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Cancer Research 67, 6863, July 15, 2007. doi: 10.1158/0008-5472.CAN-07-0483
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Nongenomic ß Estrogen Receptors Enhance ß1 Adrenergic Signaling Induced by the Nicotine-Derived Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone in Human Small Airway Epithelial Cells

Mourad Majidi1, Hussein A. Al-Wadei1, Takashi Takahashi2 and Hildegard M. Schuller1

1 Experimental Oncology Laboratory, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee and 2 Division of Molecular Carcinogenesis, Center of Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan

Requests for reprints: Hildegard M. Schuller, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN 37996. Phone: 865-974-8217; Fax: 865-974-5616; E-mail: hmsch{at}utk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Women are at higher risk for the development of lung adenocarcinoma than men; however, the mechanisms responsible for this are poorly understood. In lung adenocarcinoma cells, the estrogen receptor ß (ERß) is the predominating form. We found that 17ß-estradiol enhanced proliferation of the putative cells of origin of lung adenocarcinoma, small airway epithelial cells (HPLD1), in response to the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Reverse-phase protein microarrays combined with Western blotting revealed that NNK induced phosphorylation of ERß, an effect that involved stimulation of the adrenergic receptors ß1 (ß1AR). In transiently transfected cells, ß1AR coprecipitated with ERß, which increased with NNK treatment. ERß enhanced NNK-induced cyclic AMP accumulation as well as G{alpha}i-mediated mitogen-activated protein kinase/extracellular signal–regulated kinase (ERK) 1/2 activation. Coexpression of ß1AR and ERß activated NNK-mediated ERK1/2 cooperatively. ERß gene knockdown, as well as coexpression of the dominant negative Ras and Raf, reduced stimulation of ERK1/2 by NNK. Whereas NNK phosphorylated Akt at Thr308 and Ser473, ERß had no effect on this activity. Luciferase reporter assays showed that, in response to NNK, ERß stimulated transcription of serum responsive element (SRE) but had a very small effect on the activity of estrogen responsive element (ERE). Together, the phosphorylation of ERß, the dependence on G{alpha}i proteins, the activation of ERK1/2, and the preferential targeting of SRE over the classic ERE pathway support a role for nongenomic ERß in the development of smoking-associated lung cancer. This novel cooperation between ß1AR and ERß signaling may contribute to the prominence of lung adenocarcinoma in women. [Cancer Res 2007;67(14):6863–71]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Smoking is associated with ~90% of all lung cancer cases. Adenocarcinoma of the lung is thought to arise from the epithelial lining cells of small airways and is the most common type of lung cancer today (1, 2). The risk for the development of lung adenocarcinoma is significantly greater in women than men; however, the reasons for this gender difference are poorly understood (3).

Estrogen signaling has been shown to play an important role in lung biology and pathology (4). Although the data are still conflicting, an association between high expression levels of estrogen receptors (ER) and occurrence of pulmonary adenocarcinoma has been reported (5–10). The lung is an estrogen-responsive organ, and ERß is the predominant form in pulmonary adenocarcinoma (7). In female transgenic mice, inactivation of this receptor resulted in severe morphologic aberrations of the lung, thus revealing a crucial role for ERß in lung biogenesis (11–13). The classic genomic estrogen pathway involves mostly the association of 17ß-estradiol (E2) with nuclear ER{alpha} and ERß receptors. ERs regulate gene transcription through direct interaction with specific estrogen responsive elements (ERE) and by interaction with other transcription factors (e.g., Sp1 and AP-1) bound to their response elements (14, 15). However, recent reports have also shown estrogen action at the membrane levels. Nongenomic ER activity induces rapid activation of multiple signal transduction pathways, including the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 (16–19).

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most powerful cancer-causing agent in tobacco products. NNK induces development of lung adenocarcinoma in all laboratory rodents tested (20, 21). Its metabolites interact with DNA to form DNA methyl and pyridyloxobutyl adducts, which are thought to be crucial for its carcinogenic effects (22–24). In addition to its genotoxic effects, we have previously shown that NNK is an agonist for the adrenergic receptors ß1 and ß2 (ß1AR and ß2AR). Furthermore, we have shown that NNK stimulates proliferation of pulmonary adenocarcinoma and the putative cells of origin of this cancer, small airway epithelial cells, through a cross talk between ß1AR and epidermal growth factor receptors (EGFR; refs. 25, 26). Other reports have shown that NNK-induced ß-adrenergic signaling was associated with cell survival and growth regulation in several other non–small-cell and small-cell lung cancer cell lines and in colon cancer cells (27, 28).

To determine whether ERß directly modulates NNK intracellular signaling, we overexpressed and knocked down these receptors in the human immortalized small airway epithelial cell line HPL1D and analyzed the responses to NNK exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell line and tissue culture. The human immortalized small airway epithelial cell line HPL1D (29) was cultured at 37°C in phenol red–free Ham's F12 supplemented with 1% charcoal-stripped FCS, 5 µg/mL insulin, 5 µg/mL human transferrin, 50 nmol/L hydrocortisone hemisuccinate, 4.75 pmol/L 3,3',5'-triiodo-L-thyronine, and 50 nmol/L Na-selenite.

Chemicals and antibodies. NNK was purchased from Chemsyn Laboratories. E2 and pertussis toxin were purchased from Calbiochem. Antiestrogen ICI 182780 and adrenergic receptor antagonists atenolol and propanolol were obtained from Tocris. ERK1/2, phospho-ERK1/2 (Thr202/Tyr204) antibodies, and horseradish peroxidase–conjugated antirabbit secondary antibodies were purchased from Cell Signaling Technology. ERß, phospho-ERß (Ser87), and ER{alpha} antibodies were purchased from Santa Cruz Biotechnology. Alexa Fluor 680 probes were obtained from Molecular Probes.

Treatment protocol. Cells were starved in basal medium without serum or additives for 16 to 24 h before treatment with 100 nmol/L E2 for 10 min or 1 µmol/L NNK for 1 h. Simultaneous exposure to E2 and NNK was done by pretreating with 100 nmol/L E2 for 10 min and then removing the medium and replacing it with basal medium containing 1 µmol/L NNK for 1 h.

Transient transfection and RNA interference induction. ERß pBI-EGFP, ER{alpha} pBI-EGFP, pERE-tat-Luc, and pSRE-Luc were kindly provided by Dr. Jay Wimalasena. ß1AR cDNA was provided by Dr. R.J. Lefkowitz (Department of Biochemistry, Duke University Medical Center, Durham, NC). Cells were plated at 4 x 105 per 10-cm-diameter plate and grown for 24 h. The cells were cotransfected with 2 µg of the indicated expression vector and 500 ng of pcMV-LacZ to monitor transfection efficiency using Lipofectamine reagents (Invitrogen). Cells transfected with empty vector (pBI-cDNA) served as controls. ERß gene knockdown was accomplished by transfecting cells with 40 nmol/L ERß Stealth RNA interference (RNAi; ref. 30). Small interfering RNA (siRNA) transfection was done in the presence of Lipofectamine 2000 according to the manufacturer's protocol. The siRNA and Lipofectamine were diluted with Opti-MEMI medium (Invitrogen), combined together, and incubated for 20 min before adding the mixture to the cells. To determine whether siRNA blocked ERß expression, cells were harvested 72 h posttransfection for evaluation of ERß proteins by Western blotting. As a negative control for the effects that may be associated with siRNA delivery, we transfected cells with RNAi that has high GC duplexes (Invitrogen). The primers used to target ERß mRNA sequence for interference were sense, CCAGCAAUGUCACUAACAU, and antisense, AAGUUAGUGACAUUGCUGG.

Cell proliferation assay. The quantification of HPL1D cell proliferation was based on the measurement of bromodeoxyuridine (BrdUrd) incorporation according to manufacturer's instructions (Roche Applied Science). In 96-well plates, 10,000 cells were cultured in basal phenol red–free medium for 24 h and either left alone or exposed to 100 nmol/L or 1 µmol/L NNK for 1 h; pretreated with 100 nmol/L E2 for 10 min followed by NNK stimulation for 1 h; and pretreated with 100 nmol/L ICI 182780, or 1 µmol/L propanolol, or 1 µmol/L atenolol for 10 min before simultaneous stimulation by E2 and NNK. At the end of each treatment period, the medium was removed and replaced with basal medium without treatment agents. In parallel, cells were transfected with 40 nmol/L ERß siRNA or ER{alpha} siRNA and treated under the same condition as their untransfected counterparts. Each experiment was run twice with three samples per treatment group and BrdUrd incorporation was determined (31). The statistical significance of differences was calculated by one-way ANOVA and two-tailed t test analysis; P < 0.05 was considered significant.

Cyclic AMP immunoassay. Cells were grown until 80% to 85% confluence before transfection with 2 µg of ERß expression plasmid. Forty-eight hours posttransfection, cells were fed with phenol red–free basal medium for 24 h. Following two washes with 1x PBS, cells were treated with 1 µmol/L NNK for 1 h or pretreated with 100 nmol/L E2 for 10 min before NNK stimulation in medium containing 1 mmol/L I-methyl-3-isobutylxanthine. Then, cells were treated with 0.1 N HCl for 10 min, lysed by sonication, and samples were analyzed for cyclic AMP (cAMP) levels with a direct cAMP immunoassay kit according to the manufacturer's instructions (Assay Designs, Inc.). The statistical significance of differences was calculated by one-way ANOVA and two-tailed t test analysis; P < 0.05 was considered significant.

Reverse-phase protein microarray assay. Cells were lysed in a buffer containing 6 mol/L urea (Sigma), 65 mmol/L DTT, 2% Pharmalyte (pH 8–10.5), and 1% CHAPS (32). Samples were cleared by centrifugation at 4°C for 15 min. Approximately 60 nL of each sample were spotted onto nitrocellulose-coated glass slides (Schleicher & Schuell Bioscience) using a pin-in-ring format Affymetrix GMS 417 Arrayer (Affymetrix). Each sample was spotted in triplicate in a stepwise series of 2-fold dilutions 1, 2, 4, 8, 16, and 32. The arrayed slides were incubated with primary antibody for 1 h, washed, and reincubated in cognitive secondary antibody conjugated to Alexa Fluor 680 probe and scanned with GenePix 4000B microarray scanner (Axon Instruments). Spot images were converted to raw pixel values and analyzed by a modified version of GenePiX 5.1 software. The background was subtracted from the intensity values.

Western blot and immunoprecipitation. Equal amounts of protein were resolved on SDS-polyacrylamide gels (12% acrylamide), transferred to nitrocellulose membranes, and probed with the appropriate antibodies. For immunoprecipitation, cell lysates were incubated with 2 µg of the appropriate antibody and precleared with protein A/G-Sepharose for 2 h. Immunocomplexes were washed with 1x immunoprecipitation buffer [50 mmol/L Tris-base (pH 7.5), 5 mmol/L EDTA, 20 mmol/L ß-glycerol-phosphate, 150 mmol/L NaCl, and 1% NP40 IP]. Proteins were eluted by boiling in 2x sample buffer, separated by SDS-PAGE (12%), and then transferred to nitrocellulose membrane. Enhanced chemiluminescence (ECL-plus) was used for detection.

ERK1/2 kinase assay. Equal amounts of proteins were incubated overnight with 15 µL of agarose hydrazide beads immobilized p44/42 (Cell Signaling Technology). Immunoprecipitates were washed thrice in 100 mmol/L Tris (pH 7.5), 1% NP40, 2 mmol/L sodium orthovanadate; once in 100 mmol/L Tris (pH 7.5), 0.5 mol/L lithium chloride; and once in kinase buffer [12.5 mmol/L MOPS (pH 7.5), 12.5 mmol/L ß-glycerophosphate, 7.5 mmol/L MgCl2]. Proteins were incubated for 20 min at 30°C in a 30-µL kinase reaction containing 2-µg Elk-1 fusion protein (GST-Elk1 codons 307–428) and 10 µmol/L ATP. Proteins were separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed with anti–phospho-Elk (Ser383) antibody. After incubation with the secondary antibody, bands were visualized by enhanced chemiluminescence.

Luciferase reporter assays. Cells were cotransfected with 500 ng of pRSV-ß-galactosidase and 1 µg of ERE-Luc, or serum responsive element (SRE)-Luc cDNAs, alone or along with 2 µg ERß or 40 nmol/L ERß siRNA constructs (33). Twenty-four hours posttransfection, cells were deprived of serum for 18 h before stimulation with the indicated agents. Untransfected and transfected cells with the empty vector, either stimulated with the same agents or left alone, served as controls. Cells were harvested 24 h later and luciferase and ß-galactosidase activities were then measured using standard luciferase (Promega) and ß-galactosidase detection kits (Applied Biosystems).

Statistical analysis. All data shown as bar graphs are expressed as the mean ± SE. The statistical significance of differences was calculated by one-way ANOVA and two-tailed t test analysis (34). Two-tailed P ≤ 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 stimulates NNK-dependent BrdUrd incorporation. We have reported that NNK stimulated proliferation of the putative cells of origin of lung adenocarcinoma, small airway epithelial cells (HPLD1), via ß-adrenergic receptor signaling (25, 26). To determine whether estrogen modulates this response, we analyzed proliferation of these cells after exposure to E2 for 10 min, exposure to NNK for 1 h, and pretreatment with E2 for 10 min before exposure to NNK for 1 h. In addition, cells expressing ERß or ER{alpha} siRNA were treated in similar fashion. To determine whether siRNA blocked ERß expression, cells were analyzed for ERß proteins by Western blotting 72 h posttransfection. As a negative control for the effects that may be associated with siRNA delivery, we used oligos that consist of >50% GC content (Invitrogen). As expected, stimulation with 1 µmol/L NNK produced a 4-fold increase in BrdUrd incorporation (Fig. 1A ). Pretreatment with E2 enhanced BrdUrd levels by almost 8-fold. Exposure to 100 nmol/L ICI 182780 for 10 min had a slight reducing effect on E2 + NNK stimulation. By contrast, treatment with 1 µmol/L ß1 and ß2AR antagonist, propranolol, or the site-selective antagonist for ß1AR, atenolol, for 10 min reduced this response to E2 and NNK significantly (P < 0.05). BrdUrd incorporation in cells expressing ERß siRNA or ER{alpha} siRNA treated simultaneously with E2 and NNK was reduced to 4-fold and 7-fold, respectively (Fig. 1B). These results suggest that in these cells, E2 and NNK stimulation promoted entry into S phase at a significantly higher rate than either compound alone. As indicated by the greater effect of ER-ß silencing, this activity involves predominately ERß signaling via ß1AR.


Figure 1
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  		Figure 1. Estrogen enhances NNK-induced proliferation of HPL1D cells. Untransfected (A) and transfected (B) cells were exposed to NNK for 1 h with or without pre-exposure to E2 (10 min). Control groups consist of untransfected and untreated cells and cells transfected with RNAi negative control high-GC duplexes. After correction for the internal standard, BrdUrd incorporation was determined. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, P < 0.05, compared with control. **, P < 0.05, compared with E2 + NNK–stimulated cells. Expression levels of ER{alpha}, ERß, and actin were monitored by Western blot.

 
NNK induces phosphorylation of ERß. We focused on ERß instead of ER{alpha} because in lung cancer cells, the former seems to be the predominating form (1, 10, 11). In addition, the tested cell line expresses more of ERß proteins (data not shown). Furthermore, the proliferation results showed that interfering with ER{alpha} reduced the induction in response to E2 + NNK less than silencing of ERß. First, we used reverse-phase protein microarrays to analyze phosphorylation of ERß in response to 100 nmol/L and 1 µmol/L NNK stimulation. For validation purpose, increasing concentrations of HPL1D lysates (60 ng–1 µg) were spotted onto nitrocellulose-coated slides and probed with antimouse conjugated with Alexa Fluor 680. To emulate a cell lysate, Alexa labelings were carried out in a background of 1% bovine serum albumin. Control slides consisted of arrayed lysates without the primary antibody and unprinted slides with the primary antibody and the Alexa probe. The arrays were sensitive enough to detect 60 ng of spotted lysates. A linear increase in spot intensity was observed with increasing amounts of proteins. No immunoreactivity was observed in either control, which shows the specificity of the microarray (data not shown).

The results show that in response to 100 nmol/L and 1 µmol/L NNK exposure, phosphorylation of ERß (Ser87) was stimulated by 1.5- and 5-fold, respectively (Fig. 2A ). This response to NNK increased by 9-fold when cells were transfected with ERß plasmid. By contrast, pretreatment with 1 µmol/L atenolol for 10 min reduced phosphorylation of ERß (Ser87) by NNK significantly (P < 0.05). These findings are in accord with our previous observation that NNK stimulates cell proliferation by a mechanism that involves ligand binding of NNK to the ß1AR (25, 26).


Figure 2
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  		Figure 2. NNK stimulates ERß phosphorylation in a dose-dependent manner and induces ERß-ß1AR complex formation. A, ERß transfected and untransfected HPL1D were exposed to 100 nmol/L to 1 µmol/L NNK for 1 h with or without pre-exposure to 100 nm E2 (10 min) or 1 µmol/L atenolol (10 min). Phosphorylation of ERß in these samples was analyzed by reverse-phase proteomics, and spot images were generated with GenePix 4000B microarray scanner. In a parallel, ERß proteins were immunoprecipitated with ERß antibody, immunoblotted with p-ERß (Ser87), and analyzed with autoradiography. In addition, 250 µg of precleared protein lysates from ERß and ß1AR cotransfectants were either immunoprecipitated (IP) with anti-ERß and immunoblotted (IB) with ß1AR antibodies (B), or immunoprecipitated with ß1AR and immunoblotted with ERß antibodies (C). Columns, mean of three different experiments; bars, SD. *, P < 0.05, compared with untransfected and untreated cells (control). **, P < 0.05, compared with ERß transfectants treated with 1 µmol/L NNK. Expression levels of ERß and ß1AR were monitored by Western blot.

 
To further substantiate these observations, lysates from untransfected and ERß transfected cells were immunoprecipitated with ERß antibody, immunoblotted with p-ERß (Ser87) antibody, and analyzed for phosphorylation with Western blots. The results were similar to those obtained by the reverse-phase approach, showing the validity of the assay (Fig. 2A). Together, these findings show that NNK induced phosphorylation of ERß in a concentration-dependent manner, and that this effect required ß1AR. The rapid ERß phosphorylation and the dependency on membrane ß1AR activation suggest that nongenomic ERß modulates NNK intracellular signaling.

ERß coprecipitates with ß1AR and NNK further promotes this complex. Cells were transiently cotransfected with ERß and ß1AR plasmids and treated with 1 µmol/L NNK for 1 h. Immunoblot analysis of proteins immunoprecipitated with ERß (Fig. 2B) or ß1AR antibody (Fig. 2C) revealed that under basal conditions, both proteins coprecipitated. However, when cells were treated with NNK, complex formation between ERß and ß1AR was significantly evident (P < 0.05). These results suggest that NNK promotes ERß-ß1AR complex formation.

ERß enhances NNK-dependent cAMP activity. Previous studies have reported increase of cAMP levels as a result of membrane E2 receptor activation (35, 36). To determine whether phosphorylation of ERß by NNK used intracellular cAMP as a second messenger, we measured its levels in ERß transfectants after exposure to 100 nmol/L E2 for 10 min; 1 µmol/L NNK for 1 h; or pretreatment with E2 before stimulation with NNK. Control samples consisted of untransfected cells and empty vector–transfected cells. NNK treatment of untransfected cells increased cAMP levels by 6-fold as compared with basal levels (Fig. 3 ). Pretreatment with E2 slightly increased cAMP response to NNK. On the other hand, ERß transient expression enhanced NNK stimulation by almost 15-fold. E2 pretreatment had no significant effect on this activity. These findings show that overexpression of ERß enhanced NNK-induced cAMP activation significantly (P < 0.05).


Figure 3
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  		Figure 3. ERß enhances NNK-induced cAMP activity. Transfected and untransfected HPL1D cells were stimulated with 1 µmol/L NNK for 1 h or pre-exposed to 100 nmol/L E2 for 10 min before NNK stimulation. The levels of cAMP were evaluated at 405 nm. Columns, mean fold increase of cAMP over untreated controls in untransfected cells from three different experiments, each with one sample per group; bars, SD. *, P < 0.05, compared with untransfected and untreated cells (control). **, P < 0.05, compared with untreated ERß transfectants.

 
ERß enhances NNK-dependent ERK1/2 enzymatic activity. Recently, we have reported that in these cells, NNK activates the MAPK ERK1/2 signaling pathway via ß1AR-mediated transactivation of EGFR (26). To determine whether ERß modulates ERK1/2 response to NNK, cells were transfected with 2-µg ERß plasmid, serum starved, and then stimulated as indicated. ERK1/2 enzymatic activity was analyzed by an in vitro kinase assay. As shown by GST-Elk-1 fusion protein phosphorylation, E2 and NNK, alone, stimulated ERK1/2 by 2- and 3.5-fold, respectively (Fig. 4A ). E2 and NNK cotreatment produced an 11-fold increase. Transient expression of ERß enhanced ERK1/2 response to NNK by 9-fold.


Figure 4
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  		Figure 4. ERß stimulates NNK-induced ERK1/2 enzymatic activity. Kinase reactions and Western blot were done in anti–phospho-ERK1/2 immunoprecipitates using phospho-Elk1 as a substrate. A, ERß-transfected and untransfected HPL1D were treated with 1 µmol/L NNK for 1 h or pre-exposed to 100 nmol/L E2 for 10 min before NNK stimulation. B, cells were pretreated with 100 nmol/L pertussis toxin (PTX) for 10 min before exposure to E2 and NNK or with expressed ERß siRNA before treatment with E2 and/or NNK. C, cells cotransfected with 2-µg ERß and 2-µg ß1AR and treated as indicated. D, cells were cotransfected with 2 µg ERß, 2-µg ß1AR, and 2- µg Ras17N or RafC4 and treated as indicated. Spot images from reverse proteomic analysis and autoradiograms are shown. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, compared with untransfected and untreated cells (control). **, P < 0.05, compared with E2 + NNK–stimulated cells. ***, P < 0.05, compared with untreated cells cotransfected with ERß and ß1AR expression plasmids. Expression levels of ERß and ERK1/2 were monitored by Western blot.

 
The effects of pertussis toxin, an inhibitor for G protein G{alpha}i, and ERß gene knockdown were analyzed. We found that pertussis toxin exposure for 10 min blocked ERK1/2 response to E2 and NNK (Fig. 4B). We tested several Stealth RNAi oligos targeting different ERß mRNA sequences and found one transcript that exerted the highest level of suppression of total ERß expression. The results show that interfering with ERß transcription reduced ERK1/2 response to E2 and NNK to almost half (P < 0.05). This suggests a functional role for nongenomic ERß in NNK action on G{alpha}i-mediated ERK1/2 signaling.

Coexpression of ß1AR and ERß enhances ERK1/2 activity synergistically. Cells were cotransfected with 2-µg ERß and 2-µg ß1AR, starved for 24 h, and stimulated with 100 nmol/L E2 for 10 min or 1 µmol/L NNK for 1 h. As shown in Fig. 4C, whereas E2 treatment of ERß and ß1AR cotransfectants enhanced ERK1/2 activity by 4-fold, NNK stimulation resulted in a 12-fold increase. This cooperative effect shows the involvement of ß1AR in ERß-mediated NNK signaling.

ERß stimulation of NNK-dependent ERK1/2 requires Ras and Raf proteins. Recently, we have reported Raf overexpression in NNK-induced pulmonary adenocarcinoma in hamsters (37, 38). Therefore, we cotransfected cells with ERß and the dominant negative Ras (Ras17N) or Raf (RafC4) and analyzed the effect of NNK on ERK1/2 activity. Figure 4D shows that transient expression of Ras17N as well as RafC4 blocked activation of ERK1/2 by E2 and NNK (P < 0.05). In addition, it interfered with the ability of ERß to stimulate ERK1/2 in response to NNK. These findings indicate that ERß enhances the actions of NNK on ERK1/2 via Ras and Raf proteins.

ERß overexpression had no apparent effect on NNK-induced Akt phosphorylation. West et al. (39) have reported that in normal human airway epithelial cells, NNK activates Akt. As expected, we found that in these cells, NNK stimulated Akt phosphorylation at two sites, Thr308 and Ser473 (Fig. 5A and B ). However, pretreatment with E2 or ERß overexpression did not significantly increase this activity (P > 0.05), suggesting that in these cells, the actions of ERß on NNK-induced ERK1/2 activity do not involve the Akt pathway.


Figure 5
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  		Figure 5. ERß does not enhance NNK-induced activation of Akt. ERß-transfected and untransfected HPL1D were treated with 1 µmol/L NNK for 1 h or pre-exposed to 100 nmol/L E2 for 10 min before NNK stimulation. Proteins were immunoprecipitated with Akt antibody and immunoblotted with either p-Akt Ser473 (A) or p-Akt Thr308 (B). Blots were analyzed by autoradiography. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, compared with untransfected and untreated cells (control).

 
ERß transient expression constitutively activates G{alpha}i-mediated serum responsive element transcription but slightly increases ERE activity. Phosphorylation of Elk-1 by ERK1/2 binds and stimulates transcription of the serum responsive elements (SRE; refs. 40, 41). Therefore, we analyzed the effects of ERß overexpression and NNK stimulation on SRE transcription. Cells were either transfected with SRE-Luc reporter construct, alone or cotransfected with ERß, or cotransfected with ERß siRNA and treated with the indicated agents. The results show that in untransfected cells exposed to 10 nmol/L, 100 nmol/L, and 1 µmol/L NNK, SRE-Luc activity increased by 3-, 14-, and 17-fold, respectively (Fig. 6A ). Whereas E2 alone (100 nmol/L) produced a little more than 2-fold induction, addition of 1 µmol/L NNK stimulated this activity by 27-fold. Pertussis toxin blocked this synergy. Exposure of ERß transfectants to 10 nmol/L, 100 nmol/L, and 1 µmol/L NNK increased SRE-Luc activity by 4-, 19-, and 20-fold, respectively (Fig. 6B). ERß gene knockdown reduced NNK-induced SRE transactivation by 2-, 5-, and 8-fold.


Figure 6
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  		Figure 6. ERß stimulates NNK-dependent SRE gene expression but has no marked effect on ERE activity. HPL1D cells were cotransfected with expression plasmids and stimulated as indicated. SRE luciferase activity (A and B) and ERE luciferase activity (C and D) were measured with a luminometer. Columns, mean fold inductions of luciferase activity corrected for the internal ß-galactosidase from three independent experiments done in duplicate; bars, SD. *, P < 0.05, compared with untreated SRE-transfected cells (control). **, P < 0.05, compared with SRE-transfected cells treated with E2. ***, P < 0.05, compared with SRE-transfected cells treated with NNK and/or E2 and NNK.

 
Lastly, we assessed the effect of ERß overexpression on NNK-mediated ERE responses, the classic ligand-dependent E2-ER complexes (42). Contrary to the potent stimulation of SRE, we found a slight increase in ERE-Luc activity in response to E2 or NNK alone. This activity was not significantly enhanced by cotreatment with both agents (P > 0.05; Fig. 6C and D). Together, these findings show that the high increase of SRE transcriptional activity by E2 and NNK is regulated by G{alpha}i. The fact that ERE transcription was not markedly enhanced suggests that NNK signaling involves mostly nongenomic ERß.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the role and expression of ERs in lung tumors have been controversial for many years, recent studies have shown that ERß is the predominating form (9–11). In this investigation, we used the human small airway epithelial cell line HPL1D to determine whether ERß signaling modulates NNK-induced ß-adrenergic signaling. We found that rapid exposure to E2 significantly enhanced cell proliferation in response to NNK. The molecular mechanisms underlying this cooperative effect remain to be defined. However, the magnitude of this response after a short-term application strongly suggests the involvement of nongenomic ERß. The inhibitory effects of adrenergic receptor ß-blockers, propanolol and atenolol, on E2/NNK synergy suggest that each agent acts at a distinct set of receptors at the membrane level, NNK at ß1AR and E2 at ERß. Thus, the signaling persistence at these sites may induce ERß/ß1AR complex formation with changes in the pattern of phosphorylation. This will result in the loss of normal regulatory constraints and confer a growth advantage. Alternatively, E2 and NNK may have affinity for the same receptors, including ERß, and use the same signaling pathways to activate specific genes important for proliferation. Therefore, simultaneous treatment with E2 and NNK would produce higher proliferative responses than either compound alone. The fact that the classic antiestrogen ICI 182780 had only a slight negative effect on E2 + NNK–induced proliferation of these cells suggests the involvement of predominantly nongenomic ERß.

Next, we analyzed the activation status of ERß. We found that at nanomolar concentrations, NNK stimulated phosphorylation of ERß (Ser87) in a dose-dependent manner. This activity was markedly enhanced by ERß overexpression and substantially reduced after atenolol exposure. These findings suggest that the action of NNK on ERß is regulated by the transmembrane receptor ß1AR. This would support a role for nongenomic ERß in NNK-induced signaling activities. Perhaps, NNK-activated ß1AR recruits membrane or cytosolic ERß, and this may explain the rapid action of NNK on the latter. Nevertheless, the results clearly indicate that ß1AR contributed to the marked phosphorylation of ERß by NNK, therefore suggesting a cross talk between the two pathways. Our coprecipitation experiments clearly show that in these cells, NNK enhanced the small relative amount of ß1AR and ERß that are complexed together. Additional studies will be needed to clarify the role of ERß modulation of NNK-induced ß1AR activity. Determination of the precise stereochemistry of ß1AR/ERß interaction would be of great interest, considering the biological consequences of this interaction.

When we exposed cells to both E2 and NNK, cAMP activity increased only slightly. A possible explanation for these results is that E2 and NNK stimulate different adenylyl cyclase isozymes contributing to basal cAMP production; therefore, costimulation with both agents may not produce a synergistic accumulation of cAMP. Alternatively, cAMP increase in response to NNK was already at maximum, or non–adenylyl cyclase–dependent signaling pathways may have contributed to the observed slight increase. ERß transfectants stimulated with NNK exhibited the highest levels of cAMP, much more than their counterparts treated with E2. This suggests that the contribution of the nongenomic cAMP pathway of ERß action is significant and further strengthens the argument for the implication of nonnuclear ERß.

Recently, we have reported that NNK activates the MAPK/ERK1/2 pathway via ß1AR transactivation of EGFR in HPL1D and human pulmonary adenocarcinoma cells (26). When we transfected cells with ERß construct and applied NNK, ERK1/2 enzymatic activity increased pronouncedly. In contrast, ERß gene knockdown reduced NNK-dependent ERK1/2 activation to about half. The fact that ERß down-regulation did not completely abrogate ERK1/2 stimulation by NNK suggests that the latter targets nongenomic ERß indirectly via cross talks with other membrane receptors. One scenario would be that NNK induces bidirectional signaling between nonnuclear ERß and ß1AR, and possibly others. This would explain the synergistic activation of ERK1/2 in ß1AR and ERß cotransfectants.

The G{alpha}i inhibitor pertussis toxin interfered with the ability of E2 and NNK to activate ERK1/2 cooperatively. This indicates that E2-NNK signaling complexes operate via a G{alpha}i-dependent mechanism. Perhaps, E2 exposure leads to G{alpha}s activation, then NNK stimulation results in switching from G{alpha}s to G{alpha}i. In addition, we cannot exclude the possibility that both agents might induce sequential activation of G{alpha}s and G{alpha}i proteins. Similar mechanisms have been described during ERK1/2 activation by ß-adrenergic receptors in Chinese hamster ovary cells (43, 44).

Activating mutations in K-Ras are one of the most common genetic alterations in human lung adenocarcinoma (45–47). It is evident from the observed blocking effects of Ras17N and RafC4 that the resulting signals from E2/ERß and NNK stimulation converge onto the Ras-Raf–mediated ERK1/2 pathway.

Next, we found NNK-stimulated phosphorylation of Akt at Thr308 and Ser473; however; pretreatment with E2 or ERß overexpression did not enhance this activity. Akt is commonly phosphorylated at Thr308 and Ser473 in tumor cells through activation of Ras. This protein has been shown to contribute to NNK-induced cell proliferation in a nicotinic acetylcholine receptor–dependent manner (39, 48). Ligand-binding of NNK to nicotinic acetylcholine receptors resulting in the activation of ERK1/2 and c-myc and subsequent stimulation of cell proliferation was first reported in human small-cell lung cancer cell lines and their putative cells of origin, pulmonary neuroendocrine cells (49, 50). The high affinity of NNK to members of both the ß-adrenergic and nicotinic acetylcholine receptor families likely contributes to the exceptional carcinogenic potency of this tobacco nitrosamine. The fact that E2 did not enhance NNK-induced Akt phosphorylation suggests that ERß cooperates with ß-adrenergic but not nicotinic receptor signaling. This interpretation is in accord with a recent report showing that nicotinic receptor–mediated activation of Akt by NNK was not inhibited by ß-adrenergic antagonists (48).

The final part of this investigation analyzed the transcriptional activity of SRE and ERE gene expression using luciferase reporter gene assays. We found that E2 and NNK cotreatment stimulated SRE synergistically and that ERß overexpression increased this response significantly (P < 0.05, two-tailed t test). ERß gene knockdown reduced NNK stimulation of SRE as to about half. In contrast to SRE, transcription of ERE was slightly affected by ERß overexpression or by E2 and NNK costimulation. Because of the short duration of exposure, the involvement of G proteins, and the rapid activation of MAPK/ERK1/2, we expected a little to no effect on ERE transcription. One possible explanation for the slight increase in ERE is that once nongenomic ERßs are activated by NNK, they exert some residual actions on their nuclear counterparts. The data strongly suggest that NNK-induced ERß activation targets predominantly SRE gene expression. ERß modulates NNK activity, as evidenced by the reducing effect of ERß gene knockdown on E2-and NNK-dependent activation of SRE gene expression.

We need to mention that we have tested the actions of ER{alpha} on NNK signaling in HPL1D cells. We found that ER{alpha} overexpression stimulated NNK actions very moderately. In addition, we found that ER{alpha} gene knockdown had very small reducing effects on NNK signaling, thus suggesting that this receptor is not the major estrogen player in NNK-mediated activity in HPL1D cells.

In summary, our data have identified a novel and hitherto unknown cooperation between NNK-induced ß1AR and nongenomic ERß mitogenic signaling, leading to up-regulation of the cAMP/Ras/Raf/ERK1/2/Elk1 signaling pathway in the cell type of origin of lung adenocarcinoma. Although further investigation is required to understand how precisely estrogen and smoking can affect tumor development, the results give new mechanistic insights into the prevalence of lung adenocarcinoma in women. Our current and published data (25, 26) suggest that in a subset of human patients, small airway epithelial cells and the adenocarcinomas derived from them are under ß-adrenergic growth control and that NNK stimulates this pathway in cooperation with the ERß. The levels of expression and the sensitivity of both ß1AR and ERß can be modulated by numerous factors associated with our modern environment and lifestyle. Among such factors are numerous decongestion, cold, and asthma medicines and dietary supplements that increase intracellular cAMP and thereby sensitize the ß-adrenergic receptors. On the other hand, estrogen contraceptives or estrogen replacement therapy, as well as exposure to environmental toxicants with estrogenic activity, may further enhance signaling via stimulation of nonnuclear ERß. Depending on the exposure history, there will thus be significant interindividual variations in ß1AR and ERß signaling in response to NNK. An in-depth understanding of the molecular basis of estrogenic influence on lung adenocarcinoma development in women is urgently needed to improve strategies for the prevention and therapy of this malignancy.


   Acknowledgments
 
Grant support: National Cancer Institute grants RO1 CA088809 and RO1 CA096128.

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

We thank Dr. Jay Wimalasena (UT Medical Hospital, Knoxville, TN) and Dr. H-C.R. Wang (College of Veterinary Medicine, Knoxville, TN) for providing us with expression vectors and for technical advice.

Received 2/ 5/07. Revised 5/ 2/07. Accepted 5/10/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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