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Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405 [A. S., C. R. T., J. B., B. T. M.], and University of Connecticut School of Pharmacy, Storrs Mansfield, Connecticut 06268 [A. K. H.]
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ABSTRACT |
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-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P 
0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P 0.05). Addition of tetramethylthiourea inhibited silica-associated increases in fra-1 and proportions of cells in S-phase (P .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, i.e., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers. |
Introduction |
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-quartz, the most common silica polymorph compromising up to 95% of all sand and silt, is a group 1 human carcinogen in some occupational settings (1)
. This decision was based primarily on epidemiology and rat inhalation studies because silica does not appear to be genotoxic in a number of in vitro and in vivo assays assessing morphological transformation, gene mutations, and aneuploidy in mammalian cells, especially when the lack of appropriate controls, statistical significance, and dose-response data are considered (1)
. A more plausible mechanism of action in lung may be the stimulation of cell signaling cascades that trigger subsequent transcriptional events important in tumor development. The MAPK3 cascade consists of three arms including the ERKs, JNKs, and p38 kinases. Phosphorylation of these proteins may result in activation of a number of transcription factors that interact with regulatory domains in the promoter regions of genes that are integral to cellular responses such as proliferation, apoptosis, or inflammation. AP-1, comprised of members of the jun and fos proto-oncogene family, is a redox-sensitive transcription factor associated with the development of cell proliferation and tumor promotion (2) .
In studies here, we used a murine alveolar epithelial type II cell line (C10; Ref. 3 ), as a cell type first encountering inhaled silica particles and a progenitor cell of lung cancers, to determine pathways of activation of MAPKs in relationship to AP-1 transactivation, expression of AP-1 family members, and functional outcomes, i.e., changes in cell cycle distributions, after exposure to silica in vitro. Crocidolite asbestos, a documented carcinogenic fiber in mesothelial cells and tumor promoter in lung epithelium (4) , was used as a positive control and GBs as a nonpathogenic particle (negative control). Our findings show protracted JNK activation, AP-1-dependent (fra-1) gene expression, and S-phase increases by silica via an oxidant-dependent mechanism. Moreover, they suggest that modification of cell signaling through antioxidant strategies may be valuable in preventive and therapeutic approaches to silica-induced lung cancers.
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Materials and Methods |
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-quartz silica (Minusil) at final concentrations of 10 and 20 µg/cm2 dish, crocidolite asbestos [Na2(Fe3+)2(Fe2+)3Si8O22(OH)2] (NIEHS reference sample) at 5 µg/cm2 dish, or GBs (Particle Information Services, Inc., Kingston, WA) at 10 or 20 µg/cm2 dish. All particulates were weighed and suspended at 1 mg/ml in HBSS (Life Technologies, Inc., Grand Island, NY) before addition at final noncytolytic concentrations to culture dishes (5
, 6)
. Sham control and particulate-exposed cells were evaluated at time points from 1 h (the time required for sedimentation of particles onto cells) to 24 h thereafter.
Western Blot Analyses for Phosphorylated ERKs, JNKs, and p38 Kinases.
In these experiments, H2O2 (300 µM for 30 min) was used as a positive control for phosphorylation of ERKs, p38, or JNKs (6)
. Cells were washed three times with cold PBS, collected in lysis buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM Na3O4V, 10 mM NaF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride], vortexed for 1 min at 4°C, and centrifuged at 14,000 rpm for 20 min. The amount of protein in each sample was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Forty µg of protein in sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.1% w/v bromphenol blue] were electrophoresed in 10% SDS-polyacrylamide gels and transferred to nitrocellulose using a semidry transfer apparatus (Ellard Instrumentation, Ltd., Seattle, WA). Blots were blocked in buffer [TBS containing 5% nonfat powdered milk plus 0.05% Tween 20 (Sigma Chemical Co., St. Louis, MO)] for 1 h, washed three times for 5 min each in TBS/0.05% Tween 20, and incubated for 45 min with antibodies specific to phosphorylated proteins [phospho-38 MAPK (Thr-180/Tyr-182 at 1:1000); phospho-p44/42 MAPK (Thr-202/Tyr-204 at 1:1000); phospho-JNK (Thr-183/Tyr-185) at 1:2000; New England Biolabs, Beverly, MA]. Blots then were washed three times with TBS/0.05% Tween 20 and incubated with an antirabbit peroxidase-conjugated secondary antibody for 30 min. After washing blots three times in TBS/0.05% Tween 20, protein bands were visualized with the LumiGlo enhanced chemiluminescence detection system (Kirkgaard and Perry Laboratories, Gaithersburg, MD).
JNK1 Activity Assay.
The immunoprecipitation assay for JNK1 activity was performed as described previously using an anti-JNK1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:1000 dilution and GST-c-Jun (kindly provided by Dr. Roger Davis, University of Massachusetts, Worcester, MA) as a substrate for JNK1 (7)
. Incorporation of [
-32P]ATP into substrate was detected by autoradiography, and data were quantitated using a phosphorimager (Bio-Rad). In initial experiments to determine a role of antioxidants or iron-catalyzed oxidants in silica-induced JNK1 activity, cells were exposed to silica incubated in HBSS or HBSS containing the iron chelator, deferoxamine mesylate (Sigma; 1 mM) for 18 h (6)
. In other groups, catalase (500 units/ml; Sigma) was added to medium for 1 h (6)
, or cells were pretreated with NAC (10 mM) for 18 h (8)
prior to exposure to silica for 1 or 4 h.
Preparation of Nuclear Extracts and Electrophoretic Gel Mobility Shift Assays for AP-1.
Nuclear extracts of sham or particle-treated C10 cells were prepared as described previously (9)
. The amount of protein in each sample was determined using the Bio-Rad protein assay (Bio-Rad). One ng of a 32P-end-labeled double-stranded oligodeoxynucleotide representing the fat-specific element that contains a TRE consensus sequence (i.e., TGACTCA) was incubated with extract as described previously (10)
. The components of the AP-1 complex were identified by supershift analysis using antibodies specific for Fra-1, Fra-2, c-Fos, c-Jun, and JunD (data not shown). Autoradiograms were developed and quantitated using a Bio-Rad phosphorimager.
RNase Protection Assays.
Confluent cultures maintained in 0.5% serum-containing medium for 24 h were exposed to particulates for 8 or 24 h. Total RNA was prepared and quantitated by absorbance at 260 nm (9)
. Steady-state mRNA levels of c-jun, junB, junD, c-fos, fra-1, fra-2, and fosB, the ribosomal probe, L32, and glyceraldehyde-3-phosphate dehydrogenase were examined using the RiboQuant multiprobe RNase protection assay system and the mFos/Jun multiprobe template set (PharMingen, San Diego, CA) according to the manufacturer’s protocol. Autoradiograms were quantitated using a Bio-Rad phosphorimager. Results were normalized to expression of the housekeeping gene, L32. In some experiments, the hydroxyl radical (OH·) scavenger, TMTU (25 and 50 mM), or catalase (500 units/ml medium; Ref. 6
) was added 1 h prior to the addition of silica.
Flow Cytometry.
Flow cytometry was used to determine alterations in cell cycle kinetics induced by particulates as described previously (11)
. At 24 h after addition of particles, cells were harvested by trypsinization, resuspended at 1 x 106 cells/ml in a propidium iodide staining solution (50 µg/ml propidium iodide, 0.1% Triton X-100, and 32 µg/ml RNase A in PBS), and incubated at 37°C for 30 min before analysis of 10,000 cells/group/time point in triplicate. The distribution of cells in the various cell cycle compartments, including cells with a hypodiploid DNA content indicative of apoptosis, was determined using a Coulter Epics Elite flow cytometer and appropriate software. Apoptosis was confirmed on C10 cells cultured on glass coverslips using an antibody recognizing single-strand DNA (Apostain; Alexis, San Diego, CA; data not shown).
Measurement of Intracellular Oxidant Accumulation.
C10 cells at confluence were exposed to particles for 1, 8, or 24 h (n = 3/group/time point). After exposure to particles, H2DCFDA (10 µM; Sigma), a nonfluorescent precursor of fluorescein, was added to each dish at a final concentration of 10 nM for 30 min at 37°C. Cells then were trypsinized, washed, and resuspended in HBSS without phenol red, and fluorescence was measured using a flow cytometer (Coulter Epics Elite; Coulter Corporation, Miami, FL) at excitation and emission wavelengths of 488 and 525 nm, respectively.
Statistical Analyses.
All data were examined by ANOVA using the Student-Newman Keuls procedure to adjust for multiple pairwise comparisons between groups.
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Results |
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show dose-related increases in phosphorylated JNK1/2 proteins at 1, 4, and 8 h after addition of silica. In contrast, exposure of C10 cells to asbestos or GBs failed to show elevations in JNK phosphorylation. Silica-associated increases in phosphorylated ERKs were also noted at 4 and 8 h and were seen as well with asbestos but not GBs. Changes in phosphorylated p38 were not observed after addition of minerals or GBs. Because prolonged increases in phosphorylated JNK proteins were a unique feature of silica (in contrast to GBs or asbestos), JNK1 activity assays also were performed. Data showed dose-related increases (P 0.05) in response to silica at all time points (Fig. 2, A and B)
. At 8 h, a less striking but significant (P 0.05) increase in JNK1 activity was also observed in cells exposed to asbestos. GBs failed to cause increases in JNK1 activity at any time point. Fig. 2, C and D
, shows that AP-1 binding to DNA was increased (P 0.05) at 8 and 24 h after exposure of C10 cells to silica (10 and 20 µg/cm2). Elevations were also noted with addition of asbestos (P 0.05) but not GBs at either time point. AP-1 transactivation by silica was confirmed in a stable C10 cell line expressing the reporter plasmid, jun-luciferase (7)
. In these experiments, both silica at 20 µg/cm2 and asbestos (5 µg/cm2 dish) caused significant increases in luciferase activity (P 0.05), whereas GBs were inactive (data not shown).
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. At 8 h, increases in mRNA levels of c-jun, junB, fra-1, and c-fos were significantly increased (P 0.05) after exposure to silica at 20 µg/cm2, and elevations (P 0.05) in c-fos levels also were observed at lower concentrations of silica (10 µg/cm2 dish). Significant increases (P 0.05) in fra-1 mRNA at both concentrations of silica as well as elevations in mRNA levels of junB at 20 µg/cm2 silica persisted for 24 h. Similar patterns of early jun/fos and more protracted fra-1 gene expression were also observed after addition of asbestos. GBs failed to cause any changes in gene expression.
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On the basis of these results, we pre-exposed C10 cells to TMTU (25 and 50 mM), which can scavenge intracellular OH·, or catalase (500 units/ml medium), an extracellular scavenger of H2O2, for 1 h in an attempt to determine whether silica-induced increases in fra-1 could be altered. In comparison to sham control groups, no changes in gene expression were observed after addition of TMTU or catalase alone to cells (Fig. 4, A and B)
. However, addition of TMTU at 50 mM, but not catalase, to silica-exposed cells caused significant decreases (P 0.05) in mRNA levels of fra-1.
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0.05) in the proportions of cells in S and G2-M phases, whereas higher exposures (20 µg/cm2) resulted in elevations (P 0.05) of cells in sub-G0-G1 and S-phase. These were confirmed as apoptotic cells using the Apostain technique (data not shown). No changes in cell cycle distributions were observed after addition of GBs to cultures, whereas asbestos caused striking increases (P 0.05) in the percentage of cells in sub-G0-G1 (data not shown). The addition of TMTU alone did not alter cell cycle kinetics nor increased numbers of cells in sub-G0-G1 when added prior to silica. However, the percentage of silica-exposed cells in S-phase was significantly inhibited by TMTU (P 0.05; Fig. 4C
).
To confirm that silica causes increased production of intracellular oxidants by pulmonary epithelial cells, intracellular levels of the fluorescent product of H2DCFDA, an indicator of oxidant production, were measured. As shown in Fig. 4D
, oxidant accumulation was significantly elevated (P 0.05) in C10 cells exposed to silica (10 and 20 µg/cm2) for 1 h. In contrast to silica, asbestos (5 µg/cm2) caused protracted increases in oxidant production over a 24-h period. GBs (20 µg/cm2) were inactive at all time points.
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Discussion |
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Another novel finding here is the increased expression of jun and fos proto-oncogenes in pulmonary epithelial cells exposed to silica. These studies support our hypothesis that pathogenic particles elicit signal transduction cascades that lead to the increased expression of early-response genes and AP-1 transactivation (4 , 10) . For example, increased mRNA levels of c-jun are observed in rodent lungs after inhalation of crocidolite asbestos, which accompany early increases in proliferation of epithelial cells (14) .
In our studies, increased mRNA levels of fra-1 and junB persisted for 24 h. Moreover, Fra-1 and JunB were observed in AP-1 complexes from silica-exposed cells (data not shown). The significance of various AP-1 family members that comprise homo- and heterodimers that may govern AP-1 binding/stability, transactivation and subsequent alterations in gene expression is largely unknown, but some studies provide insight into functional roles of fos and jun family members. For example, as summarized above, overexpression of c-jun (which was elevated as well as c-fos in our studies after addition of silica or asbestos) in tracheal epithelial cells confers an increase in cell proliferation and morphological transformation (7) . junB and junD attenuate c-jun activity and suppress proliferation, respectively, in other cell models (15 , 16) . Thus, a balance between fos and jun family members may govern the outcomes of increased DNA synthesis and apoptosis observed in silica-exposed epithelial cells. Recent studies suggest a critical role of Fra-1 in the development of epithelial tumors, because expression of exogenous Fra-1 in epithelioid adenocarcinoma cells results in increased motility and invasiveness (17) . Overexpression of Fra-1 in rat fibroblasts causes anchorage-independent growth and tumors in athymic mice as well as up-regulation of its own gene (18) . Moreover, constitutive expression of c-Fos, FosB, or c-Jun in these cells leads to AP-1-dependent stimulation of fra-1 expression.
Most notably, increases in fra-1 and junB mRNA levels by silica were inhibited significantly after addition of TMTU, an OH· scavenger, to C10 cells. Because catalase did not inhibit their expression, intracellular generation of oxidants, as verified in our studies, is implicated in silica-induced increases in fra-1. These data are exciting because they provide a rationale for modulation of silica-induced proto-oncogene expression by antioxidants. In summary, results here show induction of JNK activity and proto-oncogene expression by silica via an oxidant-dependent mechanism. Increases in fra-1 and junB mRNA levels may be causally related to increased AP-1 activity and cell cycle changes in pulmonary epithelial cells exposed to silica. Thus, approaches to alter these cell signaling events may be beneficial in altering cell proliferation in silica-associated cancers and fibroproliferative lung diseases.
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FOOTNOTES |
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1 This research was supported by R01 Grants ES/HL09213 and HL39469 (to B. T. M.) and a Shannon Award from the National Cancer Institute (to C. R. T.). 
2 To whom requests for reprints should be addressed, at Department of Pathology, A145 Soule Medical Alumni Building, University of Vermont College of Medicine, Burlington, VT 05405. Phone: (802) 656-0382; Fax: (802) 656-8892; E-mail: bmossman{at}zoo.uvm.edu
3 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun-NH2-terminal amino kinase; AP-1, activator protein-1; TMTU, tetramethylthiourea; GB, glass bead; NAC, N-acetyl-cysteine; H2DCFDA, 2',7'dichlorodihy-drofluorescein diacetate; TBS, Tris-buffered saline.
Received 11/30/00. Accepted 1/18/01.
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REFERENCES |
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B-related genes and oxidant-dependent NF-B activation in vitro. Am. J. Respir. Cell Mol. Biol., 23: 182-187, 2000.This article has been cited by other articles:
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  M. V. Karamouzis, P. A. Konstantinopoulos, and A. G. Papavassiliou The Activator Protein-1 Transcription Factor in Respiratory Epithelium Carcinogenesis Mol. Cancer Res., February 1, 2007; 5(2): 109 - 120. [Abstract] [Full Text] [PDF]
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 A. Shukla, T. F. Barrett, K. I. Nakayama, K. Nakayama, B. T. Mossman, and K. M. Lounsbury Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKC{delta}-dependent pathway in murine lung FASEB J, May 1, 2006; 20(7): 997 - 999. [Abstract] [Full Text] [PDF]
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 Y. Arita, S. H. Harkness, J. A. Kazzaz, H.-c. Koo, A. Joseph, J.A. Melendez, J. M. Davis, A. Chander, and Y. Li Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L978 - L986. [Abstract] [Full Text] [PDF]
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 J. L. Kang, H. J. Jung, K. Lee, and H. R. Kim Src Tyrosine Kinases Mediate Crystalline Silica-Induced NF-{kappa}B Activation through Tyrosine Phosphorylation of I{kappa}B-{alpha} and p65 NF-{kappa}B in RAW 264.7 Macrophages Toxicol. Sci., April 1, 2006; 90(2): 470 - 477. [Abstract] [Full Text] [PDF]
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 J. OVREVIK, T. MYRAN, M. REFSNES, M. LAG, R. BECHER, R. B. HETLAND, and P. E. SCHWARZE Mineral Particles of Varying Composition Induce Differential Chemokine Release from Epithelial Lung Cells: Importance of Physico-chemical Characteristics Ann. Hyg., April 1, 2005; 49(3): 219 - 231. [Abstract] [Full Text] [PDF]
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J. Ovrevik, M. Lag, P. Schwarze, and M. Refsnes p38 and Src-ERK1/2 Pathways Regulate Crystalline Silica-Induced Chemokine Release in Pulmonary Epithelial Cells Toxicol. Sci., October 1, 2004; 81(2): 480 - 490. [Abstract] [Full Text] [PDF]
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M. S. Thibodeau, C. Giardina, D. A. Knecht, J. Helble, and A. K. Hubbard Silica-Induced Apoptosis in Mouse Alveolar Macrophages Is Initiated by Lysosomal Enzyme Activity Toxicol. Sci., July 1, 2004; 80(1): 34 - 48. [Abstract] [Full Text] [PDF]
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Q. Zhang, S. R. Kleeberger, and S. P. Reddy DEP-induced fra-1 expression correlates with a distinct activation of AP-1-dependent gene transcription in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L427 - L436. [Abstract] [Full Text] [PDF]
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 F. Gambelli, P. Di, X. Niu, M. Friedman, T. Hammond, D. W. H. Riches, and L. A. Ortiz Phosphorylation of Tumor Necrosis Factor Receptor 1 (p55) Protects Macrophages from Silica-induced Apoptosis J. Biol. Chem., January 16, 2004; 279(3): 2020 - 2029. [Abstract] [Full Text] [PDF]
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 Y. Li, Y. Arita, H.-c. Koo, J. M. Davis, and J. A. Kazzaz Inhibition of c-Jun N-Terminal Kinase Pathway Improves Cell Viability in Response to Oxidant Injury Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 779 - 783. [Abstract] [Full Text] [PDF]
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P. Adiseshaiah, S. R. Papaiahgari, H. Vuong, D. V. Kalvakolanu, and S. P. Reddy Multiple cis-Elements Mediate the Transcriptional Activation of Human fra-1 by 12-O-Tetradecanoylphorbol-13-acetate in Bronchial Epithelial Cells J. Biol. Chem., November 28, 2003; 278(48): 47423 - 47433. [Abstract] [Full Text] [PDF]
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A. Shukla, M. Jung, M. Stern, N. K. Fukagawa, D. J. Taatjes, D. Sawyer, B. Van Houten, and B. T. Mossman Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L1018 - L1025. [Abstract] [Full Text] [PDF]
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S. P. M. Reddy and B. T. Mossman Role and regulation of activator protein-1 in toxicant-induced responses of the lung Am J Physiol Lung Cell Mol Physiol, December 1, 2002; 283(6): L1161 - L1178. [Abstract] [Full Text] [PDF]
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 M. D'Alessandro, D. Russell, S. M. Morley, A. M. Davies, and E. B. Lane Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock J. Cell Sci., November 15, 2002; 115(22): 4341 - 4351. [Abstract] [Full Text] [PDF]
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 M. E. Ramos-Nino, C. R. Timblin, and B. T. Mossman Mesothelial Cell Transformation Requires Increased AP-1 Binding Activity and ERK-dependent Fra-1 Expression Cancer Res., November 1, 2002; 62(21): 6065 - 6069. [Abstract] [Full Text] [PDF]
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 X. Deng, S. Jayanthi, B. Ladenheim, I. N. Krasnova, and J. L. Cadet Mice with Partial Deficiency of c-Jun Show Attenuation of Methamphetamine-Induced Neuronal Apoptosis Mol. Pharmacol., November 1, 2002; 62(5): 993 - 1000. [Abstract] [Full Text] [PDF]
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C. B. Manning, A. B. Cummins, M. W. Jung, I. Berlanger, C. R. Timblin, C. Palmer, D. J. Taatjes, D. Hemenway, P. Vacek, and B. T. Mossman A Mutant Epidermal Growth Factor Receptor Targeted to Lung Epithelium Inhibits Asbestos-induced Proliferation and Proto-Oncogene Expression Cancer Res., August 1, 2002; 62(15): 4169 - 4175. [Abstract] [Full Text] [PDF]
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B. T. Mossman and D. C. Gruenert SV40, Growth Factors, and Mesothelioma . Another Piece of the Puzzle Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 167 - 170. [Full Text] [PDF]
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 M.-T. Lin, C.-Y. Juan, K.-J. Chang, W.-J. Chen, and M.-L. Kuo IL-6 inhibits apoptosis and retains oxidative DNA lesions in human gastric cancer AGS cells through up-regulation of anti-apoptotic gene mcl-1 Carcinogenesis, December 1, 2001; 22(12): 1947 - 1953. [Abstract] [Full Text] [PDF]
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