as Leishmania, activation of TGF- signaling actually cooperates with NF-B to positively mediate host defensive responses to bacterium NTHi. Recently, growing evidence suggests that the TGF--Smad signaling pathway regulates gene transcription either by functional cooperation with transcription factors bound to adjacent transcription factors or directly interacting with transcription factors bound to DNA response element (21-22). For instance, functional cooperation between Smad and NF-B that are bound to Smad- and TNF-response elements, respectively, activates the expression of an extracellular matrix-related gene, COL7A1 (42). In contrast, Smad3 has been shown to stimulate transcription from the HIV-1 LTR promoter via an interaction with NF-B bound to a B site (43). Similarly, a B site has also been identified as a TGF--responsive region in the 3'-downstream junB promoter region (26). Concurrent with the later cases, the TGF--Smad signaling pathway mediates NTHi-induced MUC2 transcription also via a B site in the 5'-upstream MUC2 promoter region, providing the first identification of a functional cooperation 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 between NF-B and TGF- signaling pathways. It should be noted that, in contrast to the potent activation of NF-B induced by overexpression of p65 and Smad3/4, both alone and in concert with one another, the NF-B transactivation potential of p50 is rather weak. In addition, NF-B activation induced by co-expression of p65 with Smad4 is more potent than that induced by co-transfection of p65 with Smad3. Our data are consistent with the recent report by Lopez-Rovira et al. (26) showing that overexpression of Smad3 and Smad4 enhances transactivation of NF-B and co-expression of Smad3 or Smad4 together with the NF-B subunit p65 further increases those responses. Additionally, they showed that co-expression of Smad3 or Smad4 with NF-B p52 also further greatly enhances NF-B activation. Thus, it seems necessary to further explore the involvement of NF-B p52 in the functional cooperation between NF-B and TGF- signaling involved in NTHi-induced MUC2 transcription.

In conclusion, our studies demonstrate that bacterium NTHi utilizes the TGF- receptor type I/II-Smad3/4 signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKK/-IB pathway to induce NF-B-dependent MUC2 mucin transcription, a primary innate defensive response for mammalian airways. Functional cooperation of NF-B p65/p50 with TGF--Smad3/4 is likely involved in mediating NTHi-induced MUC2 transcription. These studies provide evidence for the first time that demonstrate the activation of TGF- receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF- signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

	ACKNOWLEDGEMENTS

We thank A. S. Baldwin for p65 and p50 expression plasmids and J. Massague for mutant mink cell lines DR26 and R1B.

	FOOTNOTES

^* This work was supported in part by grants from the National Institutes 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 Veterans Affairs Medical Research Service (to J. G. and Y. S. K.), American Cancer 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.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M206883200

	ABBREVIATIONS

The abbreviations used are: NTHi, nontypeable Haemophilus influenzae; ELISA, enzyme-linked immunosorbent assay; SBE, Smad-binding element; TGF-, transforming growth factor-; COPD, chronic obstructive pulmonary diseases; OM, otitis media; R-Smad, receptor-activated Smad; TR, TGF- receptor; CAPE, caffeic acid phenethyl ester; HM3, human colon epithelial cell line; HMEEC-1, human middle ear epithelial cell line; NHBE, primary human bronchial epithelial cell; PAI-1, plasminogen activator inhibitor-1; MUC, mucin; TLR, Toll-like receptor.

	REFERENCES

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