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(The FASEB Journal. 2006;20:2093-2101.)
© 2006 FASEB

Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of {alpha}7 nicotinic receptor in oral keratinocytes

Juan Arredondo*, Alexander I. Chernyavsky*, David L. Jolkovsky{ddagger}, Kent E. Pinkerton{dagger} and Sergei A. Grando*,1

* Department of Dermatology and

{dagger} Center for Health and the Environment, University of California, Davis; and

{ddagger} Section of Periodontics, School of Dentistry, University of California, Los Angeles, California, USA

1Correspondence: Department of Dermatology, University of California Davis Medical Center, 3301 C St., Ste. 1400, Sacramento, CA 95816, USA. E-mail: sagrando{at}ucdavis.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly {alpha}7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into {alpha}7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of {alpha}7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the {alpha}7 antagonist {alpha}-bungarotoxin ({alpha}BTX) or transfected with anti-{alpha}7 small interfering RNA (siRNA-{alpha}7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated {alpha}7-dependent up-regulation of STAT-3. Targeted mutation of the {alpha}7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by {alpha}BTX or siRNA-{alpha}7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from {alpha}7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral {alpha}7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.—Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of {alpha}7 nicotinic receptor in oral keratinocytes.


Key Words: nicotinic ACh receptors • gene expression • oral cancer • periodontal therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE USE OF TOBACCO products is associated with increased incidence of periodontal disease and poor response to periodontal therapy (1 2 3) . Frequent users of tobacco products also have an increased risk for developing oral cancer (4 , 5) . Among >4000 chemicals present in tobacco smoke, nicotine, which was originally thought to be responsible only for tobacco addiction, is now recognized for the modulation of key cellular proteins and processes involved in the pathobiological effects of tobacco in non-neuronal locations (6 7 8) . Indeed, nicotine has been shown to promote survival of many cell types, including head and neck cancer cells (9) . Binding of nicotine to the cell membrane modulates the expression of a diverse set of genes that may be broadly categorized into four groups: transcription factors, protein processing factors, RNA binding proteins, and plasma membrane-associated proteins (10) . Nicotine stimulation rapidly increases extracellular signal-regulated kinase (ERK) activation, tyrosine and serine phosphorylation of signal transducer and activator of transcription (STAT)-1 and STAT-3, and p38 mitogen-activated protein kinase (11) .

Nicotine displaces the local cytotransmitter ACh from the nicotinic ACh receptors (nAChRs) expressed by oral keratinocytes (KCs). The nAChRs play important roles in regulating non-neuronal cells (12 , 13) . The keratinocyte nAChRs can be composed of the {alpha}3, {alpha}5, {alpha}7, {alpha}9, ß2, ß4 subunits (14 15 16) . The heteromeric channels can be composed of {alpha}3, {alpha}5, ß2, and ß4 subunits, e.g., {alpha}3(ß2/ß4) ± {alpha}5, and homomeric channels can be composed by several {alpha}7 or {alpha}9 subunits. Both {alpha}7 and non-{alpha}7 nAChRs can mediate pathobiologic effects of tobacco and nicotine on KCs (14 , 15 , 17) . Emerging evidence indicates that in addition to the agonist nicotine, nAChRs can also be stimulated by the nicotine-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine (18 , 19) .

Several studies have identified {alpha}7 nAChR as the principal receptor subtype mediating the effects of tobacco products and pure nicotine on epithelial cells (19 20 21 22 23 24) . {alpha}7 nAChR is essential for a sustained turnover of the mucocutaneous epithelium in humans (25) . The genomic effects downstream of {alpha}7 nAChR can be represented by activation of tyrosine hydroxylase and dopamine ß-hydroxylase gene expression (26) , whereas the nongenomic pathways involve regulation of protein phosphorylation (23 , 27) . The end point effects of signaling via {alpha}7 nAChR may include an appearance of the transformed cellular phenotype manifested as a disappearance of contact inhibition, loss of dependence on exogenous growth factors, and attenuated apoptosis induced by various proapoptotic stimuli (18) . The {alpha}7 nAChR is also expressed in distinct types of human cancer and its activation accelerates tumors progression, indicating that the {alpha}7 nAChR-coupled signaling may play an important role in the development of tobacco-related cancers (28 29 30) .

The signaling mechanism(s) mediating downstream effects of nAChR are not yet fully understood. The {alpha}7 nAChR acts through different intracellular transduction pathways to protect or kill cells (31) . {alpha}7 forms a receptor/channel that has a high relative permeability to Ca2+ (32 , 33) and is inhibited with high affinity by {alpha}-bungarotoxin ({alpha}BTX) (32 , 34) . Activation of {alpha}7 nAChR leads to an increase in the concentration of free cytosolic Ca2+ (35 , 36) due to activation of Ca2+-induced Ca2+ release from intracellular stores, triggered by influx through both {alpha}7 channels and voltage-gated Ca2+ channels (37 , 38) . The pathway mediating {alpha}7 signaling in human epidermal cells involves activation of calcium/calmodulin-dependent protein kinase II (CaM kinase II), conventional isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K), and recruitment of Rac/Cdc42 (39) . Agonists of {alpha}7 nAChR can also activate Akt, which depends on phosphatidylinositol-3-kinase (18 , 40) . In various types of epithelial cells, {alpha}7 nAChR can also utilize the Ras/Raf/MEK/ERK signaling pathway (19 , 24 , 41 42 43) . Indeed, the activation of the ERK/MAP kinase pathway promotes gene expression, cell proliferation, and cell survival (44) . Most recently, the genomic effect of nicotine brought about via {alpha}7 nAChR in a non-neuronal cell has been linked to the JAK-2/STAT-3 signaling (45) . The tyrosine kinase JAK-2 that activates the transcription factor STAT-3 was recruited and phosphorylated after nAChR activation by nicotine (45) . Thus, the genomic effect of nicotine requires the ability of phosphorylated STAT-3 to bind and transactivate its DNA response elements.

To gain a mechanistic insight into the mode of pathophysiologic action of nicotine in the epithelium lining the upper portion of the digestive tract, in this study we investigated the signaling pathways downstream of {alpha}7 nAChR that can mediate effects of aged and diluted sidestream cigarette smoke (ADSS) on oral KCs. Results of experiments demonstrated that nuclear transactivation of STAT-3 requires simultaneous activation of two complementary pathways. While activation of the Ras/Raf-1/MEK1/ERK pathway provided for up-regulated expression of STAT-3, the recruitment of JAK-2 was required for STAT-3 phosporylation and homodimer formation. The similarity of the effects observed with exposures to equivalent doses of pure nicotine argued that nicotine is the major component of ADSS responsible for the observed signaling events. Nicotine-induced JAK-2/STAT-3 activation could be prevented by functional inactivation of {alpha}7 nAChR with {alpha}BTX or anti-{alpha}7 small interfering RNA (siRNA-{alpha}7), suggesting that oral {alpha}7 nAChR may provide a novel molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and transfection reagents
The {alpha}7 antagonist {alpha}Btx and nicotine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The noncompetitive inhibitor of the Ras acceptor protein manumycin A (46) , the cRaf-1 kinase inhibitor GW5074 [5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone] (47) , the JAK-2 inhibitor AG 490, the cell-permeable, potent and selective inhibitor of MEK "MEK inhibitor I" (48) , and a less specific MEK inhibitor U0126 (49) were from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Plasmids encoding the constitutively active MEK1 (CA-MEK) with 2 point mutations (S218E and S222E) and a deletion of amino acid residues 31–52, the dominant negative MEK1 mutant (DN-MEK) that contains 3 point mutations (K97R, S218A, and S222A) and thus could neither be phosphorylated by its activators nor phosphorylate its downstream effectors ERKs, and the wild-type (WT) control MEK1 were purchased from Biomyx Technology (San Diego, CA, USA). siRNA-{alpha}7 was designed and custom synthesized by Dharmacon (Lafayette, CO, USA). The target sequences for the human CHRNA7 mRNA (NM_000746) gene was 5'-GGACAGAUCACUAUUUACA-3'. The negative control siRNA targeting luciferase gene with the target sequence 5'-CGTACGCGGAATACTTCGA-3' used in all RNA inhibition experiments was also purchased from Dharmacon.

Culturing and transfecting human KCs
Normal human KCs were obtained from attached gingiva (this study has been approved by the University of California Davis Human Subjects Review Committee), as detailed elsewhere (14) . Briefly, attached gingival samples were cut into 3–4 mm pieces and incubated overnight in a humidified atmosphere with 5% CO2 at 37°C in 0.06% trypsin (Sigma-Aldrich) in minimum essential medium 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, Gaithersburg, MD, USA). Individual KCs were isolated by gentle pipetting, followed by centrifugation, and 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 (17) . 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 experimentally determined by Western blot 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.

{alpha}7 Knockout murine KCs
The {alpha}7–/– and {alpha}7+/+ murine KCs were obtained as reported elsewhere (17) from oral mucosa of 2- to 4-day-old Acra7-deficient ({alpha}7 null) mice generated as described previously (50) . This study was approved by University of California Davis Committee on the Use of Animals in Research. The cells were grown at 37°C and 5% CO2 in 25 cm2 Falcon culture flasks using the cell culture techniques optimized for mouse KCs (51 , 52) . The genotyping of the {alpha}7 mutant mice was performed by PCR analysis of mouse tail DNA, as detailed elsewhere (25) . The {alpha}7+/+ littermates of {alpha}7–/– mice were used as controls.

Tobacco smoke/nicotine exposure experiments
The monolayers of human KCs grown to ~80% confluence in 6-well plates (Corning Glass Works) were incubated for 24 h in KGM pre-exposed to ADSS as a surrogate of tobacco smoke or containing an equivalent concentration of nicotine (10 µM). The KGM was exposed to ADSS in the chambers of a sidestream smoke exposure system (53) . Briefly, a TE-10 smoking machine (Teague Enterprises, Davis, CA, USA) burned 2RF4 reference cigarettes (Tobacco and Health Research Institute, University of Kentucky) that had been temperature and humidity conditioned to generate ADSS. Each 2RF4 cigarette delivers ~0.8 mg of nicotine (53) . 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/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 (17) . 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 STAT-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 were 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 assay
In-cell Western assay (LI-COR Lincoln, NE, USA) was performed as described in detail elsewhere (54) . 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 a primary antibody (Ab) to STAT-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After that, the cells were washed and stained with a secondary goat anti-rabbit Alexa Fluor®680 (1:5,000 dilution; Molecular Probes, Eugene, OR, USA). The protein expression was then quantitated using the Odyssey Imaging System (LI-COR).

Gel mobility shift assay
In accordance with established protocol (55) , the nuclear extract was obtained from experimental and control KCs grown in 6-well plates. The cells were released from the dish bottom by trypsinization, resuspended in cold (4°C) TBS (Bio-Rad, Hercules, CA, USA), pelleted by centrifugation, and lysed with 10% Nonidet P-40/400 µl buffer A [10 mM Na-HEPES (pH 7.9) 10 mM KCl, 0.1 mM EDTA (pH 8.0) 0.1 mM EGTA (pH 8.0), 1 mM DTT, and 0.5 mM PMSF]. The nuclei were pelleted by centrifugation and resuspended in buffer B [20 mM Na-HEPES (pH 7.9), 400 mM KCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 1 mM DTT, 0.5 mM PMSF, and 10% glycerol]. The gel mobility shift assay was performed as described in detail elsewhere (56) . Briefly, the oligonucleotide 5'-GATCCTTCTGGGAATTCCTAGATC-3' supplied by the Operon (Alameda, CA, USA) was labeled with Digoxigenin and its complement. The gel shift reaction buffer consisted of 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol. Nuclear extracts containing 10 µg of protein were incubated with 0.5 ng of Digoxigenin-labeled STAT-1 oligonucleotide 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 0.5 x Tris, boric acid, EDTA and blotted overnight at 4°C in 1 x sodium chloride/sodium citrate buffer onto the Positive Charge Nylon Membranes, followed by UV cross-linking, and Digoxigenin detection with anti-Digoxigenin-activating protein monoclonal antibody (mAb) and NBT/BCIP Nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate toluidine salt as a substrate (all from Roche, Indianapolis, IN, USA).

Kinase activity assays
The MAP Kinase Erk Assay kit (Upstate, Lake Placid, NY, USA) was used to measure ERK1/2 activity in {alpha}7+/+ vs. {alpha}7–/– KCs in accordance with protocol provided by the manufacturer. The assay measures the phosphotransferase activity of MAP using a specific substrate (myelin basic protein; MBP). The phosphorylated substrate was analyzed by immunoblotting with a monoclonal phospho-specific MBP Ab. The relative density of scanned bands was determined by area integration using ImageQuant software (Molecular Dynamics). The results were expressed as integrated intensity of pixels of the spot excluding the background. The level of phosphorylation of the tyrosine kinase JAK-2 was also measured by a quantitative immunoblot, using a phospho-janus-activated kinase (JAK)-2 (Tyr1007/1008) Ab purchased from Upstate. The presence in the samples of ERK 1 and 2 as well as JAK-2 was demonstrated using corresponding rabbit polyclonal antibody (pAb) purchased from Santa Cruz Biotechnology, Inc. All antibodies were used at 1:1,000 dilution.

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
 
The role of {alpha}7 nAChR in mediating ADSS and nicotine effects on the expression of STAT-3 in KCs
To elucidate the signaling mechanisms involved in the {alpha}7 nAChR-mediated effects of nicotine toxicity on KCs, we studied transcription of the gene encoding STAT-3, an effector of the JAK-2/STAT-3 pathway activated by nicotine in macrophages (45) . By real-time PCR and In-cell Western, the KCs stimulated with ADSS or pure nicotine showed multifold increases of STAT-3 (Fig. 1 ). The up-regulated expression was more pronounced at the mRNA level. The effects of ADSS and nicotine could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the {alpha}7 antagonist {alpha}BTX or transfected with siRNA-{alpha}7 (Fig. 1) .


Figure 1
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  		Figure 1. Alterations in STAT-3 gene expression in human KCs exposed to ADSS or pure nicotine. Real time-polymerase chain reaction (A, C) and Incell Western (B, D) analysis of ADSS (A, B) and nicotine (C, D) effects on STAT-3 gene expression in human KCs. Total RNA and proteins were isolated after 24 h exposures to ADSS or 10 µM nicotine. The relative amounts of mRNA transcript and protein levels of STAT-3 were measured as described in Materials and Methods. Some cells were transfected with receptor-specific, or control, siRNA, and/or MEK1 mutants, as detailed in Materials and Methods. To standardize the analysis, gene expression ratios in the control cells (i.e., intact KCs and KCs transfected with a nonspecific siRNA in experiments with siRNA-{alpha}7) were taken as 1. The following experimental treatments were used: 3 µM manumycin A (Mnmc); 3 µM manumycin A on KCs transfected with constitutively active (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 inhibitor I (MEK-Inh); 10 µM U0126; transfection with dominant negative (DN)-MEK; transfection with the control, WT MEK1 mutant (WT-MEK); 1 µM {alpha}Btx; transfection with siRNA-{alpha}7; and cotransfection with siRNA-{alpha}7 and CA-MEK (CA-MEK+siRNA-{alpha}7), WT-MEK (WT-MEK+siRNA-{alpha}7) or DN-MEK (DN-MEK+siRNA-{alpha}7). Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n=3). Asterisks indicate significant (P<0.05) differences from intact control KCs. The pound signs indicate significant (P<0.05) differences from KCs exposed to either ADSS or nicotine alone.

These results indicated that in human KCs, activation of {alpha}7 can mediate stimulatory effects of tobacco products on gene expression of the transcription factor STAT-3.

Signaling through the Ras/Raf-1/MEK1/ERK pathway mediates the {alpha}7-dependent up-regulation of STAT-3 in KCs
Since {alpha}7 activates the Ras/Raf-1/MEK1/ERK signaling pathway coupled to regulation of gene expression in KCs and other cells (43 , 57) , we studied the ability of pathway inhibitors to abolish up-regulated expression of STAT-3 induced by ADSS or nicotine. The elevation of STAT-3 at both the mRNA and protein levels could be prevented by the Ras inhibitor manumycin A, 3 µM, and the cRaf-1 inhibitor GW5074, 0.1 µM (Fig. 1) . Neither inhibitor, however, could block up-regulation of this transcription factor in KCs transfected with CA-MEK (P<0.05) (Fig. 1) .

Evidence of the involvement of MEK1/ERK in the signaling pathway mediating the response of KCs to ADSS/nicotine was obtained in experiments with 1 µM of MEK Inhibitor I that blocked up-regulated expression of STAT-3. Similar results were obtained using 10 µM of another inhibitor U0126, but not its negative control analog U0124 (Fig. 1) . To ultimately establish the role for the MEK1/ERK-mediated signaling, we transfected KCs with DN-MEK. This MEK mutant, but not WT control MEK, inhibited the effect of ADSS/nicotine (Fig. 1) . The response of KCs to both ADSS and nicotine was also abolished if the cells were cotransfected with the WT control MEK1 and siRNA-{alpha}7. In marked contrast, cotransfection of KCs with siRNA-{alpha}7 and CA-MEK did not affect up-regulated expression of STAT-3 protein (P<0.05) (Fig. 1) .

These results indicated that {alpha}7 nAChR uniquely mediated effects of ADSS/nicotine on the expression of STAT-3 in human KCs and that downstream signaling leading to elevation of this transcription factor proceeded through the Ras/Raf-1/MEK1/ERK steps.

Targeted mutation of the {alpha}7 nAChR gene abolishes ERK1/2 activation by nicotine
To ultimately establish the role of {alpha}7 in the ERK1/2-mediated up-regulation of the STAT-3 gene expression, we performed a series of exposure experiments using single-cell type cultures of murine KCs grown from {alpha}7–/– vs. {alpha}7+/+ mice. Exposure to nicotine significantly (P<0.05) up-regulated ERK1/2 activity in {alpha}7+/+ KCs, but failed to do so in {alpha}7–/– KCs (P>0.05). The {alpha}7+/+ KCs exposed to nicotine featured > 2-fold higher ERK1/2 activity compared to {alpha}7–/– KCs (Fig. 2 ). These results provided direct evidence that the {alpha}7 nAChR-coupled pathway leads to activation of ERK1/2.


Figure 2
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  		Figure 2. Null mutation of {alpha}7 nAChR subunit abolishes nicotine-dependent activation of ERK1/2. Murine KCs were preincubated for 24 h with 10 µM of nicotine, followed by analysis of ERK1/2 activity using myelin basic protein (MBP) as specific substrate. Immunoblot was probed with the antiphospho-MBP Ab as a measure of ERK1/2 kinase activity or with anti-ERK1/2 Ab to confirm that the enzymes were present in all samples. The optical density (OD) of the bands was analyzed as described in Materials and Methods. Shown in the gel are typical results obtained in triplicate assays (n=3). Lane 1: intact {alpha}7 +/+ KCs; lane 2: intact {alpha}7 –/– KCs; lane 3: {alpha}7 +/+ KCs + nicotine; lane 4: {alpha}7 –/– KCs + nicotine; lane 5: {alpha}7 +/+ KCs without ERK substrate; lane 6: {alpha}7 –/– KCs without ERK substrate. The following ratios between ERK1/2 activities in different experimental conditions were determined: lanes 1/2 = 1.05 ± 0.1 (P>0.05); lanes 3/4 = 2.11 ± 0.2 (P<0.05); lanes 3/1 = 1.74 ± 0.1 (P<0.05); and lanes 4/2 = 1.18 ± 0.2 (P>0.05).

The role of {alpha}7 nAChR in mediating ADSS and nicotine effects on the transcriptional activity of STAT-3 in KCs
Using the gel mobility shift assay, we further investigated whether up-regulated gene expression of STAT-3 correlated with its activity in ADSS/nicotine-exposed KCs. This assay identifies the interaction of proteins with DNA, which is central to the control of many cellular processes, including DNA replication, recombination, and transcription. We found that the protein binding activity of STAT-3 induced by ADSS or pure nicotine (Fig. 3 A) correlated with the transcriptional induction observed in the quantitative mRNA and protein assays (Fig. 1) . To verify that STAT-3 activation by nicotine was mediated by the {alpha}7 nAChR subtype, we pretreated cells with {alpha}BTX or transfected them with siRNA-{alpha}7. The up-regulated STAT-3 activity could be abolished by both {alpha}BTX and siRNA-{alpha}7 (Fig. 3A ).


Figure 3
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  		Figure 3. Assessment of the effects of {alpha}7 nAChR activation on the activities of STAT-3 and JAK-2. A) Transactivation of STAT-3. The KCs were treated with ADSS or pure nicotine in the presence or absence of 10 µM of the JAK-2 inhibitor AG-490, then subjected to functional analysis of STAT-3. The STAT-3 DNA-protein complex was detected by gel mobility shift assay with nuclear extracts prepared from experimental and control KCs, as detailed in Materials and Methods. Lane 1: nicotine; lane 2: ADSS; lane 3: nicotine + {alpha}BTX; lane 4: ADSS + {alpha}BTX; lane 5: nicotine + siRNA-{alpha}7; lane 6: ADSS + siRNA-{alpha}7; lane 7: untreated control cells. B) Transactivation of JAK-2. Phosphorylation of JAK-2 was determined using antiphospho-JAK-2 (Tyr1007/1008) Ab that visualized band with an apparent MW of 130 kDa. The OD of the bands was analyzed as described in Materials and Methods. The density of 1 was assigned to control, untreated KCs. The ratio data underneath the bands are the means ± SD of the values obtained in three independent experiments. Lane 1: nicotine; lane 2: ADSS; lane 3: nicotine + {alpha}BTX; lane 4: nicotine + siRNA-{alpha}7; lane 5: ADSS + {alpha}BTX; lane 6: ADSS + siRNA-{alpha}7; lane 7: untreated control cells. Asterisks indicate significant (P<0.05) difference compared to control.

Transactivation of STAT-3 could be abolished in the presence of the JAK-2 inhibitor AG 490 (Fig. 3A ), thus establishing a causative link between the two in KCs.

To gain mechanistic insight into the signaling pathway downstream of {alpha}7 nAChR that increased transcriptional activity of STAT-3 in KCs, we investigated the phosphorylation of JAK-2 using antibodies to the phosphorylated species of this tyrosine kinase. We knew that, on the one hand, stimulation of {alpha}7 nAChR with an agonist leads phosphorylation and activation of JAK-2 physically associated with this receptor (45) and on the other, that phosphorylated JAK-2 activates its substrate STAT-3 via phosphorylation in another cell type (58) . As expected, activation of {alpha}7 nAChR with ADSS or nicotine in both cases significantly (P<0.05) increased the level of phosphorylation of JAK-2 in KCs, which could be prevented when the cells were either pretreated with {alpha}BTX or transfected with siRNA-{alpha}7 (Fig. 3B ), but not control siRNA (data not shown).

Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via an intracellular signaling downstream from {alpha}7 that proceeds via two complementary pathways: the Ras/Raf-1/MEK1/ERK cascade that culminates in an increase of the relative amount of this transcription factor, and the recruitment and activation of tyrosine kinase JAK-2 that phosphorylates it.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was designed to gain a mechanistic insight into nAChR-mediated morbidity of tobacco products in the oral cavity. We studied the effects of exposures to ADSS vs. equivalent concentration of pure nicotine on oral KCs and demonstrated for the first time that both ADSS and nicotine use {alpha}7 nAChR to mediate their effects on keratinocyte gene expression. The results revealed an involvement of Ras/Raf-1/MEK1/ERK pathway in the {alpha}7-dependent up-regulation of the transcription factor STAT-3 at both the mRNA and protein levels. The nuclear transactivation of STAT-3, however, depended on a complementary signaling step executed by the tyrosine kinase JAK-2 that was directly activated due to stimulation of {alpha}7 nAChR. Activated JAK-2 phosphorylated STAT-3, which allows it to form dimers that can translocate to the nucleus and produce biological effects. The cooperation between Ras/Raf/MEK/ERK and JAK-2/STAT-3 pathways in mediating intracellular signaling downstream of {alpha}7 nAChR is schematically depicted in Fig. 4 . This novel paradigm offers an unorthodox explanation of the intricate mechanisms of intracellular signaling mediating pathobiologic effects of nicotine in non-neuronal cells, and suggests innovative solutions to ameliorate the tobacco-related cell damage and intercede in disease pathways.


Figure 4
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  		Figure 4. Cooperation of the Ras/Raf/MEK/ERK and JAK-2/STAT-3 pathways downstream of {alpha}7 nAChR. Stimulation of {alpha}7 nAChR by either its physiological ligand ACh or nicotine (Nic) leads to alterations in gene expression due to transactivation of STAT-3 that occurs through two complementary signaling pathways coupled by this receptor. The pathway mediated by stepwise activation of Ras/Raf/MEK/ERK provides for an increased cytoplasmic concentration of STAT-3 due to an up-regulated expression, whereas activation of the tyrosine kinase JAK-2 provides for phosphorylation of STAT-3 with subsequent translocation of STAT-3 dimers to the nucleus to alter gene expression.

Activation of ERK1/2 isoforms through phosphorylation by activated MAP kinase kinase (MEK) occurs due to upstream engagement of the Ras/Raf-1 steps (59) . Indeed, the Raf/MEK/ERK cascade can be regulated independently from Ras in a PKC-dependent manner (60) . However, results of our published experiments with PMA stimulation of KCs convincingly demonstrated that the ERK1/2 activity sensitive to inhibition by manumycin and GW5074 did not result from activation of an alternative PKC-mediated pathway (43) . Definitive evidence of activation of the Ras/Raf-1/MEK1/ERK signaling through {alpha}7 nAChR was obtained in this study by demonstrating that targeted mutation of the {alpha}7 nAChR gene in {alpha}7–/– murine KCs abolishes ERK1/2 activation by nicotine.

Extracellular signal-regulated kinase (ERK; p44/p42), also called mitogen-activated protein kinase (MAPK), plays a critical role in signal transduction cascades from the cell surface to the nucleus. ERK is a serine/threonine protein kinase that responds to a diverse array of extracellular stimuli, including neurotransmitters, hormones, growth factors, and several types of stress, and regulates many important cellular tasks such as cell growth, cell movement, differentiation, proliferation, and death (44) . The Ca2+-dependent activation of the ERK1/2 cascade could proceed through a variety of upstream Ca2+-dependent kinases, including PI3K, CaM kinase II, PKC, and PKA (59) . These and other signal transduction kinases have been shown to subserve the function of {alpha}7 in KCs and other cell types (18 , 19 , 24 , 39 40 41 42) . This variety of upstream regulators explains our observation that {alpha}7–/– cells exhibited basal ERK activity. The fact that the KCs that lacked {alpha}7 nAChRs, in contrast to {alpha}7+/+ cells, failed to respond to nicotine with an increase in ERK1/2 phosphorylating activity indicates that among the subtypes of nAChRs expressed in KCs, the {alpha}7 subtype uniquely couples this effector enzyme.

Results of this study revealed a key role for JAK-2 in the {alpha}7 nAChR-dependent activation of the transcription factor involved in the physiological regulation of cell survival and death, such as STAT-3 (61) . Results of an early study provided evidence that the nicotine-induced complex formation between the {alpha}7 nAChR and the tyrosine-phosphorylated enzyme JAK-2 leads to subsequent activation of PI3K and Akt, which was associated with inhibition of both caspase-3 activity and cleavage of the DNA-repairing enzyme poly(ADP-ribose) polymerase (62) .

The comprehensive analysis of the biological function of {alpha}7 nAChR in KCs revealed its important role in sustaining normal unfolding of the genetically determined program of cell cycle progression, eventuating in cell death by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes (25) . In Acra7 homozygous mutant mice, the missing regulatory pathway causes transient changes in skin phenotype characteristic of delayed epidermal turnover. The skin of these knockout mice also features decreased amounts of the extracellular matrix proteins collagen 1{alpha}1 and elastin as well as metalloproteinase-1 (63) . Thus, identification of a nAChR subtype selective control of the gene expression responsible for acquisition of a particular cell phenotype provides a mechanistic insight into a general regulatory mechanism subserving the deleterious effects of tobacco and nicotine in the oral epithelium. Further, nicotine and its carcinogenic derivatives activating {alpha}7 nAChRs expressed in oral cells may play a key role in the pathogenesis of head and neck cancer by acting as tumor promoters that facilitate the outgrowth of cells with genetic damage (epigenetic effect).

The {alpha}7 subunit is abundantly expressed in the epithelial cells lining skin, oral mucosa, and esophagus (14) . The expression of {alpha}7 nAChR channels on the cell membrane of non-neuronal cells is modulated by exposure to nicotine (15 , 64) , which may provide a mechanism for nicotine-induced changes in gene expression (15 , 65 , 66) , proliferation (30 , 40) , apoptosis (9 , 67) , and secretion (68 , 69) in non-neuronal locations.

In this study, the dosing of nicotine was chosen to correlate well with the levels found in mucous secretions of smokers and snuffers. Previous studies have demonstrated that the maximal effects of nicotine on non-neuronal cells occur at the dose ranging from 10–8 to 10–6 M (15 , 17 , 70 71 72 73 74) . This is in keeping with the fact that tobacco smokers have saliva concentrations of nicotine as high as 1.3 µg/ml, which is > 100-fold higher than the level in the blood (75 76 77) .

Taken together, the experimental results obtained in this study identified the homomeric nAChR channel comprised by {alpha}7 subunits as a major mediator of pathobiologic effects of tobacco/nicotine in the epithelium lining the upper digestive tract, and warranted further mechanistic studies into nicotinic regulation of the gene expression leading to alterations in mucosal cell growth and function.


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

Received for publication March 20, 2006. Accepted for publication May 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bergstrom, J. (1989) Cigarette smoking as risk factor in chronic periodontal disease. Community Dent. Oral Epidemiol. 17,245-247[CrossRef][Medline]
  2. Haber, J. (1994) Cigarette smoking: a major risk factor for periodontitis. Compendium 15,10021004–1008[Medline]
  3. Mandel, I. (1994) Smoke signals: an alert for oral disease. J. Am. Dental Assoc. 125,872-878[Abstract]
  4. Johnson, G. K., Squier, C. A. (1993) Smokeless tobacco use by youth: a health concern. Ped. Dent. 15,169-174
  5. Sugar, J., Vereczkey, I., Toth, J. (1996) Some etio-pathogenetic factors in laryngeal carcinogenesis. J. Environ. Pathol. Toxicol. Oncol. 15,195-199[Medline]
  6. Benowitz, N. L. (1997) Systemic absorption and effects of nicotine from smokeless tobacco. Adv. Dental Res. 11,336-341[Abstract]
  7. Minna, J. D. (2003) Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer. J. Clin. Invest. 111,31-33[CrossRef][Medline]
  8. Conti-Fine, B. M., Navaneetham, D., Lei, S., Maus, A. D. (2000) Neuronal nicotinic receptors in non-neuronal cells: new mediators of tobacco toxicity?. Eur. J. Pharmacol. 393,279-294[CrossRef][Medline]
  9. Onoda, N., Nehmi, A., Weiner, D., Mujumdar, S., Christen, R., Los, G. (2001) Nicotine affects the signaling of the death pathway, reducing the response of head and neck cancer cell lines to DNA damaging agents. Head Neck 23,860-870[CrossRef][Medline]
  10. Dunckley, T., Lukas, R. J. (2003) Nicotine modulates the expression of a diverse set of genes in the neuronal SH-SY5Y cell line. J. Biol. Chem. 278,15633-15640[Abstract/Free Full Text]
  11. Li, J. M., Cui, T. X., Shiuchi, T., Liu, H. W., Min, L. J., Okumura, M., Jinno, T., Wu, L., Iwai, M., Horiuchi, M. (2004) Nicotine enhances angiotensin II-induced mitogenic response in vascular smooth muscle cells and fibroblasts. Arterioscler. Thromb. Vasc. Biol. 24,80-84[Abstract/Free Full Text]
  12. Lindstrom, J. (1997) Nicotinic acetylcholine receptors in health and disease. Mol. Neurobiol. 15,193-222[Medline]
  13. Grando, S. A., Kawashima, K., Wessler, I. (2003) The non-neuronal cholinergic system in humans. Life Sci. 72,2009-2012[CrossRef][Medline]
  14. Nguyen, V. T., Hall, L. L., Gallacher, G., Ndoye, A., Jolkovsky, D. L., Webber, R. J., Buchli, R., Grando, S. A. (2000) Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia. J. Dent. Res. 79,939-949[Abstract/Free Full Text]
  15. Arredondo, J., Nguyen, V. T., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. (2001) A receptor-mediated mechanism of nicotine toxicity in oral keratinocytes. Lab. Invest. 81,1653-1668[Medline]
  16. Nguyen, V. T., Ndoye, A., Grando, S. A. (2000) Novel human {alpha}9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity. Am. J. Pathol. 157,1377-1391[Abstract/Free Full Text]
  17. Arredondo, J., Chernyavsky, A. I., Marubio, L. M., Beaudet, A. L., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. (2005) Receptor-mediated tobacco toxicity: Regulation of gene expression through {alpha}3ß2 nicotinic receptor in oral epithelial cells. Am. J. Pathol. 166,597-613[Abstract/Free Full Text]
  18. West, K. A., Brognard, J., Clark, A. S., Linnoila, I. R., Yang, X., Swain, S. M., Harris, C., Belinsky, S., Dennis, P. A. (2003) Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J. Clin. Invest. 111,81-90[CrossRef][Medline]
  19. Schuller, H. M., Orloff, M. (1998) Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells. Biochem. Pharmacol. 55,1377-1384[CrossRef][Medline]
  20. Cattaneo, M. G., D’Atri, F., Vicentini, L. M. (1997) Mechanisms of mitogen-activated protein kinase activation by nicotine in small-cell lung carcinoma cells. Biochem. J. 328,499-503[Medline]
  21. Codignola, A., Tarroni, P., Cattaneo, M. G., Vicentini, L. M., Clementi, F., Sher, E. (1994) Serotonin release and cell proliferation are under the control of alpha-bungarotoxin-sensitive nicotinic receptors in small-cell lung carcinoma cell lines. FEBS Lett. 342,286-290[CrossRef][Medline]
  22. Schuller, H. M. (1989) Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem. Pharmacol. 38,3439-3442[CrossRef][Medline]
  23. Schuller, H. M., Jull, B. A., Sheppard, B. J., Plummer, H. K. (2000) Interaction of tobacco-specific toxicants with the neuronal {alpha}7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders. Eur. J. Pharmacol. 393,265-277[CrossRef][Medline]
  24. Schuller, H. M., Plummer, H. K., 3rd, Jull, B. A. (2003) Receptor-mediated effects of nicotine and its nitrosated derivative NNK on pulmonary neuroendocrine cells. Anat. Rec. 270A,51-58[Medline]
  25. Arredondo, J., Nguyen, V. T., Chernyavsky, A. I., Bercovich, D., Orr-Urtreger, A., Kummer, W., Lips, K., Vetter, D. E., Grando, S. A. (2002) Central role of {alpha}7 nicotinic receptor in differentiation of the stratified squamous epithelium. J. Cell Biol. 159,325-336[Abstract/Free Full Text]
  26. Gueorguiev, V. D., Zeman, R. J., Meyer, E. M., Sabban, E. L. (2000) Involvement of {alpha}7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine ß-hydroxylase gene expression in PC12 cells. J. Neurochem. 75,1997-2005[CrossRef][Medline]
  27. Kihara, T., Shimohama, S., Sawada, H., Honda, K., Nakamizo, T., Shibasaki, H., Kume, T., Akaike, A. (2001) {alpha}7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A ß-amyloid-induced neurotoxicity. J. Biol. Chem. 276,13541-13546[Abstract/Free Full Text]
  28. Plummer, H. K., 3rd, Dhar, M., Schuller, H. M. (2005) Expression of the alpha7 nicotinic acetylcholine receptor in human lung cells. Respir. Res. 6,29[CrossRef][Medline]
  29. Ehrhardt, A., Bartels, T., Klocke, R., Paul, D., Halter, R. (2003) Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type II cells. J. Cancer Res. Clin. Oncol. 129,71-75[Medline]
  30. Ye, Y. N., Liu, E. S., Shin, V. Y., Wu, W. K., Cho, C. H. (2004) The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. J. Pharmacol. Exp. Ther. 311,123-130[Abstract/Free Full Text]
  31. Li, Y., Papke, R. L., He, Y. J., Millard, W. J., Meyer, E. M. (1999) Characterization of the neuroprotective and toxic effects of {alpha}7 nicotinic receptor activation in PC12 cells. Brain Res. 830,218-225[CrossRef][Medline]
  32. Seguela, P., Wadiche, J., Dineley-Miller, K., Dani, J. A., Patrick, J. W. (1993) Molecular cloning, functional properties, and distribution of rat brain {alpha}7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13,596-604[Abstract]
  33. Castro, N. G., Albuquerque, E. X. (1995) {alpha}-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys. J. 68,516-524[Medline]
  34. Couturier, S., Bertrand, D., Matter, J. M., Hernandez, M. C., Bertrand, S., Millar, N., Valera, S., Barkas, T., Ballivet, M. (1990) A neuronal nicotinic acetylcholine receptor subunit ({alpha}7) is developmentally regulated and forms a homo-oligomeric channel blocked by {alpha}-BTX. Neuron 5,847-856[CrossRef][Medline]
  35. Quik, M., Philie, J., Choremis, J. (1997) Modulation of {alpha}7 nicotinic receptor-mediated calcium influx by nicotinic agonists. Mol. Pharmacol. 51,499-506[Abstract/Free Full Text]
  36. Delbono, O., Gopalakrishnan, M., Renganathan, M., Monteggia, L. M., Messi, M. L., Sullivan, J. P. (1997) Activation of the recombinant human {alpha}7 nicotinic acetylcholine receptor significantly raises intracellular free calcium. J. Pharmacol. Exp. Ther. 280,428-438[Abstract/Free Full Text]
  37. Taylor, S. C., Peers, C. (2000) Three distinct Ca2+ influx pathways couple acetylcholine receptor activation to catecholamine secretion from PC12 cells. J. Neurochem. 75,1583-1589[CrossRef][Medline]
  38. Sharma, G., Vijayaraghavan, S. (2001) Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proc. Natl. Acad. Sci. U. S. A. 98,4148-4153[Abstract/Free Full Text]
  39. Chernyavsky, A. I., Arredondo, J., Marubio, L. M., Grando, S. A. (2004) Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes. J. Cell Sci. 117,5665-5679[Abstract/Free Full Text]
  40. Tsurutani, J., Castillo, S. S., Brognard, J., Granville, C. A., Zhang, C., Gills, J. J., Sayyah, J., Dennis, P. A. (2005) Tobacco components stimulate Akt-dependent proliferation and NF{kappa}B-dependent survival in lung cancer cells. Carcinogenesis 26,1182-1195[Abstract/Free Full Text]
  41. Jull, B. A., Plummer, H. K., 3rd, Schuller, H. M. (2001) Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells. J. Cancer Res. Clin. Oncol. 127,707-717[Medline]
  42. Dajas-Bailador, F. A., Soliakov, L., Wonnacott, S. (2002) Nicotine activates the extracellular signal-regulated kinase 1/2 via the {alpha}7 nicotinic acetylcholine receptor and protein kinase A, in SH- SY5Y cells and hippocampal neurones. J. Neurochem. 80,520-530[CrossRef][Medline]
  43. Chernyavsky, A. I., Arredondo, J., Karlsson, E., Wessler, I., Grando, S. A. (2005) The Ras/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration. J. Biol. Chem. 280,39220-39228[Abstract/Free Full Text]
  44. Chang, L., Karin, M. (2001) Mammalian MAP kinase signalling cascades. Nature 410,37-40[CrossRef][Medline]
  45. De Jonge, W. J., van der Zanden, E. P., The, F. O., Bijlsma, M. F., van Westerloo, D. J., Bennink, R. J., Berthoud, H. R., Uematsu, S., Akira, S., van den Wijngaard, R. M., Boeckxstaens, G. E. (2005) Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat. Immunol. 6,844-851[CrossRef][Medline]
  46. Hara, M., Akasaka, K., Akinaga, S., Okabe, M., Nakano, H., Gomez, R., Wood, D., Uh, M., Tamanoi, F. (1993) Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc. Natl. Acad. Sci. U. S. A. 90,2281-2285[Abstract/Free Full Text]
  47. Lackey, K., Cory, M., Davis, R., Frye, S. V., Harris, P. A., Hunter, R. N., Jung, D. K., McDonald, O. B., McNutt, R. W., Peel, M. R., et al (2000) The discovery of potent cRaf1 kinase inhibitors. Bioorg. Med. Chem. Lett. 10,223-226[CrossRef][Medline]
  48. Wityak, J., Hobbs, F. W., Gardner, D. S., Santella, J. B., 3rd, Petraitis, J. J., Sun, J. H., Favata, M. F., Daulerio, A. J., Horiuchi, K. Y., Copeland, R. A., et al (2004) Beyond U0126. Dianion chemistry leading to the rapid synthesis of a series of potent MEK inhibitors. Bioorg. Med. Chem. Lett. 14,1483-1486[CrossRef][Medline]
  49. English, J. M., Cobb, M. H. (2002) Pharmacological inhibitors of MAPK pathways. Trends Pharmacol. Sci. 23,40-45[CrossRef][Medline]
  50. Orr-Urtreger, A., Goldner, F. M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J. A., Patrick, J. W., Beaudet, A. L. (1997) Mice deficient in the {alpha}7 neuronal nicotinic acetylcholine receptor lack {alpha}-bungarotoxin binding sites and hippocampal fast nicotinic currents. J. Neurosci. 17,9165-9171[Abstract/Free Full Text]
  51. Lee, Y. S., Dlugosz, A. A., McKay, R., Dean, N. M., Yuspa, S. H. (1997) Definition by specific antisense oligonucleotides of a role for protein kinase C alpha in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes. Mol. Carcinog. 18,44-53[CrossRef][Medline]
  52. Li, L., Tucker, R. W., Hennings, H., Yuspa, S. H. (1995) Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro. J. Cell. Physiol. 163,105-114[CrossRef][Medline]
  53. Teague, S. V., Pinkerton, K. E., Goldsmith, M., Gebremichael, A., Chang, S., Jenkins, R. A., Moneyhun, J. H. (1994) Sidestream cigarette smoke generation and exposure system for environmental tobacco smoke studies. Inhal. Toxicol. 6,79-93[CrossRef]
  54. Arredondo, J., Chernyavsky, A. I., Webber, R. J., Grando, S. A. (2005) Biological effects of SLURP-1 on human keratinocytes. J. Invest. Dermatol. 125,1236-1241[CrossRef][Medline]
  55. Abravaya, K., Myers, M. P., Murphy, S. P., Morimoto, R. I. (1992) The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression. Genes Dev. 6,1153-1164[Abstract/Free Full Text]
  56. Sistonen, L., Sarge, K. D., Morimoto, R. I. (1994) Human heat shock factors 1 and 2 are differentially activated and can synergistically induce hsp70 gene transcription. Mol. Cell. Biol. 14,2087-2099[Abstract/Free Full Text]
  57. Chen, B. C., Yu, C. C., Lei, H. C., Chang, M. S., Hsu, M. J., Huang, C. L., Chen, M. C., Sheu, J. R., Chen, T. F., Chen, T. L., et al (2004) Bradykinin B2 receptor mediates NF-kappaB activation and cyclooxygenase-2 expression via the Ras/Raf-1/ERK pathway in human airway epithelial cells. J. Immunol. 173,5219-5228[Abstract/Free Full Text]
  58. Sattler, M., Durstin, M. A., Frank, D. A., Okuda, K., Kaushansky, K., Salgia, R., Griffin, J. D. (1995) The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases. Exp. Hematol. 23,1040-1048[Medline]
  59. Sweatt, J. D. (2001) The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. J. Neurochem. 76,1-10[CrossRef][Medline]
  60. Genersch, E., Hayess, K., Neuenfeld, Y., Haller, H. (2000) Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways. J. Cell Sci. 113,4319-4330[Abstract]
  61. Levy, D. E., Lee, C. K. (2002) What does Stat3 do?. J. Clin. Invest. 109,1143-1148[CrossRef][Medline]
  62. Shaw, S., Bencherif, M., Marrero, M. B. (2002) Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1–42) amyloid. J. Biol. Chem. 277,44920-44924[Abstract/Free Full Text]
  63. Arredondo, J., Nguyen, V. T., Chernyavsky, A. I., Bercovich, D., Orr-Urtreger, A., Vetter, D. E., Grando, S. A. (2003) Functional role of {alpha}7 nicotinic receptor in physiological control of cutaneous homeostasis. Life Sci. 72,2063-2067[CrossRef][Medline]
  64. Zia, S., Ndoye, A., Nguyen, V. T., Grando, S. A. (1997) Nicotine enhances expression of the {alpha}3, {alpha}4, {alpha}5, and {alpha}7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells. Res. Commun. Mol. Pathol. Pharmacol. 97,243-262[Medline]
  65. Zhang, S., Day, I. N., Ye, S. (2001) Microarray analysis of nicotine-induced changes in gene expression in endothelial cells. Physiol. Genomics 5,187-192[Abstract/Free Full Text]
  66. Zhang, S., Day, I., Ye, S. (2001) Nicotine induced changes in gene expression by human coronary artery endothelial cells. Atherosclerosis 154,277-283[CrossRef][Medline]
  67. Maneckjee, R., Minna, J. D. (1994) Opioids induce while nicotine suppresses apoptosis in human lung cancer cells. Cell Growth Differ. 5,1033-1040[Abstract]
  68. Cox, M. E., Parsons, S. J. (1997) Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells. J. Neurochem. 69,1119-1130[Medline]
  69. Tang, K., Wu, H., Mahata, S. K., O’Connor, D. T. (1998) A crucial role for the mitogen-activated protein kinase pathway in nicotinic cholinergic signaling to secretory protein transcription in pheochromocytoma cells. Mol. Pharmacol. 54,59-69[Abstract/Free Full Text]
  70. Theilig, C., Bernd, A., Ramirez-Bosca, A., Gormar, F. F., Bereiter-Hahn, J., Keller-Stanislawski, B., Sewell, A. C., Rietbrock, N., Holzmann, H. (1994) Reactions of human keratinocytes in vitro after application of nicotine. Skin Pharmacol. 7,307-315[Medline]
  71. Kwon, O. S., Chung, J. H., Cho, K. H., Suh, D. H., Park, K. C., Kim, K. H., Eun, H. E. (1999) Nicotine-enhanced epithelial differentiation in reconstructed human oral mucosa in vitro. Skin Pharmacol. Applied Skin Physiol. 12,227-234
  72. Heeschen, C., Jang, J. J., Weis, M., Pathak, A., Kaji, S., Hu, R. S., Tsao, P. S., Johnson, F. L., Cooke, J. P. (2001) Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat. Med. 7,833-839[CrossRef][Medline]
  73. Joad, J. P., Pinkerton, K. E., Bric, J. M. (1993) Effects of sidestream smoke exposure and age on pulmonary function and airway reactivity in developing rats. Pediatr. Pulmonol. 16,281-288[Medline]
  74. Joad, J. P., Ji, C., Kott, K. S., Bric, J. M., Pinkerton, K. E. (1995) In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 132,63-71[CrossRef][Medline]
  75. Hoffmann, D., Adams, J. D. (1981) Carcinogenic tobacco-specific N-nitrosamines in snuff and in the saliva of snuff dippers. Cancer Res. 41,4305-4308[Medline]
  76. Lindell, G., Farnebo, L. O., Chen, D., Nexo, E., Rask Madsen, J., Bukhave, K., Graffner, H. (1993) Acute effects of smoking during modified sham feeding in duodenal ulcer patients an analysis of nicotine acid secretion gastrin catecholamines epidermal growth factor prostaglandin E-2 and bile acids. Scand. J. Gastroenterol. 28,487-494[Medline]
  77. Russell, M. A., Jarvis, M., Iyer, R., Feyerabend, C. (1980) Relation of nicotine yield of cigarettes to blood nicotine concentrations in smokers. Br. Med. J. 280,972-976[Medline]



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