1. Activation of MAPK1/2 by Estrogen and Estrogen Mimics
in a Human Lung Adenocarcinoma Cell Line
WHITNEY R. TALBOTT and MARY O. HUFF
Department of Biology, Bellarmine University, Louisville, KY USA
ABSTRACT
Recent studies support a role for estrogen and estrogen mimics in the etiology of lung cancer. In previous studies, estrogen and two estrogen mimics found in cigarette smoke, cadmium chloride and sodium arsenate, induced cellular proliferation in NCI-H1793, a human lung adenocarcinoma cell line derived from a female non-smoker. To determine if this increased proliferation involves the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, cells were treated with 100 nM concentrations of estrogen, cadmium chloride or sodium arsenate for 0 to 90 minutes. At specific time points, cell lysates were collected, and phosphorylation of MAPK1/2 was determined using immunoblot analysis. Induced phosphorylation of MAPK1/2 was observed within 10 minutes of treatment with both estrogen and cadmium chloride. Treatment with sodium arsenate induced phosphorylation within 5 minutes of treatment. In all treatment conditions, maximum phosphorylation was reached within 20 minutes followed by a decline in phosphorylation for the remaining 90 minutes. To determine if phosphorylation of MAPK1/2 involves the estrogen signaling pathway, cells were treated with estrogen, cadmium chloride, or sodium arsenate in the presence or absence of the estrogen receptor antagonist ICI 182,780. Reduction in P-MAPK after exposure to ICI supports the role of the estrogen receptor in activation of MAPK, possibly through estrogen receptors in the cell membrane.
Treatment and preparation of whole cell extracts
Cells were treated with 0.01% ethanol, 10 pM or 100 nM estrogen, 10 nM or 100 nM cadmium chloride, and 100 nM or 100 nM sodium arsenate for times as indicated on each figure. Cell lysates were prepared by washing cells twice in ice cold PBS. Cells were collected in 60 uL lysis buffer using a cell scraper. Samples were sonicated twice at low power and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant were collected and stored at -80°C. Protein concentration was determined using Bio-Rad protein assay (Hercules, CA).
Western blot analysis
Whole cell extracts (30 μg of protein) were separated on 10% polyacrylamide gels and transferred overnight onto PVDF membranes. Each membrane was washed in TTBS, and then placed in blocking buffer (5% milk in TTBS) for 1 hour at room temperature. A polyclonal antibody raised to phosphorylated MAPK (Cell Signaling Technology, Danvers, MA) was diluted to a concentration of 1:1000 in blocking buffer and allowed to incubate overnight at 4°C with shaking. The blot was washed again in TTBS. The membrane was incubated with a 1:3000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody (Pierce Biotechnology, Rockford, IL) one hour at room temperature with shaking. After washing with TTBS, the proteins were visualized using an ECL detection kit (GE Healthcare) as directed by the manufacturer. Images were captured on Amersham Hyperfilm.
To strip the blots, membranes were incubated in 100 mM В Mercaptoethanol, 2% SDS and 62.5 mM Tris pH 6.7 at 60°C for 20 min and then thoroughly washed in TTBS. The blot was placed in blocking buffer for one hour at room temperature, with shaking. It was washed again in TTBS followed by incubation in a 1:1000 dilution of p44/42 MAPK polyclonal rabbit antibody (Cell Signaling Technology) overnight at 4°C, with shaking. The membranes were washed with TTBS and incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit antibody diluted to a concentration of 1:3000 in blocking buffer. Following washes in TTBS, the proteins were visualized using an ECL detection kit (GE Healthcare) as directed by the manufacturer. Images were captured on Amersham Hyperfilm.
PMAPK1/2
MAPK1/2
100 nM Estrogen (min.)
Control EtOH E E CdCl2 CdCl2 NaArs NaArs ICI
ICI ICI ICI
PMAPK1/2
MAPK1/2
Figure 5. ICI 182,780, an estrogen receptor antagonist, reduces the phosphorylation of MAPK induced by estadiol (E2), cadmium chloride (CdCl2) and sodium arsenate (NaArs) treatment.
Figure 2. Treatment with 100 nM estrogen results in phosphorylation of MAPK within minutes of exposure. P-MAPK induction occurs at 5 minutes, is maximized at approximately 20 minutes, and decreases after 45 minutes.
CONCLUSIONS
Nanomolar concentrations of estrogen and the estrogen mimics, cadmium chloride and sodium arsenate, induced phosphorylation of MAPK1/2 within five minutes of treatment. Maximum phosphorylation of MAPK was reached 20 minutes after cells were treated with 100 nM estrogen and 100 nM cadmium chloride and 15 minutes after cells were treated with 100 nM sodium arsenate.
P-MAPK levels were greatly reduced in samples pre-treated with ICI 182,780, an estrogen receptor antagonist. This suggests that phosphorylation of MAPK by estrogen mimics involves activation of the membrane bound estrogen receptor. Future studies will determine if other proteins in the non-genomic pathway are activated by estrogen mimics to further support the involvement of the estrogen signaling pathway in the proliferative response induced by cadmium chloride and sodium arsenate.
100 nM Cadmium Chloride (min.)
PMAPK1/2
MAPK1/2
INTRODUCTION
In the United States, lung cancer is the cause of 28% of all cancer deaths. Female lung cancer incidence is on the rise, with 35 cases per 100,000. The majority of primary lung cancers are caused when lung cells are exposed to the carcinogens and tumor promoting chemicals found in cigarette smoke. Studies have shown that women have 1.5 times higher relative risk per exposure than men. This suggests a role of gender-dependent factors in the etiology of lung cancer.1 The steroid estrogen, which has a role in cell growth2-3 and differentiation4-5 may be involved. In the classic pathway, estrogen has been shown to exert its effects by binding to a nuclear receptor that directly stimulates transcription of target genes. A non-genomic pathway has also been recently elucidated. In this pathway, a membrane bound estrogen receptor activates estrogen responsive genes through a protein signaling pathway. Cigarette smoke is known to contain two environmental estrogens, sodium arsenate and cadmium chloride.6 Studies have shown that these heavy metals induce cellular proliferation, mimicking the physiological response of estrogen.4-10
The purpose of this study was to determine if the environmental estrogens, cadmium chloride and sodium arsenate, stimulate proliferation by inducing phosphorylation of mitogen-activated protein kinase (MAPK), a downstream signaling protein in the non-genomic estrogen pathway. Our results show that like β-estradiol, cadmium chloride and sodium arsenate induce MAPK phosphorylation within minutes of treatment and that this event is partially stimulated by activation of the estrogen receptor.
Figure 3. Phosphorylation of MAPK is stimulated within minutes of exposure to 100 nM cadmium chloride. P-MAPK induction occurs at 5 minutes, is maximized at approximately 20 minutes, and decreases after 30 minutes.
REFERENCES
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RESULTS
PMAPK1/2
MAPK1/2
100nM Sodium Arsenate (min.)
PMAPK1/2
MAPK1/2
METHODS
Figure 4. Treatment with 100 nM sodium arsenate results in phosphorylation of MAPK within minutes of exposure. P-MAPK induction occurs at 5 minutes, is maximized at approximately 15 minutes, and decreases after 20 minutes.
Figure 1. Phosphorylation of MAPK is induced after 15 minutes of treatment with nanomolar concentrations of estrogen (E2), cadmium chloride (CdCl2) and sodium arsenate (NaArs).
Tissue Culture and Chemicals
Cultures of the NCI-H1793 lung adenocarcinoma cell line from a female nonsmoker (American Type Culture Collection, Manassas, VA) were maintained in recommended media. Cadmium chloride, sodium arsenate, and β-estradiol were obtained from Sigma Chemicals (St. Louis, MO). ICI-182,780 was obtained from Tocris (Ellisville, MO).