Up-regulation of Expression of the Ubiquitin Carboxyl-Terminal

Hydrolase L1 Gene in Human Airway Epithelium of

Cigarette Smokers

Brendan J. Carolan,1Adriana Heguy,

1Ben-Gary Harvey,

2Philip L. Leopold,

1

Barbara Ferris,1and Ronald G. Crystal

1,2

1Department of Genetic Medicine and 2Division of Pulmonary and Critical Care Medicine, Weill Medical Collegeof Cornell University, New York, New York

Abstract

Neuroendocrine differentiation is a common feature of lungcancer and increased numbers of neuroendocrine cells andtheir peptides have been described in chronic smokers. Tounderstand the effects of cigarette smoking on the geneexpression profile of neuroendocrine cells, microarray anal-ysis with TaqMan confirmation was used to assess airwayepithelial samples obtained by fiberoptic bronchoscopy from81 individuals [normal nonsmokers, normal smokers, smokerswith early chronic obstructive lung disease (COPD), andsmokers with established COPD]. Of 11 genes considered to beneuroendocrine cell specific, only ubiquitin carboxyl-terminalhydrolase L1 (UCHL1), a member of the ubiquitin proteasomepathway, was consistently up-regulated in smokers comparedwith nonsmokers. Up-regulation of UCHL1 at the protein levelwas observed with immunohistochemical analysis of bronchi-al biopsies of smokers compared with nonsmokers. UCHL1expression was evident only in neuroendocrine cells of theairway epithelium in nonsmokers; however, UCHL1 was alsoexpressed in ciliated epithelial cells in smokers. This obser-vation may add further weight to recent observations thatciliated cells are capable of transdifferentiating to otherairway epithelial cells. In the context that UCHL1 is involved inthe degradation of unwanted, misfolded, or damaged proteinswithin the cell and is overexpressed in >50% of lung cancers,its overexpression in chronic smokers may represent an earlyevent in the complex transformation from normal epitheliumto overt malignancy. (Cancer Res 2006; 66(22): 10729-40)

Introduction

Neuroendocrine cells, flask-like cells present in sparse numbersin the airway epithelium, are believed to play an important role inlung physiology with regard to vascular control, inflammation, andresponses to hypoxia by virtue of their ability to produce andsecrete a variety of active peptides (1–3). In the healthy adulthuman lung, neuroendocrine cells are rare, with estimatednumbers of 1 per 2,500 epithelial cells, mostly concentrated inthe intrapulmonary airways (4). The majority of airway neuroen-docrine cells exist as solitary cells but some are present in clusterstermed neuroepithelial bodies (1–3). Neuroendocrine cells are

relevant to lung cancer in that f100% of small cell and 10% to 15%of non–small cell lung cancers have neuroendocrine features (5–7).Immunohistologic assessment of neuroendocrine-specific geneproducts is routinely used to assess lung tumors, and for non–small cell lung cancer, neuroendocrine features are proposed topredict responses to chemotherapy and overall survival (8–11).

One such neuroendocrine cell–specific product is ubiquitincarboxyl-terminal hydrolase L1 (UCHL1; also called protein geneproduct 9.5), a 24,000 Da peptide normally expressed in neuronsand cells of the neuroendocrine system (1–3, 12, 13). UCHL1 is amember of the ubiquitin proteasome pathway controlling intra-cellular protein degradation, functioning to maintain ubiquitinbalance by associating with ubiquitin and by releasing ubiquitinfrom tandemly conjugated ubiquitin monomers and small adductsor unfolded proteins (14). Other functions ascribed to UCHL1include ubiquitin ligase activity and stabilization of ubiquitinwithin the cell, and it may serve as a regulator of apoptosis (15–19).Immunohistochemical assessment of lung tumors shows that >50%of non–small cell lung cancers express UCHL1, correlating with amore advanced cancer stage (20, 21).

Based on the knowledge that cigarette smoking is the leadingcause of lung cancer (22), increased numbers of neuroendocrinecells are present in the airways of cigarette smokers with lungdisease (23), increased levels of neuroendocrine peptides likebombesin and calcitonin are found in biological fluids of healthysmokers (24–26), and that the UCHL1 protein is used as a markerof lung cancer (20), we asked: Does cigarette smoking up-regulatethe expression of the UCHL1 gene in the normal (i.e., nonmalig-nant) airway epithelium? To evaluate this question, large and smallairway epithelium obtained by fiberoptic bronchoscopy andbrushing from 81 individuals [phenotypically normal nonsmokersand normal smokers, as well as individuals with early andestablished chronic obstructive lung disease (COPD)] wereassessed for the expression of 11 neuroendocrine cell–specificgenes using Affymetrix microarrays with TaqMan RT-PCR confir-mation. We found that the expression of neuroendocrine cell–specific genes was not significantly altered by cigarette smokingexcept for a consistent up-regulation of expression of UCHL1 inalmost all smokers. Immunofluorescence staining of bronchialbiopsies showed that, in addition to being present in neuroendo-crine cells of nonsmokers and smokers, UCHL1 was expressed inciliated epithelial cells in smokers. In light of recent observationsin experimental animals that airway ciliated cells can trans-differentiate to nonciliated cells (27, 28), and in the context thatUCHL1 is overexpressed in many lung cancers (20), its up-regulation in response to cigarette smoking may represent one ofthe early events in the complex progression from normalepithelium to neoplastic transformation.

Requests for reprints: Ronald G. Crystal, Department of Genetic Medicine, WeillMedical College of Cornell University, 515 East 71st Street, S-1000, New York, NY 10021.Phone: 212-746-2258; Fax: 212-746-8383; E-mail: nmohamed@med.cornell.edu.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-2224

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Materials and Methods

Study population. Normal nonsmokers, healthy chronic smokers,smokers with early COPD, and smokers with established COPD wereevaluated at the Weill Cornell NIH General Clinical Research Center underprotocols approved by the Weill Cornell Medical College InstitutionalReview Board [different arrays were used to assess the total of 114 samplesfrom 81 individuals; the demographic data for each group and for each site(large and small airway epithelium) are presented in Table 1]. Writteninformed consent was obtained from each individual before enrollment inthe study. No individual in any study group had any variable that suggestedevidence of a lung malignancy. Normal nonsmokers and normal smokerswere determined to be phenotypically normal on the basis of clinical historyand physical examination, routine blood screening tests, urinalysis, chestX-ray, electrocardiogram, and pulmonary function testing. Current smokingstatus was confirmed on history, venous carboxyhemoglobin levels, andurinalysis for nicotine levels and its derivative cotinine. Smokers weredefined as having early COPD if they had a diffusion capacity for carbonmonoxide (DLCO) of <80% predicted with no evidence of airflowobstruction on pulmonary function testing and/or high-resolution com-puted tomography scanning of the chest revealed evidence of emphysema.Smokers with established COPD were defined according to Global Initiativefor Chronic Obstructive Lung Disease criteria (29).

Sampling of airway epithelial cells. Epithelial cells from the large and

small airways were sampled using fiberoptic bronchoscopy as previouslydescribed (30, 31). Smokers were asked not to smoke the evening before the

procedure. After achieving mild sedation and anesthesia of the vocal cords,

a fiberoptic bronchoscope (Pentax, EB-1530T3) was advanced to the desired

bronchus. Large airway epithelial samples were collected by gentle brushing

of the third- to fourth-order bronchi and small airway samples were

collected from 10th- to 12th-order bronchi. These cells were subsequently

collected in 5 mL of bronchial epithelium basal cell medium (Clonetics,

Walkersville, MD). An aliquot was used for cytology and differential cell

count and the remainder was processed immediately for RNA extraction.

Total cell counts were obtained using a hemocytometer, whereas differential

cell counts (epithelial versus inflammatory) were determined on sedimented

cells prepared by centrifugation (Cytospin11, Shandon Instruments,

Pittsburgh, PA) and stained with DiffQuik (Baxter Healthcare, Miami, FL).RNA extraction and microarray processing. Analyses were done

using three different Affymetrix (Santa Clara, CA) microarrays, including

HuGeneFL array (7,000 probe sets), HG-U133A array (22,000 probe sets), and

HG-U133 Plus 2.0 array (54,000 probe sets). The protocols used were as

described by the manufacturer. Total RNA was extracted from epithelial

cells using TRIzol (Invitrogen, Carlsbad, CA) followed by RNeasy (Qiagen,

Valencia, CA) to remove residual DNA. This process yielded 2 to 4 AgRNA per 106 cells. Samples analyzed using the HuGeneFL and HG-133A

microarrays were processed as previously described (30, 31), using 6 AgRNA. For samples analyzed using the HG-U133 Plus 2.0 array, an aliquot of

each RNA sample was run on an Agilent Bioanalyzer (Agilent Technologies,

Palo Alto, CA) to visualize and quantify the degree of RNA integrity. The

concentration was determined using a NanoDrop ND-1000 spectropho-

tometer (NanoDrop Technologies, Wilmington, DE). Three quality control

criteria were used for an RNA sample to be accepted for further processing:

(a) A260/A280 ratio between 1.7 and 2.3; (b) concentration within the range

of 0.2 to 6 Ag/mL; and (c) Agilent electropherogram displaying two distinct

Table 1. Study population and airway epithelial samples

Variable HuGeneFL array HG-U133A array

Large airways Large airways Small airways

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

n 9 13 5 6 5 6

Sex (male/female) 7/2 9/4 3/2 4/2 3/2 4/2

Age (y) 39 F 13 38 F 8 34 F 5 39 F 5 34 F 5 39 F 5

Race (B/W/H) 5/4/0 6/1/1 2/2/1 2/3/1 2/2/1 2/3/1Smoking history (pack-years) 0 21 F 9 0 24 F 3 0 24 F 3

Urine nicotine (ng/mL) 8 F 7 3,493 F 1,110 2 F 5 585 F 609 2 F 5 585 F 609

Urine cotinine (ng/mL) Negative 1,118 F 457 34 F 15 1,129 F 460 34 F 15 1,129 F 460Venous CO-Hb ND 4.0 F 2.0 0.5 F 0.2 4.6 F 1.8 0.5 F 0.2 4.6 F 1.8

Pulmonary function variables (% predicted)

FVC 102 F 3 102 F 3 111 F 10 109 F 18 111 F 10 109 F 18

FEV1 102 F 2 102 F 3 107 F 12 100 F 16 107 F 12 100 F 16FEV1/FVC 101 F 3 99 F 2 95 F 5 91 F 4 95 F 5 91 F 4

TLC 103 F 3 99 F 3 100 F 15 103 F 14 100 F 15 103 F 14

DLCO 88 F 2 85 F 3 92 F 11 91 F 9 92 F 11 91 F 9

Epithelial cellsTotal number recovered � 106 9.4 F 3.5 8.5 F 3.9 6.9 F 1.6 8.6 F 3.2 9.8 F 5.8 7.0 F 3.8

Percent epithelial cells 99 F 1 99 F 1 98 F 1 98 F 1 96 F 4 96 F 4

Percent inflammatory 1 F 1 1 F 1 1 F 1 1 F 1 4 F 3 4 F 4Differential cell count

Ciliated 44 F 3 46 F 4 50 F 2 43 F 3 80 F 5 75 F 6

Secretory 9 F 2 8 F 2 9 F 4 10 F 2 4 F 1 4 F 3

Basal 25 F 4 22 F 4 20 F 3 27 F 4 5 F 3 8 F 2Undifferentiated 21 F 6 24 F 3 21 F 4 20 F 2 8 F 2 9 F 3

Abbreviation: ND, not determined.

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peaks corresponding to the 28S and 18S rRNA bands at a ratio of 28S/18S of

>0.5 with minimal or no degradation. Double-stranded cDNA was

synthesized from 3 Ag of total RNA using the GeneChip One-Cycle cDNA

Synthesis kit, followed by cleanup with GeneChip Sample Cleanup Module,

in vitro transcription (IVT) reaction using the GeneChip IVT Labeling kit,and clean-up and quantification of the biotin-labeled cRNA yield by

spectrophotometric analysis. All kits were from Affymetrix. Hybridizations

to test chips and to the microarrays were done according to Affymetrix

protocols, and microarrays were processed by the Affymetrix fluidics stationand scanned with an Affymetrix GeneArray 2500 (HuGeneFL) and the

Affymetrix GeneChip Scanner 3000 7G (HG-U133A and HG-U133 Plus 2.0).

To maintain quality, only samples hybridized to test chips with a 3¶ to 5¶ratio of <3 were deemed satisfactory.

Microarray data analysis. Captured images were analyzed using

Microarray Suite version 5.0 algorithm (Affymetrix). These data were

normalized using GeneSpring version 6.2 software (Agilent Technologies)as follows: (a) per array, by dividing raw data by the 50th percentile of all

measurements; and (b) per gene, by dividing the raw data by the median

expression level for all the genes across all arrays in a data set. All HG-

U133A data and HG-U133 Plus 2.0 large airway data was log transformedbefore statistical analysis. To evaluate neuroendocrine cell–specific gene

expression in the large and small airway samples of nonsmokers, healthy

smokers, smokers with early COPD, and smokers with established COPD,

a list of known neuroendocrine cell–specific genes was established fromthe literature (1–3, 32–34). This signature transcriptome of neuroendo-

crine cells was used to assess the effects of smoking on the genome of

these cells. Expression was defined as having an Affymetrix Detection Callof Present (P call) in z50% of samples assessed by each type of

microarray.

TaqMan reverse transcription-PCR confirmation of microarrayexpression levels. TaqMan real-time reverse transcription-PCR (RT-PCR)

was done on available RNA samples from the small airways of 12 normal

nonsmokers and 10 normal smokers that had been assessed with the HG-U133 Plus 2.0 array. cDNA was synthesized from 2 Ag RNA in a 100 ALreaction volume, using the TaqMan Reverse Transcriptase Reaction kit

(Applied Biosystems, Foster City, CA), with random hexamers as primers.

Two dilutions of 1:50 and 1:100 were made from each sample and

triplicate wells were run for each dilution. TaqMan PCR reactions were

carried out using premade gene expression assays for neuroendocrine

genes from Applied Biosystems and 2 AL cDNA were used in each 25-ALreaction volume. The endogenous control was 18S rRNA and relative

expression levels were determined using the DDC t method (Applied

Biosystems) with the average value for the nonsmokers as the calibrator.

The PCR reactions were run in an Applied Biosystems Sequence Detection

System 7500.

Localization of UCHL1 in the airway epithelium. To determine which

airway epithelial cells express UCHL1, bronchial biopsies were obtainedfrom the large airway epithelium of six nonsmokers and six normal smokers

using conventional methods. Immunohistochemistry was subsequently

done on paraffin-embedded endobronchial biopsies. Sections were depar-

affinized and rehydrated through a series of xylenes and alcohol. Toenhance staining, an antigen retrieval step was carried out by microwave

treatment of the sections at 100jC for 15 minutes in citrate buffer solution

(Labvision, Fremont, CA) followed by cooling at 23jC for 20 minutes.Endogenous peroxidase activity was quenched using 0.3% H2O2 and

blocking with normal goat serum to reduce background staining. Samples

were incubated with the primary antibody at 23jC for 1 hour. For

chromogranin A (CHGA), the primary antibody was mouse monoclonal(LK2H10 + PHE5) anti-human antibody (Labvision) diluted 1:5,000 and

mouse IgG1 was the isotype control. For UCHL1 detection, the primary

antibody was rabbit polyclonal anti-human UCHL1 (Labvision) diluted

1:2,500 and rabbit IgG (DakoCytomation, Carpinteria CA) was the isotype

Table 1. Study population and airway epithelial samples (Cont’d)

HG-U133 Plus 2.0 array

Large airways Small airways

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

Early COPD

smokers

Established COPD

smokers

4 5 12 12 9 6

4/0 2/3 10/2 9/3 6/3 5/1

36 F 2 43 F 2 42 F 8 45 F 4 51 F 7 49 F 6

2/2/0 3/2/0 6/4/2 7/5/0 5/3/1 2/4/00 24 F 4 0 26 F 9 35 F 25 26 F 13

Negative 254 F 266 Negative 648 F 265 485 F 118 347 F 155

Negative 1,310 F 716 Negative 1,263 F 212 1,230 F 435 835 F 3741.7 F 0.9 3.9 F 2.7 1.7 F 0.7 3.3 F 0.9 ND 1.4 F 0.6

107 F 14 103 F 11 105 F 9 103 F 12 98 F 11 106 F 11

104 F 10 98 F 10 105 F 7 100 F 14 89 F 13 87 F 2596 F 5 94 F 8 99 F 7 97 F 7 78 F 4 66 F 14

95 F 9 94 F 17 97 F 8 96 F 15 95 F 13 107 F 20

111 F 6 90 F 7 95 F 12 94 F 10 60 F 11 82 F 19

6.5 F 0.5 4.9 F 1.0 5.3 F 1.7 6.8 F 2.2 7.3 F 1.6 5.7 F 0.5

99 F 1 99 F 1 99 F 1 99 F 1 99 F 1 96 F 1

1 F 1 1 F 1 1 F 1 1 F 1 1 F 1 4 F 1

51 F 4 42 F 3 78 F 7 75 F 10 81 F 3 71 F 4

8 F 1 10 F 2 7 F 3 7 F 3 4 F 1 9 F 4

23 F 1 27 F 2 7 F 2 9 F 4 7 F 3 2 F 117 F 3 20 F 3 8 F 4 9 F 4 7 F 1 2 F 1

Airway Epithelial UCHL1 Gene Expression

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control. To block UCHL1 antibody binding, the UCHL1 antibody was

incubated with the full-length recombinant UCHL1 protein (Labvision) at23jC for 30 minutes to saturate binding sites before being applied to sample

tissues. Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and

3, 3¶-diaminobenzidine substrate kit (Vector Laboratories) were used to

visualize antibody binding. The sections were counterstained withhematoxylin (Sigma Aldrich, St. Louis, MO) and mounted using GVA

mounting medium (Zymed, San Francisco, CA). Brightfield microscopy was

done using a Nikon Microphot microscope equipped with a Plan �40

numerical aperture (NA) 0.70 objective lens. Images were captured with anOlympus DP70 CCD camera.

Immunofluorescent staining was carried out on airway epithelial

biopsies using primary antibodies for UCHL1 and CHGA as describedabove; mouse monoclonal (ONS1A6) anti-human h IV tubulin (1/500

dilution; Biogenex, San Ramon, CA) as a marker for ciliated cells (35);

mouse monoclonal (45M1) mucin 5AC (1/200; Labvision) as a marker for

secretory cells (36); and mouse monoclonal (SH-L1) S100 A2 (1/50dilution; GeneTex, Inc. San Antonio, TX) as a marker for basal cells (37).

Following incubation with the primary antibodies at 23jC for 1 hour

in a humidified chamber, goat anti-rabbit Cy5 conjugated AffiniPure

F(ab¶)2 (Jackson Immunoresearch, West Grove, PA) at 1/100 dilutionwas used as a secondary antibody for UCHL1 and goat anti-mouse

Cy3-conjugated AffiniPure F(ab¶)2 (Jackson Immunoresearch) at 1/100

dilution was used as a secondary antibody for all other antibodies.

Fluorescence microscopy was done using a Zeiss LSM 510 LaserScanning Confocal microscope equipped with a Plan Neofluor �40 NA

0.75 objective lens. Illumination was provided by an argon laser (488 nm

line) and two helium/neon lasers (543 and 633 nm lines) with matcheddichroic mirrors and emission filters. Images were analyzed using Zeiss

LSM Image Browser version 3.1.099. Pseudocolor images were formed by

encoding Cy5 fluorescence in the green channel, Cy3 fluorescence in the

red channel, and autofluorescence in gray scale. The images were

composed by integrating five independent images collected at a step

size of 1.7 Am.Statistical analysis. For all HuGeneFL data and small airway data

analyzed on the HG-U133 Plus 2.0, P values for all comparisons were

calculated using a two-tailed t test, assuming unequal variance (Welch

t test) with the Benjamini-Hochberg multiple test correction for false-discovery rate (38), using GeneSpring software. Genes were considered

significant if the Benjamini-Hochberg corrected P value was <0.05. For

HG-U133A large and small airway data and for large airway data analyzed

on the HG-U133 Plus 2.0 microarray, P values were calculated as describedabove, but in the absence of the Benjamini-Hochberg correction. Average

expression values for neuroendocrine cell–specific genes in large and

small airway samples were calculated from normalized expression levelsfor nonsmokers, normal smokers, smokers with early COPD, and smokers

with established COPD. TaqMan data was normalized per gene by

dividing by the median expression of each gene in all samples, and

subsequently the mean and SE were calculated for normalized valuesof expression. P values for TaqMan data were calculated using the Welch

t test.

Web deposition of data. All data has been deposited in the Gene

Expression Omnibus site,3 which is curated by the National Center forBioinformatics. Accession numbers include (a) HuGeneFL accession

number GSE5056; (b) small airways HG-U133A, accession number

GSE3320, already cited in ref. 31; (c) large Airways HG-U133A accession

number GSE5057; (d) large airways HG-U133 Plus 2.0 accession numberGSE5059; and (e) small airways HG-U133 Plus 2.0 accession number

GSE5058 (31).

Table 2. Microarray assessment of neuroendocrine cell–specific genes in normal nonsmokers, normal smokers, smokers withearly COPD, and smokers with established COPD

Gene symbol %P call

HUGene FL chip Hu 133A chip HG-U133 Plus 2.0 chip

Large airways Large airways Small airways Large airways

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

Normal

nonsmokers

Normal

smokers

SGNE1 NA NA 20 0 17 50 0 0

PENK NA NA 40 17 20 17 25 40

TAC1 NA NA 0 17 20 0 0 0ASCL1 0 8 NA NA NA NA 0 20

NCAM1 NA NA 0 33 20 0 25 40

CALCB 11 23 40 0 0 33 40 0

SCG2 0 4 20 66 20 83 75 100CHGA NA NA 0 0 0 17 25 80ENO2 67 62 80 66 100 100 100 60GRP 22 85 0 17 20 33 0 60UCHL1 0 69 0 100 0 100 0 80

Abbreviation: NA, probe not on array, not applicable.

*Overall assessment of expression was based on P call z50% in at least two of the three arrays used. Fmeans probably expressed, but observed in only

one type of array; this may be dependent on different probes on the different arrays.cEarly COPD smokers—smokers with normal lung function except for abnormal DLCO.bEstablished COPD smokers—smokers with COPD as defined by the GOLD criteria (29).x‘‘No’’ indicates not expressed with a P call <50% and ‘‘Yes’’ indicates expression with a P call z50% (bold type).

3 http://www.ncbi.nlm.nih.gov/geo.

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Results

Study population. A total of 114 samples were assessed from 81study individuals (Table 1). Results were obtained using threedifferent microarrays including: (a) HuGeneFL microarray—18 largeairway samples from 9 nonsmokers and 26 large airway samplesfrom 13 normal smokers; (b) HG-U133A array—large and smallairway samples from 5 nonsmokers and 6 normal smokers; and (c)HG-U133 Plus 2.0 array—large airway samples from 4 normalnonsmokers and 5 normal smokers, and small airway samples from12 normal nonsmokers, 12 normal smokers, 9 smokers with earlyCOPD, and 6 smokers with established COPD. All individuals had nosignificant prior medical history and normal physical examinations.There were no differences between groups with regard to sex, race,or age (P > 0.05 for all comparisons). There was a statisticallysignificant difference in age in the nonsmoker group versus earlyCOPD group (P < 0.01) analyzed with the HG-U133 Plus 2.0microarray. All individuals were HIV negative with blood and urinevariables within reference ranges (P > 0.05 all comparisons).Smokers had an average smoking history of 27 F 2 pack-years.The number of cells recovered by brushing ranged from 4.9 � 106 to9.8 � 106 (Table 1). In all cases, >95% of cells recovered wereepithelial cells. The subtypes of airway epithelial cells were asexpected from the large and small airways (Table 1; ref. 29).Neuroendocrine cells were not observed in brushed airway samples.Detection of neuroendocrine gene expression in the large

and small airway epithelium. With the criteria of P call z50%,most neuroendocrine genes were not detected in the large airwayepithelium of nonsmokers [secretory granule neuroendocrinepeptide 1 (SGNE1), pro-enkephalin (PENK), tachykinin 1 (TAC1),achaete scute homologue 1 (ASCL1), neuronal cell adhesion molecule1 (NCAM1), calcitonin gene-related polypeptide b (CALCB), CHGA,gastrin releasing peptide (GRP), and UCHL1 (Table 2)]. Of the 11neuorendocrine genes evaluated, only expression of enolase 2(ENO2) was universally detected in the large airways of non-smokers. In the small airways of normal nonsmokers, 5 of the 11

neuroendocrine genes were not expressed (SGNE1, PENK, TAC1,ASCL1, CALCB , and UCHL1), one gene was equivocal (NCAM1 wasdetected in only the HG-U133 Plus 2.0 array), and four were clearlydetected [secretogranin 2 (SCG2), CHGA, ENO2 , and GRP].

In the current smokers (phenotypically normal, early COPD, andestablished COPD), expression of the neuroendocrine-specificgenes in the large and small airways was mostly consistent withthat observed in the large and small airway epithelium innonsmokers (Table 2). However, in marked contrast to the otherneuroendocrine genes, whereas UCHL1 was not detected in any ofthe large and small airway epithelial samples of the nonsmokers,UCHL1 was detected in the large and small airway epithelium ofsmokers in almost every microarray (large airway epitheliumsamples � 69% of normal smokers assessed with the HuGeneFLchip; 100% of normal smokers with HG-U133A and 80% of nor-mal smokers with HG-U133 Plus 2.0; small airway epitheliumsamples � 100% of normal smokers assessed with HG-U133A; 100%of normal smokers with HG-U133 Plus 2.0; 89% early COPDsmokers with HG-U133 Plus 2.0; and 100% of established COPDsmokers with HG-U133 Plus 2.0).

UCHL1 expression was 18.3-fold higher in normal smokerscompared with nonsmokers in the large airways analyzed with theHuGeneFL array (P < 0.01), 9.0-fold higher in large airway analyzedwith the HG-U133A array (P < 0.01), and 42.2-fold higher in largeairways analyzed with the HG-U133 Plus 2.0 array (P < 0.01). In thesmall airways, UCHL1 was 11.4-fold higher in normal smokers thannonsmokers in HG-U133A array (P < 0.01). In the HG-U133 Plus 2.0data set, UCHL1 expression was 39.3-fold higher in normal smokers(P < 0.01), 60.8-fold higher in smokers with early COPD (P < 0.01),and 38.6-fold higher in smokers with established COPD (P < 0.01).There was no significant difference in the level of expression ofUCHL1 between normal smokers and smokers with early COPD(P > 0.8) or smokers with established COPD (P > 0.9; Fig. 1).Quantitative expression of the neuroendocrine cell–specific

genes in the small airway epithelium. Of the neuroendocrine-specific genes expressed in the airway epithelium, quantitative

Table 2. Microarray assessment of neuroendocrine cell–specific genes in normal nonsmokers, normal smokers, smokers withearly COPD, and smokers with established COPD (Cont’d)

%P call

HG-U133 Plus 2.0 chip Overall assessment of expression*

Small airways Large airways Small airways

Normal

nonsmokers

Normal

smokers

Early COPD

smokersc

Established COPD

smokersb

Normal

nonsmokersxNormal

smokers

Normal

nonsmokers

Normal

smokers

Early COPD

smokers

Established COPD

smokers

17 58 56 17 No No No F* Yes No

42 25 33 17 No No No No No No

8 0 0 0 No No No No No No33 60 44 50 No No No F F Yes67 33 56 33 No No F Yes No No

0 8 0 0 No No No No No No

92 83 89 83 F Yes Yes Yes Yes Yes58 75 100 100 No F Yes Yes Yes Yes100 92 100 83 Yes Yes Yes Yes Yes Yes67 92 100 83 No Yes Yes Yes Yes Yes8 100 89 100 No Yes No Yes Yes Yes

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assessment of the relative gene expression levels showed nodifference among nonsmokers and smokers for GRP, ENO2, orSCG2 (Fig. 2; P > 0.1 for all comparisons of nonsmokers to each ofthe current smoker groups including phenotypically normalsmokers, smokers with early COPD, and smokers with establishedCOPD). There was a significant difference in expression levels ofCHGA in smokers with established COPD compared with normalnonsmokers (P < 0.04).

In marked contrast, the expression of UCHL1 was up-regulatedin smokers compared with nonsmokers in the small and largeairway epithelium in all the data sets (Fig. 1). This was true fornormal smokers compared with normal nonsmokers in the largeairway epithelial samples assessed with the HuGeneFL microarray(A ; P < 0.01); normal smokers compared with normal nonsmokers

of the large airway epithelium assessed with the HG-U133Aarray (B ; P < 0.01); normal smokers compared with normalnonsmokers of the small airway epithelium assessed with the HG-U133A array (B ; P < 0.01); normal smokers compared with normalnonsmokers of the large airway epithelium assessed with the HG-U133 Plus 2.0 array (C ; P < 0.01); and the normal smokers (P < 0.01),early COPD smokers (P < 0.01), and established COPD smokers(P < 0.05) compared with normal nonsmokers of the small airwayepithelium with the HG-U133 Plus 2.0 array (C).TaqMan RT-PCR confirmation of microarray results. To

confirm the results obtained from microarray studies, TaqManRT-PCR was carried out on RNA samples from the small airways of12 normal nonsmokers and 10 normal smokers (Fig. 3). TheTaqMan data confirmed that there was no difference in expression

Figure 1. Normalized expression levels ofthe neuroendocrine cell–specific geneUCHL1 in large and small airwayepithelium of nonsmokers and smokers.Y axis, normalized gene expression levelsfor UCHL1 . Each symbol represents anindividual. A, UCHL1 gene expressionlevels in large airways assessed usingHuGeneFL array; there are 18 samplesfrom 9 nonsmokers and 26 samples from13 normal smokers. B, UCHL1 geneexpression levels in large airway samplesassessed using HG-U133A array fromfive normal nonsmokers and six normalsmokers, and small airway samples fromfive normal nonsmokers and six normalsmokers. C, UCHL1 gene expressionlevels in large and small airway samplesassessed using HG-U133 Plus 2.0 arraywith large airway samples from 4 normalnonsmokers and 5 normal smokers, andsmall airway samples from 12 normalnonsmokers and 12 normal smokers.

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levels of other neuroendocrine-specific genes, including CHGA,GRP, ENO2, and SCG2. The TaqMan analysis also confirmed the up-regulation of UCHL1 mRNA expression in normal smokerscompared with nonsmokers (P < 0.01).Localization of UCHL1 in the airway epithelium of smokers.

Immunohistochemistry was used to assess expression of CHGAand UCHL1 in endobronchial biopsies obtained from large airwaysat bronchoscopy from six nonsmokers and six normal smokers.This analysis showed protein expression of CHGA and UCHL1 inairway epithelial cells with the typical morphology and localizationof neuroendocrine cells (Fig. 4A-C). Surprisingly, the smokers notonly had UCHL1 expression in typical neuroendocrine cells, butthere was also positive staining for UCHL1 in other epithelial cellsmore apically in the airway epithelium that was not present innonsmokers (Fig. 1E, F). To confirm the specificity of the polyclonalrabbit anti-UCHL1 antibody, a blocking step was done with full-length recombinant UCHL1 protein; this completely blocked allantibody binding on biopsy samples from smokers, demonstratingthe specificity of this polyclonal antibody for the UCHL1 epitope(not shown). Overall, although there were a greater number of cellswith a neuroendocrine morphology observed in the airway

epithelium of normal smokers compared with nonsmokers, therewere also a greater number of UCHL1-positive cells within theairway epithelium of smokers compared with nonsmokers thatwere not positive for the neuroendocrine marker CHGA. Theseadditional UCHL1-positive cells had the appearance and morphol-ogy of ciliated epithelial cells (Figs. 5 and 6). UCHL1 was confirmedto be present in ciliated airway epithelial cells in the smokers asevidenced by colocalization with the ciliated cell–specific marker hIV tubulin but not with the secretory cell marker MUC5AC. Thecolocalization of UCHL1 and h IV tubulin was almost universalthroughout the cilia with some cilia being more intensely positivefor UCHL1, whereas, as expected, all cilia stained positive for h IVtubulin. UCHL1 was not present in basal cells as evidenced bylack of colocalization with S100 A2, a marker of these cells (notshown).

Discussion

Cigarette smoking is the major risk factor associated with thedevelopment of lung cancer (22). In the present study, we show thatUCHL1, a ubiquitin thiolesterase and member of the ubiquitin

Figure 2. Normalized expression levels of the neuroendocrine cell–specific genes ASCL1, SCG2, CHGA, ENO2 , and GRP in human small airway epithelium assessedwith the HG-U133 Plus 2.0 array in 12 normal nonsmokers, 12 normal smokers, 9 individuals with early emphysema, and 6 individuals with established COPD.X axis, the neuroendocrine-specific gene; Y axis, normalized gene expression levels. Each symbol represents an individual: nonsmokers (4), normal smokers (E),early COPD ( w ), and individuals with established COPD ( ).

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proteasome pathway, is consistently up-regulated in the large andsmall airway epithelium of cigarette smokers, including normalsmokers, smokers with early COPD, and smokers with establishedCOPD. These observations are relevant to lung cancer in severalrespects. First, UCHL1 is commonly used as a marker in assessinglung cancer, with 100% of small cell cancers and 50% to 60% ofnon–small cell cancers expressing UCHL1. The expression ofUCHL1 has also been linked to outcome (20, 21). Second, the up-regulation of an enzyme linked to the degradation of misfoldedproteins is relevant to the multifactorial, complex pathogenesis oflung cancer. Third, the observation that much of the up-regulatedUCHL1 in the smoker airway epithelium was not only inneuroendocrine cells, but also in ciliated cells, together with theobservation that ciliated cells can trans-differentiate to other celltypes (27, 28), leads to the provocative question as to whether somelung cancers are derived from cells that were originally differen-tiated ciliated cells.Neuroendocrine-specific gene expression in the airway

epithelium. Using Affymetrix microarrays with TaqMan confirma-tion, assessment of gene expression of neuroendocrine-relevantgenes in the large and small airway epithelium of healthynonsmokers, phenotypically normal smokers, smokers with earlyCOPD, and smokers with established COPD showed that smokingdid not alter the expression of genes coding formost neuroendocrinepeptides at themRNA level, but therewas consistent up-regulation ofUCHL1 in smokers.

Consistent with the sparse numbers of neuroendocrine cellspresent in the airway epithelium (0.3-0.5% of airway epithelialcells; ref. 39), expression of many neuroendocrine cell–specificgenes could not be detected in the brushed samples of large andsmall airway epithelium. Using the criteria of an AffymetrixDetection Call of Present in z50% samples, the neuroendocrinegenes that were expressed (other than UCHL1 in smokers)included GRP, CHGA, neuron-specific enolase , and SCG2 . Althoughprevious studies have described increased numbers of neuroen-docrine cells in the airways and their peptides in biological fluidsof smokers with a variety of pulmonary disease states (23, 40),the data did not show increased expression of the mRNA forthese peptides, suggesting that smoking may induce the releaseof some neuroendocrine peptides rather than up-regulatingthe expression of most of the neuroendocrine cell–relevant genes(24–26).UCHL1 gene expression in the airway epithelium. The

observation that UCHL1 is up-regulated in smokers comparedwith nonsmokers is important in the context that ubiquitinationof proteins is an important control process within cells, targetingthem for localization and degradation, particularly oxidized ormisfolded proteins (41, 42). Much of the knowledge of the functionof UCHL1 is from the gracile axonal dystrophy mouse, a naturallyoccurring mutant that lacks the UCHL1 protein due to a deletionof a genomic fragment that includes exons 7 and 8 (43). Thesemice develop neurodegenerative disease and inclusions withinneuronal cells that are composed of ubiquitin and abnormalproteins (44). Under normal conditions, when proteins are taggedwith ubiquitin, they are targeted for degradation within the cell.UCHL1 acts to maintain ubiquitin levels within the cell by itsphysical association with ubiquitin, by hydrolyzing bonds torelease monoubiquitin from polyubiquitin chains and cleavingbonds between ubiquitin and small adducts before degradation bythe proteasome (14). UCHL1 also plays a role in programmed celldeath through its posttranslational regulation of apoptotic factors

(15–17, 19). In this regard, there is reduced apoptosis in the testesof UCHL1-deficient mice under cryptorchid-induced stress and inthe retina under ischemic stress (15–17). This is associated withan alteration in the balance of proapoptotic and antiapoptoticfactors, particularly of the bcl-2 family (15–17). In the context thatUCHL1 plays a role in apoptosis by altering the balance ofapoptotic factors, its up-regulation in smokers may reflectincreased cell stress, with cigarette smoke altering proteinstructures within the cell. A hereditary form of Parkinson’s diseasehas also been linked to an I93M mutation in the UCHL1 gene (45),consistent with a proposed role of UCHL1 in maintaining thebalance of normal proteins within the cell (46, 47).

UCHL1 may be a marker of cellular stress in lung cancer. Theubiquitin proteasome pathway regulates with exquisite specificitycellular processes, such as cell cycle progression, inhibition, or exe-cution of apoptosis and activation or expression of transcriptionfactors, including p53, c-Jun, and HIF1 (35, 48, 49). UCHL1 has beenshown to interact with JAB1, a Jun activation domain bindingprotein that can bind to p27Kip1, a cyclin-dependent proteinkinase inhibitor involved in cell cycle regulation (35). UCHL1associates with JAB1 in lung cancer cell lines and its overexpressionhas been linked to decreased levels of p27Kip1, which is observedin many lung cancers (35). Immunohistochemistry for UCHL1 isfrequently used in the evaluation of small cell and non–small celllung cancer (20, 21). UCHL1 is highly expressed in >50% of primarylung cancers, most lung cancer cell lines, and some nonpulmonarycancers like pancreatic and esophageal cancer (50). The level ofexpression in lung cancer increases with increasing tumor stageand this expression may occur independently of overt neuroendo-crine differentiation (20).

Although the data in the present study is insufficient todetermine the temporal role, if any, of UCHL1 in the progressionof smoking-induced neoplastic transformation, the data set ofSpira et al. (GSE994) of airway epithelium gene expression in-cludes a group of ex-smokers who had baseline levels of UCHL1.

Figure 3. Confirmation of microarray results with TaqMan real-time RT-PCR forfive neuroendocrine cell–specific genes, including CHGA, GRP, ENO2, SCG2 ,and UCHL1 . The data include samples from the small airways from 12nonsmokers and 10 smokers. X axis, neuroendocrine-specific genes evaluated;Columns, average expression levels on a logarithmic scale. Expression levelsare normalized by dividing individual values by the median expression levelfor each gene in all nonsmokers and smokers. Bars, SE. P values represent thecomparison between nonsmokers and smokers.

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This suggests that expression of UCHL1 may return to the levelof never smokers upon smoking cessation, at least in somepeople.

It is observed in this study that many of the UCHL1-positive cellsin the airway epithelium of smokers are not positive for otherneuroendocrine peptides like CHGA, and that many of the UCHL1-positive cells have the morphology of ciliated epithelial cells.Immunofluorescent staining and confocal microscopy confirmedthe presence of UCHL1 in ciliated epithelial cells where it wascolocalized with h IV tubulin. UCHL1 expression in ciliatedepithelial cells was only present in smokers, whereas it wasexpressed in neuroendocrine cells in both nonsmokers andsmokers, where it colocalized with CHGA. UCHL1 was not presentin other epithelial cells like secretory cells and basal cells, as it did

not colocalize with MUC5AC or S100 A2, markers for these cells,respectively. Ciliated epithelial cells have traditionally been thoughtof as terminally differentiated cells, incapable of further change,but recent studies point to the plasticity of these cells and the rolethey have to play in the repair process of the respiratory epithelium(27, 28). The observation that UCHL1 is up-regulated in ciliatedepithelial cells in cigarette smokers is important because UCHL1 istraditionally a marker of neuroendocrine cells and a commonlyused marker for lung cancer.

In summary, UCHL1 , a gene that is overexpressed in many lungcancers, is expressed in neuroendocrine cells of nonsmokers and inneuroendocrine and ciliated epithelial cells of smokers. In additionto reflecting the plasticity of ciliated epithelial cells in the humanairway, this may represent a very early event in the complex

Figure 4. Immunohistochemistryassessment of expression ofneuroendocrine cell–specific peptidesCHGA and UCHL1 in normal nonsmokerand normal smoker endobronchialbiopsies. A, left, CHGA, normalnonsmoker, mouse IgG1 isotypecontrol; right, CHGA, normal nonsmoker,mouse antihuman CHGA antibody.B, left, CHGA, normal smoker, mouseIgG1 isotype control; right, CHGA,normal smoker, antihuman CHGAantibody. C, CHGA, normal smoker,high-power magnification, anti-CHGAantibody. D, left, UCHL1, normal non-smoker, rabbit IgG control; right, UCHL1,normal nonsmoker, anti-UCHL1 antibody.E, left, UCHL1, normal smoker, rabbitIgG control; right, UCHL1, normal smoker,anti-UCHL1 antibody. F, UCHL1, normalsmoker, high-power magnification,anti-UCHL1 antibody. Arrows, positivestaining is indicated by the browndiaminobenzidine precipitant. All panels,bar 20 Am.

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changes that occur in the spectrum from normal cells to overtmalignancy. Further studies are required to advance the under-standing of the role of UCHL1 in epithelial cell function and in thepathogenesis of pulmonary disease and cancer.

Acknowledgments

Received 6/19/2006; revised 7/27/2006; accepted 9/25/2006.

Grant support: NIH R01 HL074326, M01RR00047, and the Will Rogers MemorialFund, Los Angeles, CA.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Tina Raman, Igor Dolgalev, and Adam Cieciuch for expert technicalassistance; Dr. Jenny Xiang from the Weill Cornell Microarray Core Facility; Lee CohenGould and the Weill Cornell Optical Microscopy Core facility for help withimmunofluorescent image acquisition; and Nahla Mohamed for help in preparingthe manuscript.

Figure 5. Confocal immunofluorescent assessment of UCHL1 expression in the airway epithelium of normal smokers. A, negative controls, rabbit IgG isotypecontrol (green ), mouse IgG1 isotype control (red), and autofluorescence (gray ). B, normal smoker, anti-UCHL1 antibody (green ), anti-CHGA antibody (red ),and autofluorescence (gray ). UCHL1 colocalizes with CHGA to neuroendocrine cells (arrows ), but is also expressed in nonneuroendocrine cells (filled arrowheads ) andnerves (open arrowheads ). C, normal smoker, anti MUC5AC antibody, and anti-UCHL1 antibody; anti-UCHL1 antibody (green ), anti-MUC5AC antibody (red).Mucin containing secretory cells (closed arrows ), UCHL1-positive airway epithelial cells (closed arrowheads ), and nerves (open arrows ). D, normal smoker, anti-h IVtubulin antibody, and anti-UCHL1 antibody; anti-h IV tubulin antibody (red) and anti-UCHL1 antibody (green ), colocalization of h IV tubulin and UCHL1 (yellow-orange,closed arrowheads). All panels, bar 20 Am.

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2006;66:10729-10740. Cancer Res   Brendan J. Carolan, Adriana Heguy, Ben-Gary Harvey, et al.   Epithelium of Cigarette Smokers

Gene in Human AirwayCarboxyl-Terminal Hydrolase L1UbiquitinUp-regulation of Expression of the

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