MUC5AC Expression through Bidirectional Communication of Notch and Pathways

This information is current as Jin Hyun Kang, Eun Hee Lee, Sung Woo Park and Il Yup of September 29, 2021. Chung J Immunol 2011; 187:222-229; Prepublished online 27 May 2011; doi: 10.4049/jimmunol.1003606

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References This article cites 61 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/187/1/222.full#ref-list-1 http://www.jimmunol.org/

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MUC5AC Expression through Bidirectional Communication of Notch and Epidermal Growth Factor Receptor Pathways

Jin Hyun Kang,* Eun Hee Lee,† Sung Woo Park,† and Il Yup Chung*

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epi- dermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jk–dependent manner. Treatment with g-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary

for the regulation of EGF receptor (EGFR)–mediated MUC5AC expression by Notch signaling. Conversely, forced expression of Downloaded from NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.

The Journal of Immunology, 2011, 187: 222–229. http://www.jimmunol.org/

ucin hypersecretion with goblet cell hyperplasia/meta- pathways. Among these, EGFR/EGFR ligands have been exten- plasia is an important pathophysiologic feature of sively studied and shown to be expressed in airway epithelium (8, M chronic airway inflammatory diseases such as severe 20, 21). They are closely associated with proliferation, differen- asthma (1), chronic obstructive pulmonary disease (2), and cystic tiation, and repair of airway epithelial cells in both normal and fibrosis (3). Normally, mucus secretion creates homeostasis for the pathological conditions (22–25). The EGFR-mediated production conducting airways by forming a physical boundary that prevents of MUC5AC in airways requires, although not exclusively, acti- entry of inhaled irritants, including microorganisms and particles. vation of the ERK pathway, because pharmacologic inhibition of by guest on September 29, 2021 However, in many chronic inflammatory airway diseases, excess the ERK pathway shuts down MUC5AC mRNA expression and mucin is produced, leading to exacerbation of the disease. Airway MUC5AC promoter activity (11). Further studies showed that mucus hypersecretion and plugging contribute significantly to the ERK activity is required for binding of specificity protein 1 to its morbidity and mortality of chronic airway diseases. Among 12 elements in the MUC5AC promoter and subsequent transcription mucin proteins known to be expressed in the respiratory tract, activation (11, 26). MUC5AC appears to be the most prominent, and also serves as the Notch plays an important role in the determination of cell fate most specific marker of goblet cells (4–7). MUC5AC is produced in multiple organ systems (27). Four Notch receptors (Notch1–4) in response to a wide variety of stimuli, including epidermal growth and five ligands (Jagged 1 and 2, and d-like 1, 3, and 4) have been factor (EGF) receptor (EGFR) ligands, inflammatory cytokines, identified in mammals to date. Notch receptor– interaction cholinergic agonists, neutrophil elastase, air pollutants, reactive via cell–cell contact is followed by two successive proteolytic oxygen species, and bacterial products (8–19). These stimuli induce cleavages of Notch, one by a disintegrin and metalloprotease 10, MUC5AC expression through both distinct and shared signaling and the other by a g-secretase, leading to the liberation of Notch intracellular domain (NICD). The released NICD translocates to *Division of Molecular and Life Sciences, College of Science and Technology, the nucleus, where it interacts with RBP-Jk/CSL to activate ex- Hanyang University, Gyeonggi-do 426-791, South Korea; and †Genome Research Center for Allergy and Respiratory Diseases, Division of Allergy and Respiratory pression of the primary target of Notch signaling such as Medicine, Soonchunhyang University Hospital, Bucheon-Si, Gyeonggi-Do 420-021, hairy and enhancer of split-1 (HES-1). HES-1 is a basic helix- South Korea loop-helix transcriptional repressor and acts as a Notch effector by Received for publication November 2, 2010. Accepted for publication April 28, 2011. negatively regulating expression of downstream target genes such This work was supported by Grant A090419 (to I.Y.C.) from the Korea Health 21 as tissue-specific transcription factors (28, 29). Although Notch R&D Project, Ministry of Health and Welfare, Republic of Korea. J.H.K. was sup- ported by BK21 from the National Research Foundation. signaling is primarily mediated by a canonical pathway through Address correspondence and reprint requests to Dr. Il Yup Chung, Division of Mo- RBP-Jk/CSL and subsequent trans activation of its downstream lecular and Life Sciences, College of Science and Technology, Hanyang University, targets, Notch engages in crosstalk with other signaling pathways, 1271 Sa-3-dong, Ansan, Gyeonggi-do 426-791, South Korea. E-mail address: [email protected] especially the EGFR and MAPK pathways. Most notable is a dy- namic interplay between the Notch and EGFR signaling pathways Abbreviations used in this article: EGF, epidermal growth factor; EGFR, EGF re- ceptor; GSI, g-secretase inhibitor; HBEC, human bronchial epithelial cell; HES-1, in the eye and wing development of fruit flies, in which the two hairy and enhancer of split-1; NICD, Notch intracellular domain; siRNA, small in- pathways act in concert to direct one phenotype or to antagonize terfering RNA. each other to produce another phenotype, depending on the de- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 velopmental context (30–32). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003606 The Journal of Immunology 223

Analysis of genetically modified animals with perturbed Notch sample using the Dual-Luciferase Assay System kit (Promega, Madison, signaling demonstrates that Notch signaling controls the fates of WI). For each transfection, relative firefly luciferase activity was normal- a wide variety of cell phenotypes in the lungs during embryonic ized to Renilla luciferase activity. development (33–37). Moreover, most of the Notch signaling Real-time PCR components, including Notch receptors, Notch ligands, and their downstream effectors, are expressed in the airway epithelium of Real-time quantitative PCR was performed in a 20 ml reaction with 0.5 ml adult humans, and some of them exhibit altered expression in cDNA, 0.8 ml of each primer (10 pM), and 10 ml SYBR Green Master Mix (Applied Biosystems, Foster City, CA), using the Applied Biosystems a diseased condition (38), suggesting active and dynamic roles of Prism 7900 Sequence Detection System (Applied Biosystems). PCR Notch signaling in adult lung tissues in addition to developing conditions were as follows: 2 min at 50˚C and 10 min at 95˚C, followed by lung. However, the functional consequence of their expression 40 cycles of 95˚C for 30 s, 60˚C for 30 s, and 72˚C for 30 s. The following remains poorly understood, especially in adult lungs. We pre- primers were used for amplification: MUC5AC, forward 59-ACTGGG- viously demonstrated that inhalation of a g-secretase inhibitor GAGAAATATCGCATG-39 and reverse 59-CTGATCAAAGGGCCTGA- TAGC-39; FOXA2, forward 59-TCTTAAGAAGACGACGGCTTCAG-39 (GSI) attenuates the cardinal features of asthma, including goblet and reverse 59-TTGCTCTCTCACTTGTCCTCGAT-39; HES-1, forward cell production, in an animal model of asthma (39). This obser- 59- GCGGACATTCTGGAAATGAC-39 and reverse 59-GATCTGGGTC- vation suggests a positive correlation between Notch signaling ATGCAGTTGG-39; and GAPDH, forward 59-CGTCTTCACCACCATG- and MUC5AC expression. In this study, we investigated the invol- GAGA-39 and reverse 59-CGGCCATCACGCCACAGTTT-39. PCR was performed in triplicate in 384-well plates. Amplification of GAPDH was vement of Notch signaling in MUC5AC expression, one of the performed to generate a standard curve to test assay sensitivity and for major phenotypes that occurs upon provocation of airway epithelial a quantification reference. The standard curve was derived from five points cells, and explored the underlying mechanism using approaches of a 2-fold cDNA dilution series. Downloaded from that inhibit or activate Notch signaling. Notch1 and HES-1 mRNA expression Materials and Methods Total mRNA was extracted from NCI-H292 cells using TRI reagent Cell culture and inhibitor treatment (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized from 2 mg total RNA using SuperScript II RNase reverse NCI-H292 cells, which are human pulmonary mucoepidermoid carcinoma transcriptase (Invitrogen) in a 20 ml reaction, including the oligo(dT) cells, were cultured in RPMI 1640 medium (Welgene) containing 10% FBS, 12–18 http://www.jimmunol.org/ primer, deoxynucleotide triphosphates (0.5 mM), MgCl2 (2.5 mM), and penicillin (100 U/ml), streptomycin (100 mg/ml), HEPES (25 mM), and DTT (5 mM). Reverse transcription was performed at 42˚C for 1 h and sodium pyruvate (1 mM). At confluence, cells were starved in RPMI 1640 followed by heat inactivation at 70˚C for 15 min. Synthesized cDNA was lacking FBS for 4 h, and incubated for 24 h in RPMI 1640 containing 1% amplified for 30 cycles with Ex Taq DNA polymerase (TAKARA, Shiga, FBS with 25 ng/ml recombinant human EGF (R&D Systems, Minneapolis, Japan). The following primers were used: Notch1, forward 59-CGTC- MN). g-Secretase inhibitors (GSIs) L-685,458 (Calbiochem, La Jolla, CA) ATCTCCGACTTCATCTAC-39 and reverse 59-GGATCAGGATCTG- and DAPT (Calbiochem), EGFR inhibitor AG1478 (Calbiochem), MEK GAAGACAC-39. Primers for RT-PCR of HES-1 and GAPDH were ones inhibitor U0126 (Calbiochem), p38 inhibitor SB203580 (Tocris, Ballwin, that were also used for real-time PCR. MO), and JNK inhibitor SP600125 (Tocris) were dissolved in DMSO. Cells were pretreated with inhibitors for 30 min prior to treatment with EGF. Western blot analysis by guest on September 29, 2021 Air–liquid interface cell culture for human bronchial epithelial Cells were lysed by incubation in RIPA buffer (50 mM Tris-Cl [pH 7.4], 0.1% NaN , 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, cell 3 1mMNa3VO4, 1 mM NaF, and protease inhibitor mixture [Calbiochem]) Primary human bronchial epithelial cells (HBECs; lot 3F1191; Cambrex for 30 min on ice. A total of 20 mg cell lysate resolved by SDS-PAGE Bio Science, Baltimore, MD) were prepared, as previously described (40). and transferred to polyvinylidene difluoride membranes. Membranes Frozen passage-1 stocks were thawed and cultured in T-flasks in bronchial were blocked with 5% nonfat dry milk, probed with anti-NICD (Abcam, epithelial cell growth medium (Cambrex Clonetics, Walkersville, MD) Cambridge, U.K.), anti–phospho-ERK1/2 ( Technology, supplemented with bovine pituitary extract, hydrocortisone (0.5 mg/ml), Beverly, MA), anti–phospho-p38 (Santa Cruz Biotechnology, Santa Cruz, recombinant human EGF (0.5 ng/ml), epinephrine (0.5 mg/ml), transferrin CA), and anti–phospho-EGFR (12A3; Santa Cruz Biotechnology), and (10 mg/ml), insulin (5 mg/ml), retinoic acid (0.1 ng/ml), triiodothyronine treated with anti-rabbit HRP-conjugated Abs (Cell Signaling Technology). (6.5 ng/ml), gentamicin sulfate (50 mg/ml), and amphotericin B (50 ng/ Anti-ERK2 (C-14; Santa Cruz Biotechnology), anti-p38 (Cell Signaling ml). Medium was replaced every 48 h until cells reached 90% confluence. Technology), anti-EGFR (13G8; Santa Cruz Biotechnology), and anti- Cells were seeded at 8.25 3 104 cells/insert on Corning Costar Transwell GAPDH (Santa Cruz Biotechnology) Abs were used as loading controls. inserts with 0.4 mm pores (Corning Life Sciences, Lowell, MA). Both the Immunostained proteins were detected with the ECL detection system apical (0.5 ml) and basolateral side (1.5 ml) were filled with differentiation (Amersham Pharmacia Biotech, Piscataway, NJ). Phospho-ERK levels medium containing 1:1 mixtures of DMEM and bronchial epithelial cell in some results were analyzed by densitometry (Image acquisition and growth medium with supplements, as above, except that triiodothyronine, analysis software, UVP) and expressed as an intensity relative to phospho- gentamicin sulfate, and amphotericin B were omitted, and penicillin, ERK1/2. streptomycin, and 50 nM all-trans retinoic acid (Sigma-Aldrich, St. Louis, MO) were added. Cells were submerged for 7 d in culture, and then the Immunocytochemistry apical medium was removed to establish an air–liquid interface for 14 d. Medium was refreshed three times weekly. Cells were grown at 37˚C in HBECs cultured at an air–liquid interface were stained for MUC5AC protein expression, as previously described (40). Cells were fixed in 4% 5% CO2 in a humidified incubator and protected from light. The apical surface of the cells was rinsed with PBS once weekly to remove accu- paraformaldehyde and embedded in paraffin, which was sliced to 4 mm. mulated mucus and debris. Both HBECs and NCI-H292 cells on slides were permeabilized in TBS containing 0.1% saponin for 15 min at room temperature. Slides were Measurement of luciferase activity treated with 0.3% H2O2-methanol for 20 min to block endogenous per- oxidase, washed twice in TBS, treated with blocking Ab for 30 min at For the reporter assay, NCI-H292 cells were transfected with RBP-jk room temperature, and incubated at 4˚C overnight with anti-MUC5AC Ab promoter–luciferase constructs (SABiosciences, Frederick, MD) using (Chemicon). After washing with TBS, slides were treated with biotinylated a Lipofectamine 2000-based transfer method (Invitrogen, Carlsbad, goat anti-mouse IgG for 30 min at room temperature, and then with avi- CA), according to the manufacturer’s specifications. In brief, cells were din–biotin peroxidase complex (Vector Laboratories, Burlingame, CA) for plated onto 12-well plates at 70–80% confluency. The cells were incubated 30 min at room temperature. Color was developed by staining with 3,39- at 37˚C with a mixture of DNA constructs and Lipofectamine 2000 in diaminobenzidine tetrachloride (Zymed Laboratories, San Francisco, CA). Opti-MEM (Invitrogen) for 12 h. The transfected cells were treated with The reaction was terminated by washing in running water until a uniform EGF in the presence or absence of GSI for 24 h. After 36 h, firefly and brown color became visible on the section. Negative control slides were Renilla luciferase activities were measured sequentially from a single incubated with isotype-matched Ab. 224 INVOLVEMENT OF NOTCH SIGNALING IN MUC5AC EXPRESSION

Expression of Notch 1 small interfering RNA A mixture of dsRNA nucleotides targeting different regions of Notch1 mRNA and negative control small interfering (si)RNA (scrambled siRNA) was obtained from Dharmacon Research (Dharmacon, Chicago, IL). For transient expression, cells were transfected with Notch1 and scrambled siRNA oligonucleotides using DharmaFECT reagent, according to the manufacturer’s protocol. NICD overexpression The cDNA encoding the NICD from Notch1 was previously described (41). A Flag epitope sequence was introduced at the N terminus of NICD, and the recombinant NICD was subcloned into pcDNA3.1. The NICD con- struct was transfected into NCI-H292 cells using Lipofectamine 2000. At 36 h posttransfection, cells were cultured in serum-free medium for 2 h, pretreated with inhibitors or neutralizing Abs (anti-EGFR or anti–TGF-a) for 30 min, and treated with EGF for 15 min. Immunoblot analysis of cell lysates determined for ERK phosphorylation and NICD expression. Statistical analysis All data were analyzed using the SPSS program for independent t tests. ,

Differences with a p value 0.05 were considered statistically significant. Downloaded from Results are expressed as mean 6 SEM.

Results Notch inhibitors block the expression of MUC5AC FIGURE 1. GSI inhibits MUC5AC mRNA expression. A, C, and D, We examined the effect of pharmacological inhibitors of Notch NCI-H292 cells were incubated with EGF in the presence or absence of L- 685,458 or DAPT for 24 h, and mRNA levels for MUC5AC, HES-1, and signaling on MUC5AC mRNA expression in NCI-H292 cells. http://www.jimmunol.org/ Treatment with EGF (25 ng/ml) increased MUC5AC mRNA ex- FOXA2 were measured by real-time PCR. GAPDH was used to normalize the levels of these transcripts. Data are shown as means 6 SEM of six pression, as determined by real-time PCR. Two different GSIs, L- independent experiments performed in triplicate (*p , 0.05, **p , 0.01 685,458 and DAPT, inhibited EGF-mediated MUC5AC mRNA compared with EGF-treated cells). B, L-685,458 prevents the EGF-medi- expression in a dose-dependent manner (Fig. 1A). To address the ated accumulation of NICD. NCI-H292 cells were treated with varying involvement of Notch signaling in EGF-mediated MUC5AC ex- concentrations of L-685,458 in the presence of EGF for 36 h, and cell pression, we determined NICD generation using an Ab specific for lysate was subjected to immunoblot analysis. The blot was probed with an Notch1-derived NICD. EGF increased NICD generation (Fig. 1B), Ab specific for NICD. GAPDH was used as the loading control. This result indicating that ligation of EGFR leads to activation of Notch is representative of five independent experiments. E, EGF stimulates Notch signaling. L-685,458 inhibited EGF-induced NICD generation in signaling in a RBP-jk–dependent manner. NCI-H292 cells were trans- by guest on September 29, 2021 a dose-dependent manner. Furthermore, L-685,458 inhibited the fected with an RBP-jk-Luc reporter. After 12 h, the cells were treated with HES-1 expression that was elevated by EGF treatment (Fig. 1C). EGF and/or GSI for 24 h. Renilla luciferase activity was used to normalize transfection efficiency. The transcription activity was expressed as a fold To test whether downregulation of MUC5AC by GSI paralleled increase over that in cells treated with medium. Data are shown as a known phenotype of goblet cell hyperplasia/metaplasia, we means 6 SEM of six independent experiments performed in duplicate. examined FOXA2 expression. Targeted disruption of the FOXA2 **p , 0.01 compared with EGF-treated cells. transcription factor gene in bronchial epithelial cells causes mucus metaplasia and increases in MUC5AC protein expression. FOXA2 culture for HBECs. Incubation of the air–liquid interface culture also inhibits activation of MUC5AC promoter (42). EGFR acti- with medium containing a low dose of EGF (0.25 ng/ml) for vation and FOXA2 expression have an inverse relationship (40). 14 d resulted in little expression of MUC5AC mRNA. After the EGF treatment slightly reduced FOXA2 mRNA expression, and differentiated cells were extensively rinsed and exposed to high this was suppressed by L-685,458 (Fig. 1D), consistent with the dose of EGF (40 ng/ml) for 36 h, MUC5AC mRNA levels finding that FOXA2 inhibits MUC5AC . To de- were increased. Addition of GSI led to inhibition of MUC5AC termine whether Notch signaling activated by EGF exerts its mRNA expression with an increase in FOXA2 mRNA level (Fig. function via RBP-jk, we used a RBP-jk-Luc reporter plasmid. 3A). Immunocytochemistry analysis showed that EGF-induced When NCI-H292 cells were transfected with the reporter and MUC5AC protein expression was also inhibited by GSI treat- treated with EGF, promoter activity increased ∼2.5-fold (Fig. 1E). ment (Fig. 3B). Collectively, these results suggest that EGF GSI treatment reduced the reporter activity to basal levels, sug- induces MUC5AC expression at least in part through activation of gesting involvement of RBP-jk in EGF-activated Notch signaling. Notch signaling, Next, we examined the effect of GSI on MUC5AC protein ex- pression, determined by immunocytochemistry. The percentage of Silencing of Notch1 reduces EGF-induced MUC5AC MUC5AC-positive cells increased from 4.1 6 0.7% to 26 6 2.8% expression following EGF stimulation. This was almost completely abolished To determine whether the selective reduction of endogenous by the EGFR inhibitor AG1478. Treatment with L-685,458 or Notch1 led to a decrease in MUC5AC expression, we used siRNA DAPT (each at 10 mM) inhibited EGF-induced MUC5AC protein oligonucleotides directed against Notch1. EGF increased Notch1 expression, similar to the inhibition by treatment with U0126, and HES-1 mRNA synthesis. When NCI-H292 cells were trans- a specific ERK inhibitor (Fig. 2). The concentrations of GSI used fected with a pool of four Notch1 siRNA oligonucleotides, EGF- did not affect the viability of the epithelial cells during culture mediated accumulation of NICD and mRNAs for Notch1 and HES- (Fig. 2A and data not shown). To evaluate the involvement of 1 was considerably inhibited, whereas scrambled siRNA oligo- Notch signaling in EGF-induced MUC5AC expression in a more nucleotides had little effect (Fig. 4A,4B). In line with these physiological setting, we implemented an air–liquid interface findings, Notch1 siRNA attenuated EGF-induced expression of The Journal of Immunology 225

basal level of active p38 was selectively reduced only by SB203580 (Fig. 5A). JNK phosphorylation was not detected in this cell type under conditions tested (data not shown). In parallel, we also tested the effects of these inhibitors on EGF-induced MUC5AC mRNA levels. Like AG1478 and U016, GSI inhibited EGF-induced MUC5AC mRNA expression, whereas inhibitors of p38 and JNK had no effect (Fig. 5C). This finding was consistent with their inhibitory effects on MUC5AC protein expression, as shown in Fig. 2. Introduction of Notch1 siRNA attenuated ERK phosphorylation in EGF-treated cells (Fig. 5D). It is noticed that Notch1 siRNA reduced the basal level of phospho-ERK in un- treated cells. These data suggest that Notch signaling is involved in EGF-mediated MUC5AC expression through regulation of the ERK pathway. Exogenous NICD expression induces ERK phosphorylation by activating EGFR As blockage of Notch signaling disturbed EGF-induced ERK

phosphorylation, we asked whether activation of Notch signaling Downloaded from itself elevated ERK phosphorylation without addition of EGF. Transfection with NICD, an active form of Notch1, clearly induced ERK phosphorylation in a dose-dependent manner, whereas an empty vector had no effect (Fig. 6A). Probing of the immunoblot with anti–phospho-EGFR revealed NICD-induced EGFR phos-

phorylation in a dose-dependent manner (Fig. 6A). NICD-induced http://www.jimmunol.org/ FIGURE 2. Blockage of Notch signaling reduces MUC5AC protein ERK phosphorylation was completely inhibited by EGFR and production. A, NCI-H292 cells were treated with AG1478, U0126, and two ERK inhibitors (Fig. 6B). These data suggest that Notch signaling different GSIs, L-685,458 and DAPT, in the presence of EGF for 36 h. All can induce ERK phosphorylation via the EGFR pathway. In inhibitors were used at a concentration of 10 mM. The cells were stained contrast, GSI had no effect on the NICD-induced ERK phos- with anti-MUC5AC. These results are representative of three independent phorylation, implying that the ERK phosphorylation results from experiments. Original magnification 3200. B, MUC5AC-positive cells an effect of the exogenous NICD. To examine the combined effect 6 were enumerated and expressed as percentages. Data are shown as means of Notch and EGFR signaling pathways, NCI-H292 cells were , SEM of three independent experiments. **p 0.01 compared with transfected with NICD, cultured for 36 h, pretreated with inhib- EGF-treated cells. itors for 30 min, and treated with EGF or left untreated for 15 min. by guest on September 29, 2021 EGF treatment induced a comparable level of ERK phosphory- MUC5AC mRNA and protein (Fig. 4C,4D). These results show lation to that induced by NICD transfection (Fig. 6B, lane 6 versus that silencing of Notch1 expression leads to downregulation of lane 2). A combination of NICD expression and EGF treatment EGF-induced MUC5AC expression, supporting the involvement augmented ERK phosphorylation approximately twice as much as of Notch signaling in EGF-induced MUC5AC expression. either alone (Fig. 6B, lane 7 versus lane 2 or lane 6). Inhibitors of both EGFR and ERK completely abolished NICD-induced ERK Notch1 silencing reduces EGF-mediated ERK phosphorylation phosphorylation (Fig. 6B). Importantly, GSI inhibited ERK It has previously been shown that activation of ERK is essential for phosphorylation by only 50%, confirming that it inhibited EGF-, EGF-mediated expression of MUC5AC (11). Hence, we examined but not NICD-mediated ERK phosphorylation as GSI itself did not whether the inhibitory effect of GSI on EGF-mediated expression inhibit the de novo synthesis of the ectopically expressed NICD of MUC5AC occurred through alteration of ERK activities. As (Fig. 6B, lane 8). This result suggests that Notch signaling is expected, EGF induced ERK phosphorylation, which was reduced essentially involved in EGF-induced MUC5AC expression. When by the MEK inhibitor U0126, but unaffected by p38 inhibitor NICD-transfected cells were incubated with neutralizing Abs to SB203580 and JNK inhibitor SP600125 (Fig. 5A). GSI severely EGFR, the NICD effect was completely nullified, whereas a con- reduced ERK phosphorylation in a dose-dependent manner (Fig. trol Ab had no effect (Fig. 6C, lane 4 versus lane 3), suggesting 5B). EGF did not induce phosphorylation of p38 MAPK, and the that activation of Notch signaling led to ERK phosphorylation

FIGURE 3. GSI inhibits EGF-induced MUC5AC mRNA and protein expression in HBECs. HBECs cultured at air–liquid interface for 14 d were washed and treated with EGF (40 ng/ml) in the presence and absence of L-685,458 (10 mM) for an additional 36 h. A, Real-time PCR analysis for MUC5AC and FOXA2 mRNA. Data are shown as means 6 SEM of eight independent experiments performed in triplicate. *p , 0.05, **p , 0.01 compared with EGF- treated cells. B, Immunocytochemistry for MUC5AC protein. MUC5AC-immunopositive cells were evident in EGF-treated cells and reduced by addition of L-685,458. Original magnification 31000. 226 INVOLVEMENT OF NOTCH SIGNALING IN MUC5AC EXPRESSION

FIGURE 4. Silencing of Notch1 reduces MUC5AC expression. NCI-H292 cells were transfected with scrambled siRNA or a pool of four Notch1 siRNA Downloaded from oligonucleotides. After 12 h, untreated and EGF-treated cells were incubated for 24 h for mRNA, and/or 36 h for protein measurement. A, Notch1 siRNA reduced generation of NICD. The blot was probed with an Ab specific for NICD. GAPDH was used as the loading control. This result is representative of three independent experiments. B, RT-PCR analysis of the expression of Notch1 and HES-1 mRNA. This result is representative of four independent experiments. C, Analysis of the expression of MUC5AC mRNA by real-time PCR. Data are shown as means 6 SEM of four independent experiments performed in triplicate. **p , 0.01 compared with EGF-treated cells transfected with scrambled siRNA. D, MUC5AC-positive cells were enumerated and expressed as percentages. Data are shown as means 6 SEM of three independent experiments. **p , 0.01 compared with EGF-treated cells transfected

with scrambled siRNA. http://www.jimmunol.org/ through EGFR activation. In contrast, anti–TGF-a slightly Discussion inhibited the NICD-mediated ERK phosphorylation (Fig. 6C, lane It is well known that the EGF/EGFR axis induces MUC5AC ex- 5). The EGFR Ab overrode the ERK phosphorylation resulting pression in lung epithelial cells (8, 11, 24, 26, 43), and that the Ras/ from the additive effect of both NICD expression and EGF Raf/ERK pathway is mainly, although not solely, responsible for stimulation, although anti–TGF-a Ab had a minimal effect on the induction of MUC5AC by EGF/EGFR (11, 26). Notch sig- ERK phosphorylation (Fig. 6C, lane 9 versus lane 10). Exogenous naling has been implicated in epithelial growth and differentiation

NICD augmented MUC5AC mRNA expression in a dose- in embryonic and adult lungs (35, 44), and a wide variety of Notch by guest on September 29, 2021 dependent manner (Fig. 6D). These results suggest that activa- receptors and their related components are abundantly expressed tion of Notch signaling induces MUC5AC expression through in adult lungs (38). It has recently been shown that ectopic Notch activation of the ERK pathway without the aid of exogenous signaling in the developing mouse lung results in more mucous EGF. cells (45); however, the functions of Notch in homeostatic and

FIGURE 5. Notch silencing reduces EGF-mediated ERK phosphorylation. A, NCI-H292 cells were pre- treated with GSI (10 mM), U0126 (10 mM), SB203580 (10 mM), or SP600125 (20 mM) for 30 min and then treated with EGF for 15 min. The blot was probed with anti–phospho-ERK1/2 Ab or anti–phospho-p38 Ab, and subsequently stripped and reprobed with anti- ERK2 Ab or anti-p38. GAPDH was used as a loading control. This result is representative of two in- dependent experiments. B, NCI-H292 cells were pre- treated with different doses of GSI for 30 min and then treated with EGF for 15 min. This result is represen- tative of two independent experiments. C, NCI-H292 cells were pretreated with inhibitors for 30 min, and then treated with EGF for 24 h. MUC5AC mRNA levels were determined by real-time PCR. Data are shown as means 6 SEM of five independent experi- ments performed in triplicate. **p , 0.01 compared with treatment with EGF alone. D, NCI-H292 cells were transfected with either scrambled siRNA or Notch1 siRNA oligonucleotides. After 36 h, cells were treated with EGF for 15 min. The blot was probed with anti–phospho-ERK1/2 Ab and reprobed with anti- ERK2 Ab. The blot is a representative of three in- dependent experiments. The Journal of Immunology 227

completely blocked by treatments that specifically interfered with EGFR activity (Fig. 6). Thus, it appears that Notch signaling induces MUC5AC expression through the following mechanism: Notch signaling activates the EGFR pathway, which in turn acti- vates the ERK pathway, leading to MUC5AC expression. From the other angle, ligation of EGF to EGFR forms a feed-forward network for MUC5AC expression by activating the Notch path- way, whose signal is integrated into the EGFR–ERK pathway, leading to MUC5AC expression (Fig. 7). A few reports have previously shown the cooperative relationship between Notch signaling and EGFR/Ras–MAPK pathways, espe- cially in maintaining neoplastic phenotypes. Activated Notch4- mediated oncogenic transformation requires ERK activation (46). In lung cancer cells, expression of Notch3 is positively correlated with EGFR expression, and dominant-active Notch3 activates ERK phosphorylation, whereas dominant-negative Notch3 inhibits ERK phosphorylation. Furthermore, Notch3 siRNA or GSI inhibits ERK phosphorylation, and the effect of Notch signaling is impeded

by both ERK and EGFR inhibitors (47–49). In addition, Notch1 Downloaded from upregulates EGFR expression through p53 (50). These results suggest that Notch signaling stimulates ERK activation at least in part through a mechanism involving the synthesis of EGFR. Our FIGURE 6. ERK phosphorylation by NICD overexpression is mediated results showed that exogenous NICD expression elicited increases through activation of EGFR. A, NCI-H292 cells were transfected with in both EGFR and ERK phosphorylations (Fig. 6A). The ERK

different doses of NICD. To eliminate artifacts resulting from transfection phosphorylation was completely inhibited by blockage of EGFR http://www.jimmunol.org/ with varying amounts of NICD expression vector, the total amount of activity, but only very weakly inhibited by blockage of synthesis of transfected DNA was equalized by supplementing with a control vector, its ligand, TGF-a (Fig. 6C), providing evidence that activation of pcDNA3.1. After 36 h, cells were lysed for Western blot analysis. The Notch signaling directly modulates EGFR activity rather than duplicated blots were probed with anti–phospho-ERK1/2 Ab and anti– synthesis of EGFR ligand. In addition, the fact that NICD, an in- phospho-EGFR and then reprobed with anti-ERK2 Ab and anti-EGFR, respectively. Exogenous NICD expression and transfection efficiency were tracellular domain of Notch, is able to induce EGFR phosphoryla- monitored by probing with anti-Flag Ab. The result shown is a represen- tion suggests that EGFR activation is unlikely to occur through tative of three independent experiments. B and C, NCI-H292 cells were a direct molecular contact between Notch1 and EGFR on the cell transfected with NICD (1 mg). After 36 h, cells were pretreated with GSI surface, but rather might be resulted from a NICD-downstream ef- (10 mM), AG1478 (10 mM), U0126 (10 mM), anti-EGFR (2 mg/ml), or fect(s). It has previously been suggested that both ligand-dependent by guest on September 29, 2021 anti–TGF-a (4 mg/ml) for 30 min and then treated with or without EGF for and ligand-independent EGFR activation pathways lead to mucin 15 min. Phospho-ERK1/2 level was normalized to total ERK level, and the synthesis (21). In ligand-dependent EGFR activation, a variety of relative intensity was analyzed by using densitometry. The result shown is stimuli of MUC5AC induces secretion of EGFR ligands, including representative of three independent experiments. D, NCI-H292 cells were cleavage of membrane-bound EGFR proligands, which in turn transfected with different doses of NICD for 36 h. The expression of activates EGFR. In contrast, ligand-independent EGFR activation MUC5AC mRNA was analyzed by real-time PCR. Data are shown as means 6 SEM of four independent experiments performed in triplicate. may occur in the absence of ligand binding by directly phosphor- *p , 0.05, **p , 0.01 compared with EGF-treated cells transfected with ylating tyrosine residues in the intracellular domain in response to scrambled siRNA. stimuli such as cigarette smoke and oxidative stress. Our data sup- port ligand-independent EGFR activation, but how Notch signaling modulates EGFR activity in a more direct manner remains to be diseased conditions are largely unknown. In the current study, we established. investigate a possible involvement of Notch signaling in the ex- There are a few reports that the EGF/Ras–MAPK pathway can pression of MUC5AC, a defining characteristic of the goblet cell activate Notch signaling. Oncogenic Ras increases levels and phenotype manifested upon insults to airway epithelial cells. Our results show that MUC5AC expression induced by EGFR acti- vation was inhibited by GSI in primary HBECs as well as lung carcinoma cells (Figs. 1–3), and that more specific inhibition of Notch signaling with siRNA led to equivalent inhibition of the EGF-mediated MUC5AC expression (Fig. 4). Conversely, when NICD was exogenously expressed in the epithelial cells, MUC5AC expression was induced even in the absence of exog- enously added EGFR ligand (Fig. 6). These findings suggest that Notch signaling is not only involved in EGF-induced MUC5AC expression, but is also itself able to induce MUC5AC expression. FIGURE 7. Proposed mechanism by which Notch signaling mediates A common key player in the activation of the Notch and EGFR MUC5AC expression. Notch signaling activates the EGFR pathway, which signaling pathways appears to be ERK: EGF-induced ERK in turn activates the ERK pathway, leading to MUC5AC expression. phosphorylation was blunted by GSI or Notch1 siRNA treatment Conversely, ligation of EGF to EGFR forms a feed-forward network for (Fig. 5), whereas exogenous NICD expression induced ERK MUC5AC expression by activating both the ERK pathway and Notch phosphorylation without the aid of EGF stimulation (Fig. 6). pathway. The latter signal is ultimately integrated into the EGFR–ERK Furthermore, NICD-induced ERK phosphorylation was nearly pathway, leading to MUC5AC expression. 228 INVOLVEMENT OF NOTCH SIGNALING IN MUC5AC EXPRESSION activity of NICD derived from Notch1, and also upregulates Notch became MUC5AC positive in both this study and others (11, 59, ligand Delta1 and presenilin1 through a p38-mediated pathway in 60). This might reflect heterogeneity in EGF responsiveness and human fibroblast and epithelial cells (51). Proinflammatory cyto- mitotic state of the cells, enabling only a fraction of the cells to kines, including TNF, IL-1b, and IFN-g, stimulate g-secretase respond to Notch signaling, leading to MUC5AC expression. activity-dependent cleavage through a JNK-dependent pathway in Novel therapies directed against a variety of targets to blunt human lung epithelial cells (52). In , the EGFR path- excess mucus production are in development (61). The EGFR– way promotes the expression of a Notch ligand, which acts as an ERK pathway plays critical roles in growth, differentiation, and inducer for cone cells (31). Thus, EGFR/Ras–MAPK pathways repair of airway epithelial cells. It is identified as a primary route can activate Notch signaling through the synthesis and elevated leading to mucin synthesis and/or goblet cell metaplasia in re- activity of molecules involved in Notch processing. Our results are sponse to a wide variety of stimuli, most of which culminate in consistent with these observations; in our study, EGF caused in- upregulation of EGFR activity (21). Our results provide evidence creased synthesis of Notch1 and the generation of NICD, and both that Notch signaling also converges into the EGFR–ERK pathway GSI and Notch1 siRNA inhibited EGF-induced ERK phosphory- to induce MUC5AC expression. Therefore, Notch signaling itself lation. By the same token, however, an important question is may be a therapeutic focus, but, more importantly, the interplay raised: how might this relate to ERK activation through the between EGFR and Notch signaling for the expression of classical EGFR–Ras pathway? The fact that GSI or Notch siRNA MUC5AC reiterates the importance of the EGFR–ERK pathway almost completely inhibited EGF-induced ERK phosphorylation as a major therapeutic target for many chronic inflammatory implies that EGF-induced ERK phosphorylation may occur pre- diseases associated with excess mucus production. In addition, dominantly through activation of Notch signaling. Thus, it is this study provides a cellular and biochemical basis for the in vivo Downloaded from puzzling that binding of EGF to EGFR causes ERK phosphory- observation that pharmacological blockage of Notch signaling lation through the somewhat indirect means of activation of reduces goblet cell production in an animal model of asthma (39). Notch signaling, despite having a more straightforward pathway in EGFR-Ras-ERK. The reason for this is not clear, although Disclosures this strategy might lead to enforcement of ERK activity through The authors have no financial conflicts of interest. a feed-forward loop with sustained influence on downstream http://www.jimmunol.org/ effectors. In this context, it is noteworthy that a recent report proposes a model in which TGF-a–induced EGFR activation References occurs in two phases (53). TGF-a binds EGFR directly, inducing 1. Lai, H. Y., and D. F. Rogers. 2010. Mucus hypersecretion in asthma: intracellular a first phase of EGFR phosphorylation that results in the activation signalling pathways as targets for pharmacotherapy. Curr. Opin. Allergy Clin. of ERK and in production of MUC5AC and CCL20. CCL20 then Immunol. 10: 67–76. 2. Hogg, J. C., F. Chu, S. Utokaparch, R. Woods, W. M. Elliott, L. Buzatu, binds to its receptor CCR6, which induces a second phase of R. M. Cherniack, R. M. 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