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Receptor-mediated tobacco toxicity: acceleration of sequential expression of 5 and 7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette 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 of Periodontics, School of Dentistry, University of California, Los Angeles, California, USA; and

Center for Health and the Environment, University of California, Davis, California, USA

¹Correspondence: Department of Dermatology, University of California, Irvine, C340 Medical Sciences I, Irvine, CA 92697, USA. E-mail: sgrando{at}uci.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca²⁺ influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca²⁺ permeability, such as 5-containing 3 nAChR and 7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the 5 and 7 expression in KCs, which could be abolished by mecamylamine and -bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: 3(β2/β4) > 3(β2/β4)5 > 7 > 7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the 3(β2/β4) to 3(β2/β4)5 nAChR transition predominantly involved protein kinase C, 3(β2/β4)5 to 7 nAChR transition—Ca²⁺/calmodulin-dependent protein kinase II and p38 MAPK, and 7 self-up-regulation—the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca²⁺ permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products. Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor-mediated tobacco toxicity: acceleration of sequential expression of 5 and 7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke.

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE COMPLEX PROCESS OF TOBACCO-induced morbidity in the oral cavity and esophagus includes direct effects of tobacco components on epithelial cells. Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) (1 2 3) and squamous cell carcinoma (4 5 6 7) . It has been estimated that in the year 2007, 34,360 new cases of cancer of the oral cavity and pharynx will occur in the United States (8) . Identification of the mechanisms leading to damage of KCs may prove useful in the prevention of tobacco toxicity. The continuous cycle of birth and death of KCs is a self-sustained process controlled, in part, by locally produced acetylcholine (ACh) through the signaling pathways that couple each type of ACh receptors to a particular cell function (reviewed in ref. 9 ). The nicotinic class of ACh receptors (nAChRs) plays important roles in regulation of growth and differentiation of oral KCs and can mediate pathobiologic effects of the smokeless tobacco and nicotine derivatives on these cells (10 11 12 13) . The nAChRs may, therefore, provide a novel molecular target to prevent, reverse, or ameliorate progression of the tobacco-related cell damage and intercede in disease pathways.

Previous studies have documented that nAChRs in oral KCs can comprise the 3, 5, 7, 9, β2, and β4 subunits (14 , 15) . The heteromeric channels can be composed of 3, 5, β2, and β4 subunits, e.g., 3(β2/β4)±5, and heteromeric channels can comprise several 7 or 9 subunits. The subunit composition of the ACh-gated nAChR channels determines the unique pattern of ion permeability and coupling to the downstream signaling pathways (16) . The repertoire of nAChRs subtypes changes during keratinocyte differentiation in the epithelium (14 , 15 , 17) . Incubation of KCs at high extracellular concentrations of Ca²⁺, which launches terminal differentiation of KCs, increases immunostaining for the 7 subunit, indicating that the expression of 7 nAChRs is differentiation dependent (18) . In contrast, the 3-containing nAChRs are present at the earliest stages of keratinocyte development, suggesting that the 3-made nAChRs play a major role in mediating the effects of nicotine at early stages of keratinocyte development. Notably, normal differentiation of KCs is associated with sequential expression 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 ). Activation of keratinocyte nAChRs elicits Ca²⁺ influx that varies in magnitude between different nAChR subtypes. The addition of 5—an auxiliary subunit that forms functional ion channels only when coexpressed with both and β subunits—to the 3β2/β4 doublet subtypes modifies the pharmacological and biophysical properties and increases Ca²⁺ permeability of the nAChR channels formed (20 21 22) . Although both 3 and 7 subunits can contribute to the nAChRs that are permeable to Ca²⁺, the ACh-gated ion channels composed of the 7 subunits have the greatest Ca²⁺ permeability (23) . Activation of the 7-containing nAChRs can increase intracellular Ca²⁺ (24 , 25) . Nicotine has been shown to elicit Ca²⁺ mobilization via activation of distinct nAChR subtypes in neurons (26) . Activation of nAChRs induces a sustained elevation of intracellular Ca²⁺ levels, which is highly dependent on the activation of voltage-operated Ca²⁺ channel, and also involves Ca²⁺ release from ryanodine and IP₃-dependent intracellular stores (27) . We have reported that nicotine induces elevation in cytosolic free Ca²⁺ in epithelial cells (18 , 28) .

In epidermal KCs, the signaling pathways downstream of 3β2 involve activation of the protein kinase C (PKC) isoform , whereas the signaling pathway coupled by 7 includes intracellular Ca²⁺, activation of Ca²⁺/calmodulin-dependent protein-kinase II (CaMKII), conventional isoforms of PKC, and phosphatidylinositol-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/ERK pathway leading to both transcriptional and translational up-regulation of the transcription factor STAT-3 and its transactivation due to JAK-2 phosphorylation (12) . Agonists of 7 nAChR can also activate Akt, which depends on PI3K (30 , 31) .

We have reported previously that smoking alters both the ligand-binding kinetics and the subunit composition of nAChRs in epithelial cells, favoring overexpression of the subunits that form the nAChR channels permeable to Ca²⁺ (28 , 32) . In epidermal KCs, chronic exposure to nicotine elicited a switch, wherein the 7 subunit containing nAChRs replaced the 3-made nAChRs (33) . This was not surprising, because while the majority of cellular receptors are down-regulated by agonists, chronic exposure to agonists of nAChRs is known to result in a paradoxical up-regulation of the expression of 7 and some other nAChR subunits (16 , 34 , 35) . In oral KCs, both ETS and pure nicotine produced similar changes in the repertoire of 3-made nAChRs, favoring overexpression of 5-containing 3β2 nAChR channels (10) . A switch in the nAChR subunit composition was associated with alterations in cell regulation and function, suggesting that agonist-dependent changes in the nAChR repertoire is a novel pathophysiological mechanism of nicotine toxicity in oral epithelium (10) . Thus, results of previous studies unequivocally demonstrated an important role of ETS/nicotine-induced alterations in the predominant subtypes of nAChRs expressed by oral KCs in mediating tobacco-related morbidity in the upper digestive tract. The pathophysiological pathways, however, remained to be elucidated.

This time-course, mechanistic study was designed to address the clinically important problem of receptor-mediated tobacco toxicity. We sought to establish the order of overexpression of nAChR subunits in human oral KCs exposed to ETS or equivalent concentration of pure nicotine and identify the signaling pathways mediating specific steps in the switches of nAChR subtypes. The results demonstrated that the pattern of nicotine-induced changes of keratinocyte nAChRs matches closely that observed during normal cell differentiation. While the levels of 3 subunit did not change significantly, both ETS and nicotine up-regulated the levels of 5 and 7 expression in a time-dependent fashion, suggesting the following sequence of autoregulation of the overexpression of nAChR subtypes: 3(β2/β4) > 3(β2/β4)5 > 7 > 7. The 3(β2/β4) to 3(β2/β4)5 nAChR transition predominantly involved PKC, 3(β2/β4)5 to 7 nAChR transition—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 in mediating self-up-regulation of 7. These results provide a mechanistic insight into the genomic effects of tobacco products on oral KCs mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca²⁺ permeability.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals and transfection reagents
The 7 antagonist -bungarotoxin (Btx), the 3 antagonist mecamylamine, and the agonist nicotine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The preferential blocker of 3β2 nAChR -conotoxins MII (CtxMII), which blocks its target receptors with an IC₅₀ of 0.5 nM and other nAChR subunit combinations with 2–4 orders of magnitude less potency (36) , was synthesized by Advanced ChemTech (Louisville, KY, USA). The PKC inhibitor Go-6976; the noncompetitive inhibitor of the Ras acceptor protein manumycin A (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; the cell-permeable, potent, and selective inhibitor of MEK, "MEK inhibitor I" (39) ; a less specific MEK inhibitor, U0126; and the inactive control U0124 (40) were from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). The cell-permeable chelator of intracellular free Ca²⁺ 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM) and the selective inhibitor of CaMKII KN-62 were from Axxora, LLC (San Diego, CA, USA). The specific inhibitor of p38 MAPK SB202190 was purchased from Calbiochem-Novabiochem. The plasmids encoding the constitutively active MEK1 (CA-MEK) with two point mutations (S218E and S222E) and a deletion of amino acid residues 31–52; the dominant negative MEK1 mutant (DN-MEK), which contains three point mutations (K97R, S218A, and S222A) and thus could be phosphorylated neither by its activators nor by its downstream effectors ERKs; and the wild-type control MEK1 (WT-MEK) were purchased from Biomyx Technology (San Diego, CA, USA). The plasmids encoding dominant negative p38 (DN-p38) and wild-type p38 (WT-p38) were from Biomyx Technology. The small interfering RNAs (siRNAs) against nAChR subunits and GATA family proteins used in this study were designed and custom synthesized by Dharmacon (Lafayette, CO, USA). The negative control siRNA (siRNA-NC) -targeting luciferase gene with the target sequence 5'-CGTACGCGGAATACTTCGA-3' that was employed in all RNA inhibition experiments was also purchased from Dharmacon.

Culturing and transfecting of oral KCs
Normal human KCs were obtained from attached gingiva. Samples of normal human attached gingiva were obtained from periodontal surgical procedures. The samples were transported to the laboratory in Minimum Essential Medium (MEM; Gibco BRL, Gaithersburg, MD, USA), freed of connective tissue and clotted blood, and washed in Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS; Gibco BRL). The attached gingival samples were cut into 3- to 4-mm pieces and incubated overnight in a humidified atmosphere with 5% CO₂ at 37°C in 0.06% trypsin (Sigma-Aldrich) in MEM supplemented with 50 µg/ml gentamicin, 50 µg/ml kanamycin sulfate, 10 U/ml penicillin G, 10 µg/ml streptomycin, and 5 µg/ml amphotericin (all from Gibco BRL). Individual KCs were isolated by gentle pipetting followed by centrifugation and 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 experiments. For transfection with siRNAs, we followed the standard protocol described in detail elsewhere (10) . Briefly, KCs were seeded at a density of 5 x 10⁴ cells per well of a 24-well plate and incubated for 16–24 h to achieve 70% confluence. To each well, increasing concentrations of siRNA duplex in the transfection solution with the TransIT-TKO transfection reagent (Mirus, Madison, WI, USA) were added, and the transfection was continued for 16 h at 37°C in a humid, 5% CO₂ incubator. On the next day, the transfection medium was replaced by KGM, and the cells were incubated for 72 h to achieve maximum inhibition of the receptor protein expression, as was experimentally determined by Western blot analysis at different time points after transfection. The siRNA transfection efficiency was also assayed using FITC-labeled luciferase GL2 duplex (Dharmacon). The same generic protocol of KCs transfection was used to express MEK1 kinase mutants. The efficacy of expression of MEK1 mutants was assessed by Western blot analysis (data not shown).

Tobacco smoke/nicotine exposure experiments
Approximately 80% confluent monolayers of intact or transfected KCs in 6-well plates (Corning Glass Works) were incubated for 24 h in the KGM pre-exposed to ETS as a surrogate of tobacco smoke or containing an equivalent concentration of nicotine (10 µM). The KGM was exposed to ETS 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 and Health Research Institute, University of Kentucky), which had been temperature and humidity conditioned to generate ETS. 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 nicotine and carbon monoxide were performed. Mean concentrations over the course of the study were 1 ± 0.07 mg/m³ of total suspended particulates, 344 ± 85 µg/m³ of nicotine, and 4.9 ± 0.7 parts per million of carbon monoxide. The experiments were performed in triplicate for both exposed and control, nonexposed cultures, and the cells from each culture were harvested and used in experiments separately. In each individual culture, 2.5 x 10⁶ viable KCs were used to extract 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 of exposure experiments using the RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) following the protocol provided by the manufacturer. Primers for the gene encoding human 5, 7, GATA-1, GATA-2, and GATA-3 were designed with the assistance of the Primer Express software version 2.0 computer program (Applied Biosystems, Foster City, CA, USA), and the service Assays-on-Design provided by Applied Biosystems. The amplification included a 2-min 50°C step required for optimal AmpErase UNG activity, an initial denaturation step for 10 min at 95°C, followed by 40 cycles consisting of 15 s at 95°C and 1 min at 60°C. Obtained gene expression values were normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to correct even minor variations in mRNA extraction and reverse transcription. The data from triplicate samples were analyzed with a sequence detector software (Applied Biosystems) and expressed as mean ± 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) was performed as described in detail elsewhere (42) . The experimental and control cells were fixed, washed, permeabilized with Triton solution, incubated with the LI-COR Odyssey Blocking Buffer, and treated for 2 h with primary antibody to 5 or 7 (Research and Diagnostic Antibodies, North Las Vegas, NV, USA) or GATA-1, GATA-2, or GATA-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After that, the cells were washed, stained with a secondary goat anti-rabbit Alexa Fluor® 680 (1:5000 dilution; Molecular Probes, Eugene, OR, USA). The protein expression was then quantitated using the Odyssey 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 and control KCs grown in 6-well plates was incubated with the digoxigenin-labeled GATA oligonucleotide 5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3' (Santa Cruz Biotechnology) and 1 µg poly dI-dC for 20 min at room temperature in gel shift reaction buffer. The DNA-protein complexes were resolved by electrophoresis through a 5% polyacrylamide gel containing 0.5x Tris, boric acid, EDTA and blotted overnight at 4°C in 1x sodium chloride/sodium citrate buffer onto the positive charge nylon membranes, followed by UV cross linking, and digoxigenin detection with anti-digoxigenin-AP monoclonal antibody and NBT/BCIP Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate toluidine salt as a substrate (all from Roche, Indianapolis, IN, USA). The presence of GATA-2 in the DNA-protein complexes was confirmed in parallel immunoblotting experiments using anti-GATA-2 antibody.

Semiquantitative immunofluorescence assay
The semiquantitative assay of fluorescence intensity with experimental and control KCs grown to confluence on glass coverslips was performed as detailed previously (28 , 43) , using computer-assisted image analysis with a software package purchased from Scanalytics (Fairfax, VA, USA). The intensity of fluorescence was calculated pixel by pixel by dividing the summation of the fluorescence intensity of all pixels by the area occupied by the pixels (i.e., segment), and then subtracting the mean intensity of fluorescence of a cell-free segment (i.e., background). For each cell culture specimen, a minimum of three different segments in at least three different microscopic fields were analyzed, and the results were compared. To visualize membrane-associated nAChR subunits, the cells were fixed for 3 min with 3% fresh depolymerized paraformaldehyde that contained 7% sucrose, so as to avoid cell permeabilization. The fixed specimens were washed and incubated overnight at 4°C with a primary anti-nAChR subunit antibody (all from Research and Diagnostic Antibodies). Binding of primary antibody was visualized by incubating the specimens for 1 h at room temperature with the appropriate secondary, FITC-conjugated goat anti-rabbit IgG antibody purchased from Pierce (Rockford, IL, USA). The specimens were examined with an Axiovert 135 fluorescence microscope (Carl Zeiss, Thornwood, NY, USA). The specificity of antibody binding was demonstrated by omitting the primary antibody or by replacing primary antibody with an irrelevant antibody of the same isotype and species as the primary antibody.

Statistical analysis
All experiments were performed in triplicate, and the results were expressed as mean ± SD. Statistical significance was determined using Student’s t test. Differences were deemed significant if the calculated P value was <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sequential overexpression of the 5-containing 3 nAChR and 7 nAChR in KCs exposed to ETS or pure nicotine
In a time-course study, we sought to establish the order of changes in the nAChR subunit expression. The relative amount of major nAChR subunits expressed on the cell surfaces of exposed vs. control KCs was measured by semiquantitative fluorescence assay. The effects of ETS and nicotine were found to be similar. Although the levels of 3 subunit did not change significantly over the course of the exposure (P>0.05), both ETS and nicotine substantially affected the levels of 5 and 7 expression (Fig. 1 ). The maximal degree of up-regulation of 5, i.e., >4-fold, was observed at 24 h, whereas that of 7 was observed at 96 h. The effects of ETS and nicotine were abolished with different efficacies by the antagonists mecamylamine, a preferential blocker of the ganglionic nAChR subtypes, such as 3-made nAChRs (44) , and Btx, the specific inhibitor 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, by mecamylamine, and that of 7, by Btx (Fig. 1) .

View larger version (12K): [in this window] [in a new window]   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 normal human gingival KCs grown to 90% confluence were exposed to ETS or 10 µM nicotine alone (1) or in combination with 50 µM mecamylamine (2) or 1 µM Btx (3) in KGM containing 0.09 mM Ca2+ for 24 or 96 h at 37°C in a humid atmosphere with 5% CO2, then fixed and stained with rabbit antibodies to 3 (A), 5 (B), or 7 (C) nAChR subunit. The relative intensity of fluorescence was measured by the semiquantitative assay detailed in Materials and Methods. *Significant (P<0.05) differences compared to the fluorescence of intact KCs (control) taken as 1. #Significant (P<0.05) differences compared to fluorescence of KCs treated with ETS or nicotine alone.

These results indicated that chronic exposure to nicotine-containing products leads to sequential changes in the repertoire of keratinocyte nAChRs, with the appearance of 5-containing 3 nAChRs followed by 7 nAChRs, which suggested that different signaling mechanisms may operate downstream of each receptor’s subtype.

The signaling pathways mediating up-regulation of 5 expression
The 3β2 nAChR subtype has been previously demonstrated to play a central role in mediating tobacco/nicotine toxicity in KCs (10) . Therefore, we studied the effect of the selective inhibitor of 3β2 nAChR CtxMII on ETS and nicotine dependent up-regulation of 5. CtxMII significantly (P<0.05) reduced up-regulation of the 5 subunit gene expression at both the 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 Btx and siRNA-7 produced only moderate inhibitory effects (P>0.05), indicating that the major pathway mediating 5 overexpression was coupled by 3-made channels, such as 3β2 nAChR.

View larger version (21K): [in this window] [in a new window]   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 were isolated after 24-h exposures to ETS or 10 µM nicotine. The relative amounts of mRNA transcript and protein levels were measured as described in Materials and Methods. Some cells were transfected with receptor-specific or control siRNA as detailed in Materials and Methods. To standardize the analysis, the gene expression ratios in the control cells (i.e., intact KCs and KCs transfected with a nonspecific siRNA in experiments with siRNA-3 and siRNA-7) were taken as 1. The following experimental treatments were used: 100 µM CtxMII, 1 µM Btx, transfection with siRNA-3 or siRNA-7, 10 µM BAPTA/AM, 10 µM KN-62, 1 µM Gö-6976, 3 µM manumycin A (Mnmc), 0.1 µM GW5074, 1 µM MEK inhibitor I (MEK Inh), and 10 µM 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.

The use of pathway inhibitors showed that the alterations of 5 expression induced by ETS and nicotine involved predominantly PKC (P<0.05) and to a lesser extent CaMKII, Ras Raf, MEK, and p38 MAPK (P>0.05) (Fig. 2) .

The signaling pathways mediating early up-regulation of 7 expression
The early events leading to up-regulated expression of 7 nAChR subunit were studied in KCs exposed to ETS or nicotine for 24 h. As expected from results of immunofluorescence assay (Fig. 1) , the 24-h exposure to ETS or nicotine alone in both cases produces a severalfold increase in the relative amounts of mRNA and protein of 7 nAChR subunit detected by real-time PCR and in-cell Western analysis, respectively (Fig. 3 ). Transfection of KCs with siRNA-3 resulted in a significantly (P<0.05) lower level of 7 mRNA or protein, compared to the high levels found in KCs incubated with ETS or nicotine given alone (Fig. 3) . Functional inhibition of the 3-made nAChRs containing 5 subunit, i.e., 3(β2/β4)5, due to transfection with siRNA-5 also reduced the degree of 7 overexpression, albeit statistically insignificantly (P>0.05). The up-regulation of 7 expression was significantly (P<0.05) reduced in the presence of the chelator of intracellular free Ca²⁺ BAPTA/AM, the inhibitor of CaMKII KN-62, the p38 inhibitor SB202190, as well in KCs transfected with DN-p38, but not in the control cells transfected with WT-p38 (Fig. 3) .

View larger version (21K): [in this window] [in a new window]   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 proteins were isolated after 24-h exposures to ETS or 10 µM nicotine. The relative amounts of mRNA transcript and protein levels were measured 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 µM BAPTA/AM, 10 µM KN-62, 1 µM Gö-6976, 3 µM Mnmc, 0.1 µM GW5074, 1 µM MEK Inh, 10 µM 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.

These results indicated that regulation of the overexpression of 7 nAChR caused by ETS/nicotine starts from activation of non-5 3 nAChRs, such as 3β2, and proceeds through the 5-containing nAChRs. The major intracellular biochemical events are apparently elicited by elevation of intracellular free Ca²⁺ and involve CaMKII and p38 MAPK.

The signaling pathways mediating late up-regulation of 7 expression
To characterize signaling events leading to overexpression of 7 due to chronic stimulation of KCs with tobacco products, the cells were exposed for 72 h to ETS or nicotine alone, after which the incubation was continued for an additional 24 h in the presence of nicotinic antagonists or pharmacologic inhibitors of signaling kinases. The significant (P<0.05) inhibition of 7 expression was achieved in the presence of Btx, whereas the inhibitory effect of CtxMII did not reach significance (P>0.05) (Fig. 4 ). These results indicated that overexpression of 7 subunit in KCs chronically exposed to nicotine-containing products results chiefly from an autoregulatory mechanism executed through the 7 receptor.

View larger version (16K): [in this window] [in a new window]   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 µM nicotine for 72 h and then exposed for an additional 24 h in the presence of 100 µM CtxMII, 1 µM Btx, 1 µM Gö-6976, 3 µM Mnmc, 0.1 µM GW5074, 1 µM MEK Inh, 10 µM SB202190, 10 µM Akt inhibitor VIII (Akt Inh), or 10 µM AG-490. The relative amounts of mRNA transcript and protein 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.

To characterize downstream signaling that could mediate self-up-regulation of 7 nAChR in KCs exposed to ETS or nicotine, we used inhibitors of the pathways that had been previously shown to subserve function of 7 nAChR in KCs (12 , 29) and other types of 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-dependent overexpression 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 regulation of the 7 nAChR subunit remains to be fully understood, it has been reported that several transcription factors, including Sp1, control expression of the 7 gene in rats (47) , and that Sp1 interacts and cooperates with GATA family transcription factors to regulate expression of various genes in a variety of cells types (48 49 50 51 52 53) . Therefore, we compared the effects of gene silencing of GATA-1, -2 and -3 on expression of the 7 gene at the mRNA and protein levels in KCs exposed to ETS or nicotine for 96 h. The results of both real time-PCR and in-cell Western analyses of 7 gene expression in KCs transfected with siRNA-GATA-1, siRNA-GATA-2, siRNA-GATA-3 revealed that knockdown of GATA-2 produced statistically significant (P<0.05) inhibition (Fig. 5 ).

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects of gene silencing of GATA-1, -2, and -3 on the autoregulation of 7 overexpression. Real time-PCR (A) and in-cell Western (B) analyses of 7 gene expression in KCs transfected with siRNA-GATA-1, siRNA-GATA-2, siRNA-GATA-3, or normal control siRNA (siRNA-NC) and then treated with ETS or 10 µM nicotine for 96 h. The relative amounts of mRNA transcript and protein levels of 7 were measured as described in the caption to Fig. 2 . *Significant (P<0.05) differences from control, nontransfected 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 in mediating downstream signaling of 7 nAChR that leads to self-up-regulation of this receptor.

The Ras/Raf-1/MEK1/ERK pathway activated by ETS and nicotine through 7 nAChR leads to up-regulation of GATA-2 expression
Since the Ras/Raf/MEK pathway had been shown in previous studies to mediate the 7 signaling that controls expression/activity of transcription factors such as STAT-3 and NF-B (11 , 12) , we sought to identify the pathway downstream of 7 nAChR that activates GATA-2, thus allowing self-up-regulation of 7 in KCs chronically exposed to ETS or nicotine. Both ETS and nicotine produced a severalfold increase in the mRNA and protein levels of GATA-2 (Fig. 6 ). Pretreatment of KCs with Btx or transfection with siRNA-7 in both cases significantly (P<0.05) reduced these effects. The Ras inhibitor manumycin A, and the cRaf-1 inhibitor GW5074 also abolished GATA-2 up-regulation. Neither inhibitor, however, could affect the up-regulation caused by transfection of KCs with CA-MEK (Fig. 6) . Both MEK inhibitor I and U0126 blocked up-regulated expression of this transcription factor, indicating to an upstream involvement of the MEK1/ERK steps. To confirm involvement of MEK1/ERK steps, the KCs were transfected with DN-MEK, which inhibited the effects of ETS/nicotine. Cotransfection with siRNA-7 and WT-MEK, but not CA-MEK, also abolished the ETS/nicotine effects on GATA-2 (Fig. 6) .

View larger version (72K): [in this window] [in a new window]   Figure 6. Effects of 7 inhibitors and Ras/Raf/MEK/ERK pathway modifiers on the expression of GATA-2 in KCs exposed to ETS or pure nicotine. The real-time PCR (A, C) and in-cell Western (B, D) analyses of ETS (A, B) and nicotine (C, D) effects on the GATA-2 gene expression in human KCs incubated in the medium pre-exposed to ETS or containing 10 µM nicotine. The relative amounts of mRNA transcript and protein levels of GATA-2 were measured as described in Materials and Methods. The gene expression ratios in the control cells (i.e., intact KCs and KCs transfected with a control siRNA in experiments with siRNA-7) were taken as 1. The following experimental treatments were used: 3 µM Mnmc; 3 µM manumycin A on KCs transfected with CA-MEK (CA-MEK+Mnmc); 0.1 µM GW5074; 0.1 µM GW5074 on the CA-MEK transfected KCs (CA-MEK+GW5074); 1 µM MEK-Inh; 10 µM U0126; transfection with DN-MEK; transfection with the control, wild-type MEK mutant (WT-MEK); 1 µM 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.

These results indicated that ETS/nicotine-dependent self-up-regulation of 7 is regulated through the Ras/Raf-1/MEK1/ERK pathway stimulating expression of the GATA-2 transcription factor.

Activation of 7 nAChR by ETS and nicotine elevates the transcriptional activity of GATA
Having found that GATA-2 is involved in the autoregulation of overexpression of 7 nAChR in exposed KCs, we sought to demonstrate that activation of 7 nAChR with ETS and nicotine leads to activation of the transcriptional activity of GATA-2. Using the gel mobility shift assay, we measured the protein-binding activity of GATA. Both ETS or nicotine caused transactivation of the transcription factor, which could be abolished by pretreating the KCs with Btx or transfecting them with siRNA-7 (Fig. 7 ). In the DNA-protein complexes, GATA-2 was visualized by GATA-2 antibody (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 7. Regulation of GATA activity. The KCs were treated with ETS or pure nicotine either in the presence of 1 µM Btx or after transfection with siRNA-7, and then subjected to functional analysis of GATA. The electrophoretic mobility shift assay was used to detect the DNA protein complex in the nuclear extracts prepared from experimental and control KCs, as detailed in Material and Methods. Lane 1: nicotine; lane 2: ETS; lane 3: nicotine + Btx; lane 4: ETS + Btx; lane 5: nicotine + siRNA-7; lane 6: ETS + siRNA-7; lane 7: untreated control cells.

These results ultimately demonstrated involvement of the Ras/Raf-1/MEK1/ERK/GATA-2 signaling cascade in self-up-regulation of 7 in KCs chronically exposed to nicotine-containing products.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study was designed to elucidate the mechanism of nAChR-mediated toxicity of tobacco products on oral epithelium. The results demonstrated for the first time that exposure of oral KCs to either ETS or pure nicotine in both cases resulted in stepwise alterations in repertoire of the keratinocyte nAChRs expressed on the cell membrane. An overexpression of the 5-containing 3 nAChRs was followed by that of 7 nAChRs and involved different signaling mechanisms downstream of each receptor subtype. The up-regulation of 5 was predominantly mediated by PKC; the transition from 5-containing 3 nAChRs to 7 nAChRs was elicited by elevation of intracellular free Ca²⁺ and also involved CaMKII and p38 MAPK. The self-up-regulation of 7 subunit depended on Akt and JAK-2 and required transactivation of the GATA-2 transcription factor through the Ras/Raf-1/MEK1/ERK pathway. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed by us (10 11 12 , 32) and other workers (1 2 3 , 54) in KCs exposed to tobacco products.

The switches in the structure and function of nAChRs normally occur in the course of cell and tissue development (55 , 56) . In a variety of cells, differentiation is associated with up-regulation of 7 subunit, whereas expression of other subunits may remain unchanged (57 , 58) . In the stratified epithelium lining of the skin and oral mucosa, 7 is predominantly expressed by differentiated KCs, in contrast to 5, which is found in partially differentiated KCs, and 3 is predominantly expressed in immature basal cells (14 , 17 , 59) . Predominant expression of 7 in mature KCs maybe related to an important role the 7 nAChR plays in differentiation of the stratified squamous epithelium (60) . Exposures to tobacco products also can alter nAChR structure and function in both neuronal and non-neuronal cells (10 , 28 , 32 , 61 , 62) . Chronic administration of nicotine increases the density of neuronal cholinergic nicotinic receptors in cells and in rodent brains, and similar increases have been reported in brains from human smokers (63) . The effect of nicotine can be reproduced by the agonist carbachol and some other nicotinic ligands (64 65 66) . Interestingly, 7 is the principal nAChR subunit that was found to be up-regulated (67 , 68) . In this study, we observed that stimulations of KCs with ETS/nicotine produce differential effect on different nAChR subunits. While both 5 and 7 were up-regulated, 3 expression remained unchanged. This is in keeping with an early report that chronic nicotine exposure differentially affects the function of human neuronal nAChR subtypes (69) . Chronic administration of nicotine even at high doses did not increase all nicotinic receptor subtypes, with 3 subunit being particularly resistant to the nicotine-induced change (70) . The effects of tobacco products on specific nAChR subunits, however, may vary from one cell type to another (71 72 73) . Some minor quantitative differences between the effects of ETS and nicotine alone on autoregulation of nAChR subtype expression and signaling pathways may be explained by the fact that tobacco smoke contains many bioactive ingredients other than nicotine that may potentially alter a pattern of nAChR gene expression in KCs.

Similarly to transition from 3 to 7 nAChRs during normal keratinocyte differentiation in the epithelium, the ETS/nicotine-dependent switch from 3 to 7 nAChRs in exposed KCs involved an intermediate step when the cells overexpressed 5 containing nAChRs. Therefore, it appears that up-regulation of 7 nAChR starts from activation of non-5 3 nAChRs, such as 3β2, and then proceeds through the 5-containing nAChRs. A transient expression of 5 mRNA has been recently demonstrated during cortical and hippocampal development (74) . The 5 subunit is unique because it modifies numerous characteristics of existing functional nAChRs, but it does not form functional nAChRs when expressed alone or with β nicotinic subunits (75) . The function of 5 subunit is believed to be limited to a modulatory role, altering numerous characteristics of the nAChR channels formed (22 , 76 , 77) . An important role of 5 in the functional properties on nAChRs has been documented in various cell types, and it has been suggested that 5-containing nAChR are involved in the modulation of colonic inflammation (78) . The coexpression of 5 with either the 3β2 or 3β4 nAChR increases receptor desensitization, increases Ca²⁺ permeability, and specifically increases ACh sensitivity of the 3β2 nAChR (20) , which may be responsible for the biological effect. It is noteworthy that the early intracellular biochemical events that elicit 7 up-regulation include elevation of intracellular free Ca²⁺ and activation of CaMKII and p38 MAPK.

The results obtained in this study revealed that different signaling pathways mediate downstream signaling of distinct nAChR subtypes in KCs exposed to ETS/nicotine. In other cell types, too, the mechanisms leading to up-regulation of 3, 5, and 7 nAChRs can be different (35 , 79) . For instance, 3 and 7 nAChRs are modulated differently in hippocampal neurons and SH-SY5Y cells, with 3 being regulated by CaMKII and 7 also by L-type Ca²⁺ channels (79) . In KCs exposed to ETS/nicotine, the up-regulation of 5 expression was predominantly mediated by PKC and to a lesser extent by CaMKII and other signal transducers. In contrast, the downstream signaling mediating self-up-regulation of 7 nAChR in exposed KCs involved previously characterized 7 coupled pathway Ras/Raf/MEK/ERK, as well as Akt and JAK-2 (12 , 30 , 31 , 80) . We also found that signaling along the 7-coupled pathway increased expression of and activated the signal transduction factor GATA-2. Other pathways also can be involved. In a recent study, nicotine-induced up-regulation of human 7 receptors was potentiated by modulation of cAMP and PKC (81) . Apparently, protein kinase A can be engaged as well (82) . The differences in the signaling pathways employed by each keratinocyte nAChR subtype observed in this study help explain a plethora of signaling mechanisms downstream of nAChRs expressed in different cell types.

The results obtained in this study, taken together with growing evidence of engagement of signaling kinases and phosphatases in the intracellular biochemical events mediating nicotinic effects of ACh in different cells, suggest that nAChRs elicit downstream signaling not only due to modulation of cell membrane permeability to ions. It is well known that 7 forms a receptor or channel that has a high relative permeability to Ca²⁺ (23 , 83) and that activation of 7 nAChR leads to an increase in the concentration of free cytosolic Ca²⁺ (24 , 25) . This may be due to activation of Ca²⁺-induced Ca²⁺ release from intracellular stores, triggered by influx through both 7 channels and voltage-gated Ca²⁺ channels (84 , 85) . In addition, our pilot studies have recently demonstrated that the mechanism of action of 7 nAChR in KCs may include a direct activation or inactivation of signaling kinases and phosphatases (86) . This is not surprising, because both tyrosine kinases and phosphotyrosine phosphatases can associate with 7 nAChR subunit and some other nAChR subunits in large multimeric complexes (87 88 89 90 91) . It was proposed that Src-associated protein tyrosine phosphatases function in early signaling events emanating from the nAChR that regulates cell function and that the Src to phosphotyrosine phosphatase ratio determines the functional state of associated nAChR (89) . The nAChR subunits 3-5 and β2 exhibit a positive interaction with the G-protein subunits G_(o) and Gβ (92) . Therefore, we propose that coupling of nAChR subunits to signaling kinases and/or phosphatases represents a novel function of subunit proteins that mediate ETS/nicotine effects on epithelial cells.

In conclusion, chronic exposures to nicotine products lead to sequential changes in the repertoire of keratinocyte nAChRs. This may contribute to pathobiologic effects of tobacco products in the stratified epithelium lining the upper digestive tract, because nAChRs are involved in a variety of cellular events, including malignant transformation (reviewed in ref. 93 ). Future studies should be directed toward identification of the specific cellular activities regulated by each major nAChR subtype expressed in the cell types targeted by tobacco toxicity and elucidation of the specific signaling pathways mediating biological effects of nAChR in these cells.

	ACKNOWLEDGMENTS

 
We thank Orla Cagney, Katrina M. Arredondo, and Pamela Sahourieh for excellent technical assistance. This work was supported by the U.S. National Institutes of Health, grants CA117327, DE14173, and ES014384, and a research grant from the Flight Attendant Medical Research Institute to S.A.G.

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

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