EGF Shifts Human Airway Basal Cell Fate Toward a Smoking-Associated Airway Epithelial Phenotype

EGF shifts human airway basal cell fate toward a smoking-associated airway epithelial phenotype

Renat Shaykhiev1, Wu-Lin Zuo1, IonWa Chao, Tomoya Fukui, Bradley Witover, Angelika Brekman, and Ronald G. Crystal2

Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065

Edited* by Michael J. Welsh, Howard Hughes Medical Institute, Iowa City, IA, and approved May 29, 2013 (received for review February 19, 2013)

The airway epithelium of smokers acquires pathological phenotypes, phosphorylation, indicative of EGFR receptor activation, has including basal cell (BC) and/or goblet cell hyperplasia, squamous been observed in airway epithelial cells exposed to cigarette metaplasia, structural and functional abnormalities of ciliated cells, smoke in vitro (26, 27). decreased number of secretoglobin (SCGB1A1)-expressing secretory Based on this knowledge, we hypothesized that smoking- cells, and a disordered junctional barrier. In this study, we hypoth- induced changes in the EGFR pathway are relevant to the EGFR- ﬁ esized that smoking alters airway epithelial structure through dependent modi cation of BCs toward the abnormal differentia- modiﬁcation of BC function via an EGF receptor (EGFR)-mediated tion phenotypes present in the airway epithelium of smokers. mechanism. Analysis of the airway epithelium revealed that EGFR is In this study, we provide evidence that although EGFR is expressed predominantly in BCs, smoking induces expression of enriched in airway BCs, whereas its ligand EGF is induced by smoking EGF in ciliated cells of the airway epithelium, and activation of in ciliated cells. Exposure of BCs to EGF shifted the BC differentiation – airway BCs with EGF skews their differentiation from the nor- program toward the squamous and epithelial mesenchymal transi- mal mucociliary pathway toward the squamous and epithelial– tion-like phenotypes with down-regulation of genes related to cilio- mesenchymal transition (EMT)-like phenotypes with decreased genesis, secretory differentiation, and markedly reduced junctional epithelial junctional barrier integrity. barrier integrity, mimicking the abnormalities present in the airways of smokers in vivo. These data suggest that activation of EGFR in Results

airway BCs by smoking-induced EGF represents a unique mechanism Enrichment of EGFR Gene Expression in Airway Basal Cells. Signaling PHYSIOLOGY whereby smoking can alter airway epithelial differentiation and through various ErbB family receptors in polarized epithelia, barrier function. including that in the airways, is determined, at least in part, by segregated distribution of the receptors and their ligands in ei- airway epithelial barrier | progenitor cell | cigarette smoking ther apical or basolateral membrane domains (28, 29). We hypothesized that an additional compartmentalization could be he normally differentiated human airway epithelium is a provided by the heterogeneous expression of the ErbB family Tpseudostratified layer composed of apically positioned dif- receptors and their ligands between the BC and BC-derived ferentiated ciliated and secretory cells, intermediate columnar differentiated cell populations, such as ciliated and secretory cells, and basal cells (BCs) located just above the basement cells, contributing to the luminal compartment. Microarray-based membrane (1, 2). BCs are the stem/progenitor cells for the air- gene expression analysis of the complete large airway epithelium way epithelium. Normal BC function includes generation of air- (LAE) compared with the LAE-derived BCs from healthy non- way basal, secretory, and ciliated cells (3–5). Cigarette smoking is smokers revealed compartmentalized expression of the EGFR associated with dramatic changes in the airway epithelial archi- family receptors in the normal human airway epithelium in vivo A tecture, inducing BC hyperplasia, mucus overproduction, and (Fig. 1 ). fi squamous metaplasia (6, 7). Squamous metaplasia represents Consistent with previous immunohistochemistry ndings (20, a histological lesion characterized by conversion to a stratified 22, 29), EGFR gene expression was much greater compared with A epithelium comprised of flattened squamous cells replacing the other ErbB receptors and was enriched in BCs (Fig. 1 ). In differentiated cell populations (6, 7). Other smoking-induced contrast, expression of ERBB4 was barely detectable in BCs, but fi changes include structural and functional abnormalities of ciliated was signi cantly greater in differentiated airway epithelium (Fig. A cells (8, 9), disorganization of cell–cell junctions leading to in- 1 ). These differences were related to cell differentiation status creased permeability of the airway epithelial barrier (10–12), and and not to the culture itself, given that expression of neither fi decreased numbers of secretoglobin (SCGB) 1A1-expressing se- EGFR nor ERBB4 varied signi cantly during the expansion A cretory cells (13). Persistence of these changes in the context of phase, when BCs were grown as submerged cultures (Fig. S1 B chronic cigarette smoking is associated with decreased mucociliary and ). However, EGFR gene expression was dramatically decreased and ERBB4 expression was markedly increased during clearance, pathogen colonization, and development of smoking- – associated lung disorders, chronic obstructive pulmonary disease, the BC differentiation phase in the air liquid interface (ALI) – model, recapitulating the expression pattern observed in the and lung cancer (6, 7, 14 16). A–C Altered BC function might be one possible mechanism of airway epithelium in vivo (Fig. S1 ). Enrichment of EGFR expression in the BC population was smoking-induced dysregulation of airway epithelial differentia- fi tion. Consistent with this concept are the observations of in- further con rmed through immunohistochemistry analysis of creased numbers of BCs in the airways of smokers (6, 7), overexpression of BC markers keratin (KRT) 5, KRT14, and tumor protein (TP) 63 in squamous lesions (6, 17), and the Author contributions: R.S., W.-L.Z., and R.G.C. designed research; R.S., W.-L.Z., I.C., T.F., ability of BCs to form squamous epithelium in vitro (18). A B.W., and A.B. performed research; R.G.C. contributed new reagents/analytic tools; R.S., molecular mechanism contributing to smoking-induced changes W.-L.Z., and R.G.C. analyzed data; and R.S. and R.G.C. wrote the paper. in BC function could be activation of EGF receptor (EGFR), The authors declare no conflict of interest. a member of the ErbB (v-erb-b oncogene homolog) family of *This Direct Submission article had a prearranged editor. tyrosine kinase receptors (19). EGFR is enriched in cells of the 1R.S. and W.-L.Z. contributed equally to this work. basal layer of various epithelial tissues, including the airways 2To whom correspondence should be addressed. E-mail: [email protected]. (20–22), and has been implicated in regulation of epithelial ho- edu. meostasis and tissue repair, production of host defense media- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tors, and production of mucin (23–25). Increased EGFR tyrosine 1073/pnas.1303058110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1303058110 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Basal cells A 150 * Basal cells C

Complete airway KRT5 epithelium EGFR 100

N.S. N.S. 50 *

Normalized expression 0 D Basal ALI (days) EGFR ERBB2 ERBB3 ERBB4 cells 8 28 28 Fig. 1. Enrichment of EGFR in airway BCs. (A) Nor- B malized expression of the ErbB family receptors in the Airway epithelium in vivo Airway brushes EGFR complete differentiated LAE of healthy nonsmokers = GAPDH (n 21) and LAE-derived BCs of healthy nonsmokers (n = 4) based on the microarray analysis. N.S., not significant. *P < 0.05. (B) Localization of EGFR in the human airway epithelium. (Left) EGFR immunohisto- E chemistry of the LAE biopsy samples. (Right, Top)EGFR ALI d28 immunocytochemistry of an airway epithelial brushing KRT5 KRT5 sample. (Right, Middle and Bottom) Immunofluores- EGFR EGFR cence colocalization of EGFR and KRT5 in the airway epithelial brushings. (C) Representative immunofluorescence image of BCs cultured from the LAE stained for EGFR and KRT5. (D) Western blot analysis of EGFR protein expression in airway BCs at baseline and in BC- Apical side KRT5 KRT5 derived airway epithelium generated from BCs after EGFR EGFR 8 d and 28 d of culture in ALI. GAPDH expression is shown as a loading control. (E) Immunofluorescence analysis of cytopreparations of airway epithelial cells generated from BCs after 28 d of culture in ALI double-stained for EGFR and KRT5; two representative images are shown. (Scale bars: 20 μm.)

LAE biopsy specimens and freshly isolated LAE brushed cells To determine whether cigarette smoke can directly induce (Fig. 1B). Immunofluorescence analysis of the airway epithelial EGF in the differentiated airway epithelium, we applied ciga- brushings obtained from different donors (Fig. 1B and Fig. S2A) rette smoke extract (CSE) to the differentiated airway epithe- revealed that >80% of EGFR-expressing cells were positive for lium generated from BCs in ALI culture. For modeling of BC marker keratin KRT5, and that >70% of BCs expressed repeated prolonged smoking exposure, CSE was added from the EGFR (Fig. S2B). The latter finding points toward heterogeneity apical side for 24 h every other day over a 2-wk period. Signifi- of BCs in terms of EGFR protein expression. This heteroge- cant up-regulation of EGF gene expression was observed in neous pattern of EGFR expression was preserved in BCs cul- differentiated airway epithelial cells stimulated with CSE (Fig. tured from freshly isolated airway epithelium (Fig. 1C and Fig. 2E). The levels of EGF protein released from the apical surface S2C). EGFR protein level decreased progressively when BCs were significantly higher than those detected in the basolateral generated differentiated airway epithelium in the ALI culture compartment of the ALI culture (Fig. 2F). Given that ciliated (Fig. 1D), where EGFR expression was restricted to a subset of cells are the predominant cell population contributing to the KRT5-expressing cells, that is, BCs (Fig. 1E). apical compartment of the ALI-differentiated airway epithelium (4, 30), this observation is in agreement with the in vivo data Smoking-Induced EGF Expression in Ciliated Cells of the Airway showing that ciliated cells are the major source of EGF in the Epithelium. To assess whether cigarette smoking is associated airway epithelium of smokers. with abnormal expression of the EGFR family receptors and ligands in the airway epithelium, we compared expression of EGF Alters Basal Cell Phenotype and Differentiation. To evaluate these genes in the LAE of healthy smokers and healthy non- whether EGF is capable of activating EGFR in airway BCs, we smokers. The data demonstrate that in the LAE of smokers, performed Western blot analysis and receptor tyrosine kinase expression of genes encoding EGFR, ErbB2, and TGF-α (an (RTK) protein array analysis, which confirmed that EGF induces EGFR ligand) was down-regulated (Fig. S3A). In contrast, ex- phosphorylation of EGFR (Fig. 3A), but not of other receptor pression of EGF, a major EGFR ligand usually expressed in the tyrosine kinases (Fig. S4), in BCs. EGF altered the appearance surface airway epithelium at relatively low levels (20, 29), was of airway BCs from their normal cuboidal shape toward an significantly up-regulated in the complete LAE of smokers, but elongated morphology with apparent loss of intercellular con- not in the BCs (Fig. 2A). Genome-wide correlation analysis re- tacts (Fig. 3B and Fig. S5A). Such morphology, typical of motile vealed strong coexpression of EGF with the genes related to the epithelial cells undergoing EMT (31), was observed in >30% of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent oxi- EGF-treated BCs, compared with <1% in the control group dative stress response (Fig. S3B). EGF up-regulation in the airway (Fig. S5B). epithelium of smokers was further confirmed by RNA-sequencing To study the effect of EGF on the airway BC differentiation, (RNA-Seq) analysis (Fig. 2B). Consistent with this observation, we took advantage of the ALI model, in which BCs generate immunohistochemistry analysis revealed increased EGF protein a fully differentiated polarized airway epithelium (4, 21, 32). Our expression in the airway epithelium of smokers (Fig. 2C and Fig. analysis, performed in the middle (day 14) of the differentiation S3C) with selective enrichment in ciliated cells, the major differ- process, revealed that EGF broadly modifies the BC differenti- entiated cell population of the airway epithelium (1), which ation program by inducing expression of KRT6, KRT14, invo- composed the majority (84.5 ± 15.6%) of EGF-expressing cells in lucrin (IVL), and stratifin (SFN), all of which are related to the airway epithelium of smokers (Fig. 2D). various aspects of epithelial injury and squamous differentiation

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1303058110 Shaykhiev et al. Downloaded by guest on October 2, 2021 A C D EGF mRNA expression Nonsmokers Smokers EGF+ cells (microarray) (% total EGF+ cells) 6 p<0.03 020406080100 Fig. 2. EGF expression in differentiated airway ep- 5 Ciliated ithelial cells. (A) Normalized EGF gene expression in Secretory p<0.005 4 the indicated groups based on microarray analysis. Basal 3 N.S. Intermediate (B) EGF expression levels, in reads per kilobase per 2 million (RPKM) mapped reads, in the complete air- 1 p<0.04 =

) way epithelium of healthy nonsmokers (n 4) and

E -3 0.14 p<0.03 Normalized expression 0 healthy smokers (n = 6), based on RNA-Seq analysis. Non- Smokers Non- Smokers 0.10 smokers smokers (C) Immunohistochemistry analysis of EGF protein Basal cells Airway 0.06 expression in LAE biopsy samples from healthy epithelium Normalized nonsmokers (Left) and smokers (Right). (Scale bars: B 0.02 EGF mRNA expression (x10 expression 0 20 μm.) (D) Contribution (%) of different cell pop- Control 1% 2% (RNA-Seq) CSE ulations to EGF-expressing cells detected in the air- p<0.01 way epithelium by immunohistochemistry as shown 1 Apical side 0.8 F 25 p<0.02 in C and in Fig. S3C.(E) Normalized expression of the p<0.001 0.6 20 EGF gene after stimulation of the ALI-differentiated ) 2 RPKM airway epithelium with CSE (1% and 2%) from the 0.4 15 Basal side 0.2 apical side every other day for 14 d compared with 10 N.S. 0 N.S. unstimulated cells. (F) ELISA-based comparison of Non- Smokers 5

EGF (pg/cm EGF levels in the apical and basolateral supernatants smokers 0 Airway epithelium 012 012 of ALI-differentiated airway epithelial cells after CSE(%) stimulation with CSE as described in E.

(16, 17), as well as CD44, a gene often associated with epithelial a remarkable increase in the number of cells expressing squa- stem cell phenotype (33) (Fig. 3C). This was accompanied by up- mous cell markers KRT14 and IVL and the EMT marker VIM regulation of genes indicative of EMT, including the epithelial (Fig. 3 D and E and Figs. S8 and S9 A and B). In the airway >

transcriptional repressor snail homolog 2 (SNAI2, also known as epithelium derived from EGF-treated BCs, 20% of cells PHYSIOLOGY SLUG), and the mesenchymal marker vimentin (VIM) (Fig. 3C). expressed IVL or VIM, compared with <5% in the control group In contrast, EGF down-regulated the physiological BC marker (Fig. 3F). The vast majority of cells expressing IVL or VIM in the + KRT5 and genes relevant to specialized differentiated airway EGF-treated group (>95% and >90%, respectively) were KRT5 epithelial cell populations, such as the ciliated cell-related genes (Fig. S10), suggesting that BCs undergo differentiation toward forkhead box J1 (FOXJ1) and dynein, axonemal, intermediate the squamous and EMT-like phenotypes in response to EGF. − chain 1 (DNAI1) and the secretory cell-related markers mucin This was accompanied by a decreased proportion of KRT5 (i.e., (MUC) 5B and SCGB1A1 (Fig. 3C). The effect of EGF on the nonbasal) cells in the EGF-treated group (Fig. 3F), pointing to- expression of many of these genes was detectable starting during ward a reduction in the differentiated cell frequency. Although the first week of ALI and remained significant throughout the the overall absolute numbers of ciliated and secretory cells were differentiation process (Fig. S6). In contrast to basolateral not statistically different between the groups (data not shown), stimulation, which allows activation of EGFR expressed on BCs, the airway epithelium generated from the EGF-treated BCs apical application of EGF did not induce similar gene expression contained multiple areas characterized by loss of ciliated and changes in differentiation-related pathways (Fig. S7A). SCGB1A1-secreting cells (Fig. S9 C and D). Consistent with the gene expression data, immunofluorescence analysis and morphological evaluation of cytopreparations EGF-Treated BCs Generate a More Permeable Airway Epithelium. The and sections of the airway epithelium generated over 28 d in ALI apical junctional barrier, composed of tight and adherens junctions, culture demonstrated that EGF treatment of BCs resulted in is dysregulated by cigarette smoking (10–12). Gene expression

Fig. 3. Modulation of the BC phenotype and airway A B Control EGF C *p<0.05 epithelial differentiation by EGF. (A) Western blot EGF (min) **p<0.01

5 15 30 Control analysis showing expression of phosphorylated EGFR ***p<0.001 p-EGFR (p-EGFR) (Upper) and total EGFR (Lower) in airway

BCs stimulated with EGF for 5, 15, or 30 min vs. EGFR unstimulated (control) BCs. (B) Representative mi- crographs of airway BCs after 48 h of stimulation with 10 ng/mL EGF (Right) vs. unstimulated control D Control EGF BCs (Left). (Scale bars: 20 μm.) (C) EGF modulation of IVL IVL the normalized expression of genes related to various KRT5 KRT5 Control EGF aspects of airway epithelial differentiation: CD44, KRT5, KRT6A, KRT6B, KRT14, IVL, SFN, SNAI2, VIM, FOXJ1, DNAI1, MUC5B, and SCGB1A1. The data were gen- KRT14 KRT14 Fold-change erated during 14 d of ALI culture stimulated with KRT5 KRT5 E 10 ng/mL EGF (n = 4) from the basolateral side vs. Control EGF F100 Control = IVL IVL unstimulated controls (n 4). (D and E) Immuno- EGF p<0.05 KRT5 KRT5 80 ﬂuorescence analysis of cytopreparations (D) and sections (E) of the airway epithelium samples dif- VIM VIM 60 p<0.01 p<0.05 KRT5 KRT5 ferentiated during 28 d of ALI culture of BCs stimu- 40 VIM VIM % cells lated with 10 ng/mL EGF (Right) vs. unstimulated KRT5 KRT5 20 controls (Left) for the expression of squamous cell 0 markers IVL or KRT14 and mesenchymal/EMT marker IVL+ VIM+ KRT5- + + − VIM. Each sample was costained for the BC marker KRT5. (Scale bars: 20 μm.) (F) Frequency of IVL ,VIM , and KRT5 cells in the airway epithelium samples differentiated during 28 d of ALI from EGF-treated vs. untreated control BCs, as shown in D and E and in Figs. S8 and S9.

Shaykhiev et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 analysis of EGF-treated BCs during their differentiation in ALI distinct membrane-associated signaling domains along the apical revealed significant down-regulation of genes relevant to the and basolateral surfaces separated by tight and adherens junc- establishment of epithelial polarity and apical junction forma- tions between adjacent epithelial cells (34). This mechanism tion, including cadherin 1 (or E-cadherin) (CDH1), tight junc- provides separation of receptors from their ligands usually po- tion protein (TJP) 1 and 3, claudin (CLDN) 3 and 8, occludin sitioned at the opposite membrane poles, preventing aberrant (OCLN), epithelial polarity complexes par-3 partitioning de- receptor activation under steady-state conditions but allowing fective homolog (PARD3) and par-6 partitioning defective ho- receptor activation in response to injury when the junctional molog (PARD6B), and tumor suppressor phosphatase and barrier is disrupted (29), as occurs in the airway epithelium of tensin homolog (PTEN) (Fig. 4A). Of note, all of these genes cigarette smokers (10–12). Originally observed in Madin-Darby were found to be down-regulated in our previous study of gene canine kidney (MDCK) cells (28), polarized distribution of the expression in the airway epithelium of smokers in vivo (12). EGFR and its ligands along the basal-apical axis of the epithelial Consistent with the gene expression data, EGF-treated BCs cell membrane has been reported in airway epithelial cells as well generated airway epithelia with markedly reduced transepithelial (20, 29, 35). electrical resistance (Rt), a measure of epithelial junctional bar- Our findings in the present study extend this concept by rier integrity (34), compared with the control group (Fig. 4B). demonstrating an additional level of EGF-EGFR compartmen- The addition of EGF to differentiated cells (i.e., from the apical talization in the human airway epithelium that is sensitive to the side) did not result in either suppression of the epithelial junction/ smoking-induced injury. Our analysis revealed that EGFR is polarity genes or reduced Rt compared with unstimulated control normally enriched in BCs, a population of cells located imme- cells (Fig. S7B). The negative effect of EGF application to BCs diately above the basement membrane and containing stem/ on Rt during airway epithelial differentiation was prevented when progenitor cells of the airway epithelium (3–5), whereas its li- the EGFR-selective tyrosine kinase inhibitor AG1478 was added gand EGF is expressed in the surface airway epithelium at barely to the basolateral compartment before the EGF treatment (Fig. detectable levels but is significantly induced by smoking in cili- 4C). Finally, significantly increased paracellular flux of the api- ated cells, a cell population contributing to the differentiated cally added dextran to the basolateral media was observed in the luminal compartment of the airway epithelium (1). airway epithelium derived in ALI from the EGF-treated BCs, Cigarette smoking-induced oxidative stress has recently been indicative of decreased barrier function (Fig. 4D). linked to EGFR activation in human lung epithelial cells in vitro (36–38). Consistent with this concept, the present study revealed Discussion a strong correlation between EGF expression and the genes re- The airway epithelium of smokers is disordered with BC hy- lated to the Nrf2-dependent oxidative stress response in the perplasia, squamous metaplasia, goblet cell hyperplasia/meta- airway epithelium of smokers previously reported by our group plasia and mucus overproduction, altered structure and function (39). Thus, in conjunction with previous observations of com- of ciliated cells, decreased numbers of SCGB1A1-expressing promised junctional barrier integrity in the airway epithelium of secretory cells, and dysregulation of the apical junctional com- smokers (10–12), our present data suggest that smoking can plex (AJC) controlling epithelial permeability (6–15). Our data activate physiologically silenced EGF-EGFR signaling in the demonstrate that EGF is produced by ciliated cells in response airway epithelium by inducing EGF production by ciliated cells to smoking and modifies the differentiation capacity of EGFR- as part of the oxidative stress response and potentially allowing expressing airway BCs, leading to the development of altered access of EGF to the EGFR-enriched BC compartment con- differentiation phenotypes similar to those present in the airway taining stem/progenitor cells of the airway epithelium. Our epithelium of smokers (Fig. S11). analysis revealed that not all BCs express EGFR to the same degree, suggesting that distinct subpopulations of airway BCs EGF-EGFR Segregation in the Airway Epithelium and Smoking. A may exist, each with a unique sensitivity to smoking-associated major feature of the differentiated airway epithelium is its po- tissue damage and a unique contribution to smoking-induced larized organization, that is, compartmentalized distribution of disordering of the airway epithelial phenotype.

A CDH1 B 4000 TJP1 3500 Control TJP3 3000 ) CLDN3 2 2500 CLDN8 2000 Fig. 4. EGF-treated BCs generate a more permeable airway OCLN 1500 epithelial barrier. (A) EGF modulation of the expression of PARD3 (Ohm x cm EGF all p<0.05 1000 * genes related to the AJC and epithelial polarity: CDH1, TJP1, vs control TJP3, CLDN3, CLDN8, OCLN, PARD3, PARD6B, and PTEN. The PARD6B 500 Transepithelial resistance Transepithelial ** PTEN data were generated during 14 d of ALI culture of BCs stimu- 0 *** -3 -2 -1 0 2 4 6 8 10121416182022242628 lated with 10 ng/mL EGF (n = 4) vs. unstimulated controls = Fold-change EGF vs control ALI days (n 4). (B) Rt of the airway epithelium generated from BCs stimulated with EGF (10 ng/mL) vs. unstimulated control cells at C D 40 different time points of the ALI culture. (C) Rt of the airway *** Control *** 35 epithelium generated at day 14 of ALI culture from BCs treated EGF * ** 30 with media only (control), DMSO (vehicle for AG1478), EGFR

) ** μ 2 25 inhibitor AG1478 (10 m), and EGF (10 ng/mL) with or without μ 20 a 1-h pretreatment with AG1478 (10 m) every second day ﬂ 15 starting on day 0 of ALI. (D) Mean uorescence intensity 10 * detected in the basolateral chamber of ALI at different time

(Ohm x cm (Ohm x 5 points after apical application of FITC-dextran to the airway epithelium samples differentiated in ALI from EGF-treated or

(Ex 485nm, Em 485nm, (Ex 538nm) 0 Fluorescence intensity d0 d1 d2 d3 control BCs. In B–D, asterisks indicate statistically signiﬁcant Transepithelial resistance resistance Transepithelial Control DMSO AG- EGF EGF+ Incubation time differences at given time points between EGF-stimulated and 1478 AG1478 control groups: *P < 0.05; **P < 0.01; ***P < 0.005.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1303058110 Shaykhiev et al. Downloaded by guest on October 2, 2021 Similar to other smoking-associated molecular changes in the of the EMT were induced by EGF in airway BCs, including ac- airway epithelium (12, 39), EGF expression varied considerably, quisition of the elongated shape, up-regulation of the key EMT- demonstrating marked up-regulation in a subset of smokers. inducing transcription factor SNAI2/SLUG, and induction of Given that smoking-induced pathological changes in the airway VIM, an intermediate filament characteristic of mesenchymal cells epithelium, including squamous metaplasia (7), and smoking- and migrating epithelial cells (31, 47). Up-regulation of VIM was associated diseases, such as chronic obstructive pulmonary dis- observed after basolateral stimulation of BCs with EGF as early as ease and lung cancer, develop in <25% of smokers (40, 41), and within 3 d of BC differentiation in ALI, long before the appear- considering the capability of EGF to alter airway epithelial ance of differentiated cells, and was not induced in differentiated phenotype demonstrated in the present study, it is possible that airway epithelium after apical application of EGF, suggesting that increased expression of EGF in the airway epithelium defines BCs undergo EGF-mediated EMT-like changes. a distinct subset of smokers susceptible to the development of In further support of the BC origin of EGF-induced EMT-like smoking-related airway pathology. phenotype, VIM-expressing cells, which appeared in the airway epithelium derived from EGF-treated BCs, were found to EGF-Modified BCs as a Cellular Origin of Squamous Metaplasia. Air- coexpress BC marker KRT5. This finding argues against transi- way BCs are stem/progenitor cells that normally can differentiate tion toward a mesenchymal cell type and supports the concept into ciliated and secretory cells (3–5). Using an ALI model of BC that EGF shifts BCs toward an EMT-like, rather than a classical differentiation (4, 21, 30, 32), we found that exposure of BCs to EMT, phenotype. Parallel acquisition of squamous and EMT- EGF during their differentiation in this culture system signifi- like phenotypes by EGF-treated BCs points toward the common cantly decreases the ability of BCs to generate normal mucociliary BC origin, coordinated EGFR-mediated mechanism, and po- airway epithelium and skews differentiation toward the squamous tentially integrated roles of these two processes in the suppres- phenotype, characterized by up-regulation of various genes as- sion of normal airway epithelial differentiation. sociated with squamous differentiation and increased numbers Acquisition of the EMT-like phenotype was accompanied by of cells expressing squamous cell markers. Among these genes are a broad down-regulation of the genes encoding various AJC IVL and KRT14, which have low expression in the normal sur- components, including epithelial-specific CDH1, tight junction face airway epithelium but are markedly up-regulated in BCs genes, PARD-family polarity complexes, and tumor suppressor undergoing squamous differentiation (16, 17). Of note, >95% of PTEN, critical for epithelial morphogenesis (48), recently shown squamous cells in the airway epithelium generated from the to be a part of the smoking-sensitive AJC transcriptional network EGF-treated BCs coexpressed BC marker KRT5, suggesting in the airway epithelium (12). Consistent with this, airway epi- that these cells are the result of directly EGF-mediated BC thelium generated from the EGF-treated BCs exhibited decreased PHYSIOLOGY differentiation toward the squamous phenotype. In further sup- barrier integrity, manifested by significantly reduced Rt and in- port of the BC origin of EGF-induced squamous metaplasia, up- creased paracellular permeability. A similar pattern of AJC-re- regulation of the squamous differentiation program in response lated features is down-regulated in the airway epithelium of to basolateral EGF stimulation was detected at day 14 of ALI, smokers in vivo (12), suggesting that smoking-associated alteration when few differentiated cells are normally present (21, 30). of the airway epithelial barrier integrity may be related, at least in Consistent with this idea are the findings that squamous meta- part, to the EMT-like phenotype induced by EGF in airway BCs. plasia commonly develops as a consequence of smoking-induced EGF-induced impairment of the airway epithelial junctional BC hyperplasia (6, 7), airway BCs can give rise to squamous barrier provides a mechanism whereby ciliated cell-derived EGF airway epithelium in vitro (18), and expression of EGF and the may access EGFR-expressing BCs and sustain EGFR-mediated number of EGFR-expressing cells are increased within squamous modification of airway epithelial differentiation. This persistent lesions in an in vivo model of airway injury (42). Given that BC activation via the EGF-EGFR pathway may be responsible squamous metaplasia is one of the early major smoking-induced for the progressive alteration of airway architecture and sub- lesions in the airway epithelium linked to smoking-induced car- sequent development of preneoplastic lesions. Consistent with cinogenesis (6, 7), and that EGFR is commonly overexpressed this concept, EMT-like changes have been linked to acquisition and activated in squamous cell lung carcinomas, a subtype of non- of the stem cell/tumor-initiating phenotype (33), and EGF has small cell lung carcinoma (NSCLC) associated with smoking (43), been shown to induce EMT in carcinoma cells (49, 50). In ad- it is possible that EGF-activated BCs contribute to the devel- dition, persistent EGFR activation and acquisition of the EMT opment of the early premalignant squamous lesions in the airway phenotype may eventually lead to the development of resistance epithelium of smokers. Consistent with this concept, cells ex- to EGFR tyrosine kinase inhibitors (26, 51). pressing KRT14, a squamous-associated gene induced by EGF in In summary, our findings demonstrate that EGFR expression airway BCs in our study, are enriched in the non-small cell lung is enriched in airway BCs, whereas its ligand EGF is induced by carcinoma tissue in association with smoking status (17). smoking in ciliated cells of the airway epithelium. EGF skews the In the present study, similar to squamous metaplasia in the airway BC fate toward abnormal, squamous and EMT-like airways of smokers, characterized by the progressive disappear- phenotypes with marked suppression of the normal differentia- ance of normal differentiated cells contributing to the luminal tion features, similar to the morphological changes seen in the epithelial compartment (7), the squamous phenotype in the airway epithelia of smokers in vivo. Selective targeting of the airway epithelium generated in vitro from EGF-treated BCs was EGF-EGFR pathway in airway BCs may be a useful strategy for accompanied by broad suppression of the genes associated with preventing the development of smoking-induced lung disorders, ciliated and secretory cell differentiation, including transcription including lung cancer, at early stages. factor FOXJ1, which is critical for ciliogenesis (44); structural cilia gene DNAI1 (45); nonmucous secretory cell-associated Methods gene SCGB1A1 (2); and serous secretory cell gene MUC5B (46). Airway Epithelium and BCs. LAE from healthy nonsmokers (n = 21) and Consistent with these observations, the presence of EGF during healthy smokers (n = 31) was collected by bronchoscopic brushing of the the differentiation of rat tracheal epithelial cells in vitro has been third- to fourth-order bronchi as described previously (12). Subjects were shown to be inversely correlated with the number of ciliated cells recruited under a protocol approved by the Weill Cornell Medical College generated (32). Institutional Review Board, with written informed consent obtained from each volunteer before enrollment in the study. Cells were processed and BCs EGF-Modified BCs and EMT. EGF-mediated differentiation of BCs isolated as described previously (12). toward the squamous phenotype was accompanied by molecular changes associated with the EMT, a process characterized by the Gene Expression Analysis. Total RNA was extracted, and double-stranded loss of some of differentiated epithelial features and acquisition of cDNA and microarray analyses were performed using HG-U133 Plus 2.0 the mesenchymal-like phenotype (31). Several key characteristics microarrays as described previously (12). In in vitro experiments, expression of

Shaykhiev et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 selected genes was analyzed by TaqMan real-time RT-PCR using specificTaq- Analysis of Protein Expression. Western blot analysis of the total and phos- Man gene expression assays (Applied Biosystems) (12). RNA-Seq analysis of the phorylated EGFR protein was performed as described in SI Methods. Human airway epithelium samples was performed as described previously (52). phospho-RTK antibody Proteome Profiler Array (R&D Systems) analysis was performed according to the manufacturer’s protocol. Immunohistochemistry In Vitro Studies. The capacity of BCs to generate differentiated airway epi- and immunofluorescence analyses were performed using previously repor- theliuminthepresenceorabsenceofEGF(10ng/mL;Sigma-Aldrich)wasassessed ted protocols (12, 21), as described in SI Methods. using the ALI model (21). The CSE stimulations were carried out as described in SI Methods. Levels of EGF released after CSE stimulation were determined using ACKNOWLEDGMENTS. We thank B.-G. Harvey, R. J. Kaner, and A. E. Tilley a commercially available DuoSet ELISA Development Kit for EGF (R&D Systems) ’ for help in obtaining the large airway epithelium samples; B. Ferris for help according to the manufacturer s instructions. The effect of EGF on BC mor- with the studies; and D. N. McCarthy and N. Mohamed for help with phology and EGFR activation was assessed as described in SI Methods. Epithelial manuscript preparation. This study was supported in part by National barrier integrity was assessed by measuring Rt and paracellular permeability Institutes of Health Grants P50 HL084936 and 1R01 HL107882. R.S. is using an FITC-dextran flux assay, as described in SI Methods. supported in part by the Parker B. Francis Foundation.

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