Receptor-mediated tobacco toxicity: acceleration of sequential expression of 5 and 7 nicotinic...

The FASEB Journal • Research Communication

Receptor-mediated tobacco toxicity: acceleration ofsequential expression of �5 and �7 nicotinicreceptor subunits in oral keratinocytes exposed tocigarette smoke

Juan Arredondo,* Alexander I. Chernyavsky,* David L. Jolkovsky,† Kent E. Pinkerton,‡

and Sergei A. Grando*,1

*Department of Dermatology, University of California, Irvine, California, USA; †Section ofPeriodontics, School of Dentistry, University of California, Los Angeles, California, USA; and ‡Centerfor Health and the Environment, University of California, Davis, California, USA

ABSTRACT Tobacco products and nicotine alter thecell cycle and lead to squamatization of oral keratino-cytes (KCs) and squamous cell carcinoma. Activation ofnicotinic acetylcholine receptors (nAChRs) elicits Ca2�

influx that varies in magnitude between differentnAChR subtypes. Normal differentiation of KCs isassociated with sequential expression of the nAChRsubtypes with increasing Ca2� permeability, such as�5-containing �3 nAChR and �7 nAChR. Exposure toenvironmental tobacco smoke (ETS) or an equivalentconcentration of nicotine accelerated by severalfold the�5 and �7 expression in KCs, which could be abolishedby mecamylamine and �-bungarotoxin with differentefficacies, suggesting the following sequence of auto-regulation of the expression of nAChR subtypes:�3(�2/�4) > �3(�2/�4)�5 > �7 > �7. This conjec-ture was corroborated by results of quantitative assaysof subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfer-ing RNAs. The genomic effects of ETS and nicotineinvolved the transcription factor GATA-2 that showed amultifold increase in quantity and activity in exposedKCs. Using protein kinase inhibitors and dominantnegative and constitutively active constructs, we charac-terized the principal signaling cascades mediating aswitch in the nAChR subtype. Cumulative results indi-cated that the �3(�2/�4) to �3(�2/�4)�5 nAChR tran-sition predominantly involved protein kinase C, �3(�2/�4)�5 to �7 nAChR transition—Ca2�/calmodulin-dependent protein kinase II and p38 MAPK, and �7self-up-regulation—the p38 MAPK/Akt pathway, andJAK-2. These results provide a mechanistic insight intothe genomic effects of ETS and nicotine on KCs andcharacterize signaling pathways mediating autoregula-tion of stepwise overexpression of nAChR subtypeswith increasing Ca2� permeability in exposed cells.These observations have salient clinical implications,because a switch in the nAChR subunit composition canbring about a corresponding switch in receptor func-tion, leading to profound pathobiologic effects ob-served in KCs exposed to tobacco products. Arre-

dondo, J., Chernyavsky, A. I., Jolkovsky, D. L.,Pinkerton, K. E., Grando, S. A. Receptor-mediatedtobacco toxicity: acceleration of sequential expressionof �5 and �7 nicotinic receptor subunits in oral kera-tinocytes exposed to cigarette smoke. FASEB J. 22,1356–1368 (2008)

Key Words: nicotinic acetylcholine receptors � Akt � PKC � p38� JAK-2 � GATA-2

The complex process of tobacco-induced morbidityin the oral cavity and esophagus includes direct effectsof tobacco components on epithelial cells. Tobaccoproducts and nicotine alter the cell cycle and lead tosquamatization of oral keratinocytes (KCs) (1–3) andsquamous cell carcinoma (4–7). It has been estimatedthat in the year 2007, 34,360 new cases of cancer of theoral cavity and pharynx will occur in the United States(8). Identification of the mechanisms leading to dam-age of KCs may prove useful in the prevention oftobacco toxicity. The continuous cycle of birth anddeath of KCs is a self-sustained process controlled, inpart, by locally produced acetylcholine (ACh) throughthe signaling pathways that couple each type of AChreceptors to a particular cell function (reviewed in ref.9). The nicotinic class of ACh receptors (nAChRs) playsimportant roles in regulation of growth and differenti-ation of oral KCs and can mediate pathobiologic effectsof the smokeless tobacco and nicotine derivatives onthese cells (10–13). The nAChRs may, therefore, pro-vide a novel molecular target to prevent, reverse, orameliorate progression of the tobacco-related cell dam-age and intercede in disease pathways.

Previous studies have documented that nAChRs inoral KCs can comprise the �3, �5, �7, �9, �2, and �4

1 Correspondence: Department of Dermatology, Universityof California, Irvine, C340 Medical Sciences I, Irvine, CA92697, USA. E-mail: [email protected]

doi: 10.1096/fj.07-9965.com

1356 0892-6638/08/0022-1356 © FASEB

subunits (14, 15). The heteromeric channels can becomposed of �3, �5, �2, and �4 subunits, e.g., �3(�2/�4)��5, and heteromeric channels can comprise sev-eral �7 or �9 subunits. The subunit composition of theACh-gated nAChR channels determines the uniquepattern of ion permeability and coupling to the down-stream signaling pathways (16). The repertoire ofnAChRs subtypes changes during keratinocyte differen-tiation in the epithelium (14, 15, 17). Incubation ofKCs at high extracellular concentrations of Ca2�, whichlaunches terminal differentiation of KCs, increases im-munostaining for the �7 subunit, indicating that theexpression of �7 nAChRs is differentiation dependent(18). In contrast, the �3-containing nAChRs arepresent at the earliest stages of keratinocyte develop-ment, suggesting that the �3-made nAChRs play amajor role in mediating the effects of nicotine at earlystages of keratinocyte development. Notably, normaldifferentiation of KCs is associated with sequentialexpression of the nAChR subtypes with increasing Ca2�

permeability (14, 17).The nAChR subtypes are differentially coupled to

specific sources of Ca2� (reviewed in ref. 19). Activa-tion of keratinocyte nAChRs elicits Ca2� influx thatvaries in magnitude between different nAChR subtypes.The addition of �5—an auxiliary subunit that formsfunctional ion channels only when coexpressed withboth � and � subunits—to the �3�2/�4 doublet sub-types modifies the pharmacological and biophysicalproperties and increases Ca2� permeability of thenAChR channels formed (20–22). Although both �3and �7 subunits can contribute to the nAChRs that arepermeable to Ca2�, the ACh-gated ion channels com-posed of the �7 subunits have the greatest Ca2� per-meability (23). Activation of the �7-containing nAChRscan increase intracellular Ca2� (24, 25). Nicotine hasbeen shown to elicit Ca2� mobilization via activation ofdistinct nAChR subtypes in neurons (26). Activation ofnAChRs induces a sustained elevation of intracellularCa2� levels, which is highly dependent on the activationof voltage-operated Ca2� channel, and also involvesCa2� release from ryanodine and IP3-dependent intra-cellular stores (27). We have reported that nicotineinduces elevation in cytosolic free Ca2� in epithelialcells (18, 28).

In epidermal KCs, the signaling pathways down-stream of �3�2 involve activation of the protein kinaseC (PKC) isoform �, whereas the signaling pathwaycoupled by �7 includes intracellular Ca2�, activation ofCa2�/calmodulin-dependent protein-kinase II (CaMKII),conventional isoforms of PKC, and phosphatidylinosi-tol-3-kinase (PI3K) (29). The signaling downstream of�7 evoked by nicotine or environmental tobacco smoke(ETS) in oral KCs involves the Ras/Raf-1/MEK1/ERKpathway leading to both transcriptional and transla-tional up-regulation of the transcription factor STAT-3and its transactivation due to JAK-2 phosphorylation(12). Agonists of �7 nAChR can also activate Akt, whichdepends on PI3K (30, 31).

We have reported previously that smoking alters both

the ligand-binding kinetics and the subunit composi-tion of nAChRs in epithelial cells, favoring overexpres-sion of the subunits that form the nAChR channelspermeable to Ca2� (28, 32). In epidermal KCs, chronicexposure to nicotine elicited a switch, wherein the �7subunit containing nAChRs replaced the �3-madenAChRs (33). This was not surprising, because whilethe majority of cellular receptors are down-regulated byagonists, chronic exposure to agonists of nAChRs isknown to result in a paradoxical up-regulation of theexpression of �7 and some other nAChR subunits (16,34, 35). In oral KCs, both ETS and pure nicotineproduced similar changes in the repertoire of �3-madenAChRs, favoring overexpression of �5-containing�3�2 nAChR channels (10). A switch in the nAChRsubunit composition was associated with alterations incell regulation and function, suggesting that agonist-dependent changes in the nAChR repertoire is a novelpathophysiological mechanism of nicotine toxicity inoral epithelium (10). Thus, results of previous studiesunequivocally demonstrated an important role of ETS/nicotine-induced alterations in the predominant sub-types of nAChRs expressed by oral KCs in mediatingtobacco-related morbidity in the upper digestive tract.The pathophysiological pathways, however, remainedto be elucidated.

This time-course, mechanistic study was designed toaddress the clinically important problem of receptor-mediated tobacco toxicity. We sought to establish theorder of overexpression of nAChR subunits in humanoral KCs exposed to ETS or equivalent concentration ofpure nicotine and identify the signaling pathways me-diating specific steps in the switches of nAChR sub-types. The results demonstrated that the pattern ofnicotine-induced changes of keratinocyte nAChRsmatches closely that observed during normal cell dif-ferentiation. While the levels of �3 subunit did notchange significantly, both ETS and nicotine up-regu-lated the levels of �5 and �7 expression in a time-dependent fashion, suggesting the following sequenceof autoregulation of the overexpression of nAChRsubtypes: �3(�2/�4) � �3(�2/�4)�5 � �7 � �7. The�3(�2/�4) to �3(�2/�4)�5 nAChR transition pre-dominantly involved PKC, �3(�2/�4)�5 to �7 nAChRtransition—CaMKII and p38 MAPK, and �7 self-up-regulation—the p38 MAPK/Akt pathway and JAK-2.The transcription factor GATA-2 played a key role inmediating self-up-regulation of �7. These results pro-vide a mechanistic insight into the genomic effects oftobacco products on oral KCs mediating autoregula-tion of stepwise overexpression of nAChR subtypes withincreasing Ca2� permeability.

MATERIALS AND METHODS

Chemicals and transfection reagents

The �7 antagonist �-bungarotoxin (�Btx), the �3 antagonistmecamylamine, and the agonist nicotine were purchased

1357NICOTINIC RECEPTOR-MEDIATED TOBACCO TOXICITY

from Sigma-Aldrich (St. Louis, MO, USA). The preferentialblocker of �3�2 nAChR �-conotoxins MII (�CtxMII), whichblocks its target receptors with an IC50 of 0.5 nM and othernAChR subunit combinations with 2–4 orders of magnitudeless potency (36), was synthesized by Advanced ChemTech(Louisville, KY, USA). The PKC inhibitor Go-6976; the non-competitive inhibitor of the Ras acceptor protein manumycinA (37); the cRaf-1 kinase inhibitor GW5074 [5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone] (38);the JAK-2 inhibitor AG 490; the Akt inhibitor VIII; thecell-permeable, potent, and selective inhibitor of MEK, “MEKinhibitor I” (39); a less specific MEK inhibitor, U0126; andthe inactive control U0124 (40) were from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). The cell-permeablechelator of intracellular free Ca2� 1,2-bis(2-aminophe-noxy)ethane-N,N,N�,N�-tetraacetic acid tetrakis(acetoxym-ethyl) ester (BAPTA/AM) and the selective inhibitor ofCaMKII KN-62 were from Axxora, LLC (San Diego, CA,USA). The specific inhibitor of p38� MAPK SB202190 waspurchased from Calbiochem-Novabiochem. The plasmids en-coding the constitutively active MEK1 (CA-MEK) with twopoint mutations (S218E and S222E) and a deletion of aminoacid residues 31–52; the dominant negative MEK1 mutant(DN-MEK), which contains three point mutations (K97R,S218A, and S222A) and thus could be phosphorylated neitherby its activators nor by its downstream effectors ERKs; and thewild-type control MEK1 (WT-MEK) were purchased fromBiomyx Technology (San Diego, CA, USA). The plasmidsencoding dominant negative p38 (DN-p38) and wild-type p38(WT-p38) were from Biomyx Technology. The small interfer-ing RNAs (siRNAs) against nAChR subunits and GATA familyproteins used in this study were designed and custom synthe-sized by Dharmacon (Lafayette, CO, USA). The negativecontrol siRNA (siRNA-NC) -targeting luciferase gene with thetarget sequence 5�-CGTACGCGGAATACTTCGA-3� that wasemployed in all RNA inhibition experiments was also pur-chased from Dharmacon.

Culturing and transfecting of oral KCs

Normal human KCs were obtained from attached gingiva.Samples of normal human attached gingiva were obtainedfrom periodontal surgical procedures. The samples weretransported to the laboratory in Minimum Essential Medium(MEM; Gibco BRL, Gaithersburg, MD, USA), freed of con-nective tissue and clotted blood, and washed in Ca2�- andMg2�-free phosphate-buffered saline (PBS; Gibco BRL). Theattached gingival samples were cut into 3- to 4-mm pieces andincubated overnight in a humidified atmosphere with 5%CO2 at 37°C in 0.06% trypsin (Sigma-Aldrich) in MEMsupplemented with 50 �g/ml gentamicin, 50 �g/ml kanamy-cin sulfate, 10 U/ml penicillin G, 10 �g/ml streptomycin, and5 �g/ml amphotericin (all from Gibco BRL). Individual KCswere isolated by gentle pipetting followed by centrifugationand were grown at 37°C and 5% CO2 in 25 cm2 or 75 cm2

Falcon culture flasks (Corning Glass Works, Corning, NY,USA) in serum-free keratinocyte growth medium (KGM;Gibco BRL) containing 0.09 mM Ca2� until use in experi-ments. For transfection with siRNAs, we followed the standardprotocol described in detail elsewhere (10). Briefly, KCs wereseeded at a density of 5 104 cells per well of a 24-well plateand incubated for 16–24 h to achieve 70% confluence. Toeach well, increasing concentrations of siRNA duplex in thetransfection solution with the TransIT-TKO transfection re-agent (Mirus, Madison, WI, USA) were added, and thetransfection was continued for 16 h at 37°C in a humid, 5%CO2 incubator. On the next day, the transfection medium wasreplaced by KGM, and the cells were incubated for 72 h toachieve maximum inhibition of the receptor protein expres-

sion, as was experimentally determined by Western blotanalysis at different time points after transfection. The siRNAtransfection efficiency was also assayed using FITC-labeledluciferase GL2 duplex (Dharmacon). The same generic pro-tocol of KCs transfection was used to express MEK1 kinasemutants. The efficacy of expression of MEK1 mutants wasassessed by Western blot analysis (data not shown).

Tobacco smoke/nicotine exposure experiments

Approximately 80% confluent monolayers of intact or trans-fected KCs in 6-well plates (Corning Glass Works) wereincubated for 24 h in the KGM pre-exposed to ETS as asurrogate of tobacco smoke or containing an equivalentconcentration of nicotine (10 �M). The KGM was exposed toETS in the chambers of a sidestream smoke exposure system(41). Briefly, a TE-10 smoking machine (Teague Enterprises,Davis, CA) burned 2RF4 reference cigarettes (Tobacco andHealth Research Institute, University of Kentucky), which hadbeen temperature and humidity conditioned to generateETS. Each 2RF4 cigarette delivers 0.8 mg of nicotine (41).Each cigarette was smoked under rigid conditions of 1 puff(35 ml volume for 2 s duration)/min over a period of 8 min.Daily measurements of total suspended particulates of nico-tine and carbon monoxide were performed. Mean concen-trations over the course of the study were 1 � 0.07 mg/m3 oftotal suspended particulates, 344 � 85 �g/m3 of nicotine,and 4.9 � 0.7 parts per million of carbon monoxide. Theexperiments were performed in triplicate for both exposedand control, nonexposed cultures, and the cells from eachculture were harvested and used in experiments separately. Ineach individual culture, 2.5 106 viable KCs were used toextract total RNA and proteins.

Real-time PCR assay

The assay was performed as described previously (10). Briefly,total RNA was extracted from cultured KCs at the end ofexposure experiments using the RNeasy® Mini Kit (Qiagen,Valencia, CA, USA) following the protocol provided by themanufacturer. Primers for the gene encoding human �5, �7,GATA-1, GATA-2, and GATA-3 were designed with the assis-tance of the Primer Express software version 2.0 computerprogram (Applied Biosystems, Foster City, CA, USA), and theservice Assays-on-Design provided by Applied Biosystems. Theamplification included a 2-min 50°C step required for opti-mal AmpErase UNG activity, an initial denaturation step for10 min at 95°C, followed by 40 cycles consisting of 15 s at 95°Cand 1 min at 60°C. Obtained gene expression values werenormalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to correct even minorvariations in mRNA extraction and reverse transcription. Thedata from triplicate samples were analyzed with a sequencedetector software (Applied Biosystems) and expressed asmean � sd of mRNA in question relative to that of GAPDH.

In-cell Western blot assay

In-cell Western blot assay (LI-COR Lincoln, NE, USA) wasperformed as described in detail elsewhere (42). The exper-imental and control cells were fixed, washed, permeabilizedwith Triton solution, incubated with the LI-COR OdysseyBlocking Buffer, and treated for 2 h with primary antibody to�5 or �7 (Research and Diagnostic Antibodies, North LasVegas, NV, USA) or GATA-1, GATA-2, or GATA-3 (Santa CruzBiotechnology, Santa Cruz, CA, USA). After that, the cellswere washed, stained with a secondary goat anti-rabbit AlexaFluor® 680 (1:5000 dilution; Molecular Probes, Eugene, OR,

1358 Vol. 22 May 2008 ARREDONDO ET AL.The FASEB Journal

USA). The protein expression was then quantitated using theOdyssey Imaging System (LI-COR).

Gel mobility shift assay

The assay was performed as described previously by us (12).Briefly, the nuclear extract obtained from experimental andcontrol KCs grown in 6-well plates was incubated with thedigoxigenin-labeled GATA oligonucleotide 5�-CAC TTG ATAACA GAA AGT GAT AAC TCT-3� (Santa Cruz Biotechnol-ogy) and 1 �g poly dI-dC for 20 min at room temperature ingel shift reaction buffer. The DNA-protein complexes wereresolved by electrophoresis through a 5% polyacrylamide gelcontaining 0.5 Tris, boric acid, EDTA and blotted overnightat 4°C in 1 sodium chloride/sodium citrate buffer onto thepositive charge nylon membranes, followed by UV crosslinking, and digoxigenin detection with anti-digoxigenin-APmonoclonal antibody and NBT/BCIP Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate toluidine salt as a sub-strate (all from Roche, Indianapolis, IN, USA). The presenceof GATA-2 in the DNA-protein complexes was confirmed inparallel immunoblotting experiments using anti-GATA-2 an-tibody.

Semiquantitative immunofluorescence assay

The semiquantitative assay of fluorescence intensity withexperimental and control KCs grown to confluence on glasscoverslips was performed as detailed previously (28, 43),using computer-assisted image analysis with a software pack-age purchased from Scanalytics (Fairfax, VA, USA). Theintensity of fluorescence was calculated pixel by pixel bydividing the summation of the fluorescence intensity of allpixels by the area occupied by the pixels (i.e., segment), andthen subtracting the mean intensity of fluorescence of acell-free segment (i.e., background). For each cell culturespecimen, a minimum of three different segments in at leastthree different microscopic fields were analyzed, and theresults were compared. To visualize membrane-associatednAChR subunits, the cells were fixed for 3 min with 3% freshdepolymerized paraformaldehyde that contained 7% sucrose,so as to avoid cell permeabilization. The fixed specimens were

washed and incubated overnight at 4°C with a primaryanti-nAChR subunit antibody (all from Research and Diag-nostic Antibodies). Binding of primary antibody was visual-ized by incubating the specimens for 1 h at room temperaturewith the appropriate secondary, FITC-conjugated goat anti-rabbit IgG antibody purchased from Pierce (Rockford, IL,USA). The specimens were examined with an Axiovert 135fluorescence microscope (Carl Zeiss, Thornwood, NY, USA).The specificity of antibody binding was demonstrated byomitting the primary antibody or by replacing primary anti-body with an irrelevant antibody of the same isotype andspecies as the primary antibody.

Statistical analysis

All experiments were performed in triplicate, and the resultswere expressed as mean � sd. Statistical significance wasdetermined using Student’s t test. Differences were deemedsignificant if the calculated P value was �0.05.

RESULTS

Sequential overexpression of the �5-containing �3nAChR and �7 nAChR in KCs exposed to ETS orpure nicotine

In a time-course study, we sought to establish the orderof changes in the nAChR subunit expression. Therelative amount of major nAChR subunits expressed onthe cell surfaces of exposed vs. control KCs was mea-sured by semiquantitative fluorescence assay. The ef-fects of ETS and nicotine were found to be similar.Although the levels of �3 subunit did not changesignificantly over the course of the exposure (P�0.05),both ETS and nicotine substantially affected the levelsof �5 and �7 expression (Fig. 1). The maximal degreeof up-regulation of �5, i.e., �4-fold, was observed at24 h, whereas that of �7 was observed at 96 h. The

Figure 1. Relative amounts of �3, �5, and �7 nAChR subunits in KCs exposed to ETS or pure nicotine for 24 or 96 h. The normalhuman gingival KCs grown to 90% confluence were exposed to ETS or 10 �M nicotine alone (1) or in combination with 50�M mecamylamine (2) or 1 �M �Btx (3) in KGM containing 0.09 mM Ca2� for 24 or 96 h at 37°C in a humid atmosphere with5% CO2, then fixed and stained with rabbit antibodies to �3 (A), �5 (B), or �7 (C) nAChR subunit. The relative intensity offluorescence was measured by the semiquantitative assay detailed in Materials and Methods. *Significant (P�0.05) differencescompared to the fluorescence of intact KCs (control) taken as 1. #Significant (P�0.05) differences compared to fluorescenceof KCs treated with ETS or nicotine alone.


effects of ETS and nicotine were abolished with differ-ent efficacies by the antagonists mecamylamine, a pref-erential blocker of the ganglionic nAChR subtypes,such as �3-made nAChRs (44), and �Btx, the specificinhibitor of the central subtype of neuronal nAChRs,such as �7-made channels (45). At these time points,up-regulation of �5 was blocked, in the most part, bymecamylamine, and that of �7, by �Btx (Fig. 1).

These results indicated that chronic exposure tonicotine-containing products leads to sequentialchanges in the repertoire of keratinocyte nAChRs, withthe appearance of �5-containing �3 nAChRs followedby �7 nAChRs, which suggested that different signalingmechanisms may operate downstream of each recep-tor’s subtype.

The signaling pathways mediating up-regulation of �5expression

The �3�2 nAChR subtype has been previously demon-strated to play a central role in mediating tobacco/nicotine toxicity in KCs (10). Therefore, we studied theeffect of the selective inhibitor of �3�2 nAChR�CtxMII on ETS and nicotine dependent up-regulationof �5. �CtxMII significantly (P�0.05) reduced up-regulation of the �5 subunit gene expression at boththe mRNA and protein levels (Fig. 2). A significant(P�0.05) decrease of �5 expression was also observed

in KCs transfected with siRNA-�3. The �7 blocker �Btxand siRNA-�7 produced only moderate inhibitory ef-fects (P�0.05), indicating that the major pathway me-diating �5 overexpression was coupled by �3-madechannels, such as �3�2 nAChR.

The use of pathway inhibitors showed that the alter-ations of �5 expression induced by ETS and nicotineinvolved predominantly PKC (P�0.05) and to a lesserextent CaMKII, Ras Raf, MEK, and p38 MAPK (P�0.05)(Fig. 2).

The signaling pathways mediating early up-regulationof �7 expression

The early events leading to up-regulated expression of�7 nAChR subunit were studied in KCs exposed to ETSor nicotine for 24 h. As expected from results ofimmunofluorescence assay (Fig. 1), the 24-h exposureto ETS or nicotine alone in both cases produces aseveralfold increase in the relative amounts of mRNAand protein of �7 nAChR subunit detected by real-timePCR and in-cell Western analysis, respectively (Fig. 3).Transfection of KCs with siRNA-�3 resulted in a signif-icantly (P�0.05) lower level of �7 mRNA or protein,compared to the high levels found in KCs incubatedwith ETS or nicotine given alone (Fig. 3). Functionalinhibition of the �3-made nAChRs containing �5 sub-unit, i.e., �3(�2/�4)�5, due to transfection with

Figure 2. Alterations in the �5 nAChR subunit gene expression in KCs exposed to ETS or pure nicotine for 24 h. Real time-PCR(A) and in-cell Western (B) analyses of ETS and nicotine effects on �5 gene expression in KCs. Total RNA and proteins wereisolated after 24-h exposures to ETS or 10 �M nicotine. The relative amounts of mRNA transcript and protein levels weremeasured as described in Materials and Methods. Some cells were transfected with receptor-specific or control siRNA as detailedin Materials and Methods. To standardize the analysis, the gene expression ratios in the control cells (i.e., intact KCs and KCstransfected with a nonspecific siRNA in experiments with siRNA-�3 and siRNA-�7) were taken as 1. The following experimentaltreatments were used: 100 �M �CtxMII, 1 �M �Btx, transfection with siRNA-�3 or siRNA-�7, 10 �M BAPTA/AM, 10 �M KN-62,1 �M Go-6976, 3 �M manumycin A (Mnmc), 0.1 �M GW5074, 1 �M MEK inhibitor I (MEK Inh), and 10 �M SB202190.Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n�3). *Significant(P�0.05) differences from control KCs. #Significant (P�0.05) differences from KCs exposed to either ETS or nicotine alone.


siRNA-�5 also reduced the degree of �7 overexpres-sion, albeit statistically insignificantly (P�0.05). Theup-regulation of �7 expression was significantly(P�0.05) reduced in the presence of the chelator ofintracellular free Ca2� BAPTA/AM, the inhibitor ofCaMKII KN-62, the p38 inhibitor SB202190, as well inKCs transfected with DN-p38, but not in the controlcells transfected with WT-p38 (Fig. 3).

These results indicated that regulation of the overex-pression of �7 nAChR caused by ETS/nicotine startsfrom activation of non-�5 �3 nAChRs, such as �3�2,and proceeds through the �5-containing nAChRs. Themajor intracellular biochemical events are apparentlyelicited by elevation of intracellular free Ca2� andinvolve CaMKII and p38 MAPK.

The signaling pathways mediating late up-regulationof �7 expression

To characterize signaling events leading to overexpres-sion of �7 due to chronic stimulation of KCs withtobacco products, the cells were exposed for 72 h toETS or nicotine alone, after which the incubation wascontinued for an additional 24 h in the presence ofnicotinic antagonists or pharmacologic inhibitors ofsignaling kinases. The significant (P�0.05) inhibitionof �7 expression was achieved in the presence of �Btx,whereas the inhibitory effect of �CtxMII did not reachsignificance (P�0.05) (Fig. 4). These results indicatedthat overexpression of �7 subunit in KCs chronicallyexposed to nicotine-containing products results chiefly

from an autoregulatory mechanism executed throughthe �7 receptor.

To characterize downstream signaling that couldmediate self-up-regulation of �7 nAChR in KCs ex-posed to ETS or nicotine, we used inhibitors of thepathways that had been previously shown to subservefunction of �7 nAChR in KCs (12, 29) and other typesof cells (30, 31, 46). The inhibitors of Ras, Raf, MEK,p38 MAPK, Akt, and JAK-2 produced significant(P�0.05) inhibition of ETS- or nicotine-dependentoverexpression of �7 at both mRNA and protein levels(Fig. 4).

The role of GATA-2 in mediating the late up-regulation of �7 expression

Although the exact mechanism of transcriptional reg-ulation of the �7 nAChR subunit remains to be fullyunderstood, it has been reported that several transcrip-tion factors, including Sp1, control expression of the�7 gene in rats (47), and that Sp1 interacts andcooperates with GATA family transcription factors toregulate expression of various genes in a variety of cellstypes (48–53). Therefore, we compared the effects ofgene silencing of GATA-1, -2 and -3 on expression ofthe �7 gene at the mRNA and protein levels in KCsexposed to ETS or nicotine for 96 h. The results of bothreal time-PCR and in-cell Western analyses of �7 geneexpression in KCs transfected with siRNA-GATA-1,siRNA-GATA-2, siRNA-GATA-3 revealed that knock-down of GATA-2 produced statistically significant(P�0.05) inhibition (Fig. 5).

Figure 3. Alterations in the �7 nAChR subunit gene expression in KCs exposed to ETS or pure nicotine for 24 h. Real time-PCR(A) and in-cell Western (B) analyses of ETS and nicotine effects on �7 gene expression in exposed KCs. Total RNA and proteinswere isolated after 24-h exposures to ETS or 10 �M nicotine. The relative amounts of mRNA transcript and protein levels weremeasured and analysis was performed as described in the caption to Fig. 2. The following experimental treatments were used:transfection with siRNA-�3 or siRNA-�5, 10 �M BAPTA/AM, 10 �M KN-62, 1 �M Go-6976, 3 �M Mnmc, 0.1 �M GW5074, 1 �MMEK Inh, 10 �M SB202190, and transfection with DN-p38 or WT-p38. *Significant (P�0.05) differences from control KCs.#Significant (P�0.05) differences from KCs exposed to either ETS or nicotine alone; n � 3.


Thus, GATA-2 was found to play an important role inmediating downstream signaling of �7 nAChR thatleads to self-up-regulation of this receptor.

The Ras/Raf-1/MEK1/ERK pathway activated by ETSand nicotine through �7 nAChR leads toup-regulation of GATA-2 expression

Since the Ras/Raf/MEK pathway had been shown inprevious studies to mediate the �7 signaling that con-trols expression/activity of transcription factors such asSTAT-3 and NF- B (11, 12), we sought to identify thepathway downstream of �7 nAChR that activatesGATA-2, thus allowing self-up-regulation of �7 in KCschronically exposed to ETS or nicotine. Both ETS andnicotine produced a severalfold increase in the mRNAand protein levels of GATA-2 (Fig. 6). Pretreatment ofKCs with �Btx or transfection with siRNA-�7 in bothcases significantly (P�0.05) reduced these effects. TheRas inhibitor manumycin A, and the cRaf-1 inhibitorGW5074 also abolished GATA-2 up-regulation. Neitherinhibitor, however, could affect the up-regulationcaused by transfection of KCs with CA-MEK (Fig. 6).Both MEK inhibitor I and U0126 blocked up-regulatedexpression of this transcription factor, indicating to anupstream involvement of the MEK1/ERK steps. Toconfirm involvement of MEK1/ERK steps, the KCs weretransfected with DN-MEK, which inhibited the effectsof ETS/nicotine. Cotransfection with siRNA-�7 andWT-MEK, but not CA-MEK, also abolished the ETS/nicotine effects on GATA-2 (Fig. 6).

These results indicated that ETS/nicotine-dependentself-up-regulation of �7 is regulated through the Ras/

Raf-1/MEK1/ERK pathway stimulating expression ofthe GATA-2 transcription factor.

Activation of �7 nAChR by ETS and nicotine elevatesthe transcriptional activity of GATA

Having found that GATA-2 is involved in the autoreg-ulation of overexpression of �7 nAChR in exposed KCs,we sought to demonstrate that activation of �7 nAChRwith ETS and nicotine leads to activation of the tran-scriptional activity of GATA-2. Using the gel mobilityshift assay, we measured the protein-binding activity ofGATA. Both ETS or nicotine caused transactivation ofthe transcription factor, which could be abolished bypretreating the KCs with �Btx or transfecting them withsiRNA-�7 (Fig. 7). In the DNA-protein complexes,GATA-2 was visualized by GATA-2 antibody (data notshown).

These results ultimately demonstrated involvementof the Ras/Raf-1/MEK1/ERK/GATA-2 signaling cas-cade in self-up-regulation of �7 in KCs chronicallyexposed to nicotine-containing products.

DISCUSSION

This study was designed to elucidate the mechanism ofnAChR-mediated toxicity of tobacco products on oralepithelium. The results demonstrated for the first timethat exposure of oral KCs to either ETS or purenicotine in both cases resulted in stepwise alterations inrepertoire of the keratinocyte nAChRs expressed onthe cell membrane. An overexpression of the �5-

Figure 4. Alterations in the �7 nAChR subunit gene expression in KCs exposed to ETS or pure nicotine for 96 h. Real time-PCR(A) and in-cell Western (B) analyses of �7 gene expression in KCs treated with ETS or 10 �M nicotine for 72 h and then exposedfor an additional 24 h in the presence of 100 �M �CtxMII, 1 �M �Btx, 1 �M Go-6976, 3 �M Mnmc, 0.1 �M GW5074, 1 �M MEKInh, 10 �M SB202190, 10 �M Akt inhibitor VIII (Akt Inh), or 10 �M AG-490. The relative amounts of mRNA transcript andprotein levels of �7 were measured as described in the caption to Fig. 2. *Significant (P�0.05) differences from control KCs.#Significant (P�0.05) differences from KCs exposed to either ETS or nicotine alone; n � 3.


containing �3 nAChRs was followed by that of �7nAChRs and involved different signaling mechanismsdownstream of each receptor subtype. The up-regula-tion of �5 was predominantly mediated by PKC; thetransition from �5-containing �3 nAChRs to �7nAChRs was elicited by elevation of intracellular freeCa2� and also involved CaMKII and p38 MAPK. Theself-up-regulation of �7 subunit depended on Akt and

JAK-2 and required transactivation of the GATA-2 tran-scription factor through the Ras/Raf-1/MEK1/ERKpathway. These observations have salient clinical impli-cations, because a switch in the nAChR subunit com-position can bring about a corresponding switch inreceptor function, leading to profound pathobiologiceffects observed by us (10–12, 32) and other workers(1–3, 54) in KCs exposed to tobacco products.

The switches in the structure and function ofnAChRs normally occur in the course of cell and tissuedevelopment (55, 56). In a variety of cells, differentia-tion is associated with up-regulation of �7 subunit,whereas expression of other subunits may remain un-changed (57, 58). In the stratified epithelium lining ofthe skin and oral mucosa, �7 is predominantly ex-pressed by differentiated KCs, in contrast to �5, whichis found in partially differentiated KCs, and �3 ispredominantly expressed in immature basal cells (14,17, 59). Predominant expression of �7 in mature KCsmaybe related to an important role the �7 nAChR playsin differentiation of the stratified squamous epithelium(60). Exposures to tobacco products also can alternAChR structure and function in both neuronal andnon-neuronal cells (10, 28, 32, 61, 62). Chronic admin-istration of nicotine increases the density of neuronalcholinergic nicotinic receptors in cells and in rodentbrains, and similar increases have been reported inbrains from human smokers (63). The effect of nico-tine can be reproduced by the agonist carbachol andsome other nicotinic ligands (64–66). Interestingly, �7is the principal nAChR subunit that was found to beup-regulated (67, 68). In this study, we observed thatstimulations of KCs with ETS/nicotine produce differ-ential effect on different nAChR subunits. While both�5 and �7 were up-regulated, �3 expression remainedunchanged. This is in keeping with an early report thatchronic nicotine exposure differentially affects thefunction of human neuronal nAChR subtypes (69).Chronic administration of nicotine even at high dosesdid not increase all nicotinic receptor subtypes, with �3subunit being particularly resistant to the nicotine-induced change (70). The effects of tobacco productson specific nAChR subunits, however, may vary fromone cell type to another (71–73). Some minor quanti-tative differences between the effects of ETS and nico-tine alone on autoregulation of nAChR subtype expres-sion and signaling pathways may be explained by thefact that tobacco smoke contains many bioactive ingre-dients other than nicotine that may potentially alter apattern of nAChR gene expression in KCs.

Similarly to transition from �3 to �7 nAChRs duringnormal keratinocyte differentiation in the epithelium,the ETS/nicotine-dependent switch from �3 to �7nAChRs in exposed KCs involved an intermediate stepwhen the cells overexpressed �5 containing nAChRs.Therefore, it appears that up-regulation of �7 nAChRstarts from activation of non-�5 �3 nAChRs, such as�3�2, and then proceeds through the �5-containingnAChRs. A transient expression of �5 mRNA has beenrecently demonstrated during cortical and hippocam-

Figure 5. Effects of gene silencing of GATA-1, -2, and -3 on theautoregulation of �7 overexpression. Real time-PCR (A) andin-cell Western (B) analyses of �7 gene expression in KCstransfected with siRNA-GATA-1, siRNA-GATA-2, siRNA-GATA-3, or normal control siRNA (siRNA-NC) and thentreated with ETS or 10 �M nicotine for 96 h. The relativeamounts of mRNA transcript and protein levels of �7 weremeasured as described in the caption to Fig. 2. *Significant(P�0.05) differences from control, nontransfected KCs. #Sig-nificant (P�0.05) differences from KCs exposed to eitherETS or nicotine alone; n � 3.


pal development (74). The �5 subunit is unique be-cause it modifies numerous characteristics of existingfunctional nAChRs, but it does not form functionalnAChRs when expressed alone or with � nicotinicsubunits (75). The function of �5 subunit is believed tobe limited to a modulatory role, altering numerous

characteristics of the nAChR channels formed (22, 76,77). An important role of �5 in the functional proper-ties on nAChRs has been documented in various celltypes, and it has been suggested that �5-containingnAChR are involved in the modulation of colonicinflammation (78). The coexpression of �5 with either

Figure 6. Effects of �7 inhibitors and Ras/Raf/MEK/ERK pathway modifiers on the expression of GATA-2 in KCs exposed toETS or pure nicotine. The real-time PCR (A, C) and in-cell Western (B, D) analyses of ETS (A, B) and nicotine (C, D) effectson the GATA-2 gene expression in human KCs incubated in the medium pre-exposed to ETS or containing 10 �M nicotine. Therelative amounts of mRNA transcript and protein levels of GATA-2 were measured as described in Materials and Methods. Thegene expression ratios in the control cells (i.e., intact KCs and KCs transfected with a control siRNA in experiments withsiRNA-�7) were taken as 1. The following experimental treatments were used: 3 �M Mnmc; 3 �M manumycin A on KCstransfected with CA-MEK (CA-MEK�Mnmc); 0.1 �M GW5074; 0.1 �M GW5074 on the CA-MEK transfected KCs (CA-MEK�GW5074); 1 �M MEK-Inh; 10 �M U0126; transfection with DN-MEK; transfection with the control, wild-type MEK mutant(WT-MEK); 1 �M �Btx; transfection with siRNA-�7; and cotransfection with siRNA-�7 and CA-MEK (CA-MEK�siRNA-�7),WT-MEK (WT-MEK�siRNA-�7), or DN-MEK (DN-MEK�siRNA-�7). *Significant (P�0.05) differences from intact control KCs.#Significant (P�0.05) differences from KCs exposed to either ETS or nicotine alone.


the �3�2 or �3�4 nAChR increases receptor desensiti-zation, increases Ca2� permeability, and specificallyincreases ACh sensitivity of the �3�2 nAChR (20),which may be responsible for the biological effect. It isnoteworthy that the early intracellular biochemicalevents that elicit �7 up-regulation include elevation ofintracellular free Ca2� and activation of CaMKII andp38 MAPK.

The results obtained in this study revealed thatdifferent signaling pathways mediate downstream sig-naling of distinct nAChR subtypes in KCs exposed toETS/nicotine. In other cell types, too, the mechanismsleading to up-regulation of �3, �5, and �7 nAChRs canbe different (35, 79). For instance, �3 and �7 nAChRsare modulated differently in hippocampal neurons andSH-SY5Y cells, with �3 being regulated by CaMKII and�7 also by L-type Ca2� channels (79). In KCs exposedto ETS/nicotine, the up-regulation of �5 expressionwas predominantly mediated by PKC and to a lesserextent by CaMKII and other signal transducers. Incontrast, the downstream signaling mediating self-up-regulation of �7 nAChR in exposed KCs involvedpreviously characterized �7 coupled pathway Ras/Raf/MEK/ERK, as well as Akt and JAK-2 (12, 30, 31, 80). Wealso found that signaling along the �7-coupled path-way increased expression of and activated the signaltransduction factor GATA-2. Other pathways also canbe involved. In a recent study, nicotine-induced up-regulation of human �7 receptors was potentiated bymodulation of cAMP and PKC (81). Apparently, pro-tein kinase A can be engaged as well (82). The differ-ences in the signaling pathways employed by eachkeratinocyte nAChR subtype observed in this study helpexplain a plethora of signaling mechanisms down-stream of nAChRs expressed in different cell types.

The results obtained in this study, taken togetherwith growing evidence of engagement of signalingkinases and phosphatases in the intracellular biochem-ical events mediating nicotinic effects of ACh in differ-

ent cells, suggest that nAChRs elicit downstream signal-ing not only due to modulation of cell membranepermeability to ions. It is well known that �7 forms areceptor or channel that has a high relative permeabil-ity to Ca2� (23, 83) and that activation of �7 nAChRleads to an increase in the concentration of freecytosolic Ca2� (24, 25). This may be due to activation ofCa2�-induced Ca2� release from intracellular stores,triggered by influx through both �7 channels andvoltage-gated Ca2� channels (84, 85). In addition, ourpilot studies have recently demonstrated that the mech-anism of action of �7 nAChR in KCs may include adirect activation or inactivation of signaling kinases andphosphatases (86). This is not surprising, because bothtyrosine kinases and phosphotyrosine phosphatases canassociate with �7 nAChR subunit and some othernAChR subunits in large multimeric complexes (87–91). It was proposed that Src-associated protein tyrosinephosphatases function in early signaling events emanat-ing from the nAChR that regulates cell function andthat the Src to phosphotyrosine phosphatase ratiodetermines the functional state of associated nAChR(89). The nAChR subunits �3-�5 and �2 exhibit apositive interaction with the G-protein subunits G(o)�and G�� (92). Therefore, we propose that coupling ofnAChR subunits to signaling kinases and/or phospha-tases represents a novel function of subunit proteinsthat mediate ETS/nicotine effects on epithelial cells.

In conclusion, chronic exposures to nicotine prod-ucts lead to sequential changes in the repertoire ofkeratinocyte nAChRs. This may contribute to pathobio-logic effects of tobacco products in the stratified epi-thelium lining the upper digestive tract, becausenAChRs are involved in a variety of cellular events,including malignant transformation (reviewed in ref.93). Future studies should be directed toward identifi-cation of the specific cellular activities regulated byeach major nAChR subtype expressed in the cell typestargeted by tobacco toxicity and elucidation of thespecific signaling pathways mediating biological effectsof nAChR in these cells.

We thank Orla Cagney, Katrina M. Arredondo, and PamelaSahourieh for excellent technical assistance. This work wassupported by the U.S. National Institutes of Health, grantsCA117327, DE14173, and ES014384, and a research grantfrom the Flight Attendant Medical Research Institute toS.A.G.

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Received for publication October 5, 2007.Accepted for publication November 8, 2007.


Receptor-mediated tobacco toxicity: acceleration of sequential expression of 5 and 7 nicotinic...

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