* Laboratory of Computational Biology and Risk Analysis, NIEHS, Research Triangle Park, North Carolina, 27709; College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996; and
Laboratory of Molecular Carcinogenesis, NIEHS, Research Triangle Park, North Carolina, 27709
Received July 7, 2004; accepted August 30, 2004
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ABSTRACT |
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Key Words: early growth response 1; mRNA stabilization; cell transformation; 2,3,7,8-tetrachlorodibenzo-p-dioxin; lung epithelial cells.
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INTRODUCTION |
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One gene whose expression appeared to be significantly altered by TCDD was transcription factor EGR1. EGR1 (also known as NGFI-A, TIS8, Krox-24, and Zif268) is a member of the immediate early gene family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to signals such as mitogens, growth factors, and stress stimuli (Liu et al., 1996; Svaren et al., 1996
). EGR1 is reported to possess pro-tumorigenic activity in some cases, and is also considered a tumor suppressor gene (Calogero et al., 2001
; Liu et al., 1998
). EGR1 can act as an anti-tumorigenic protein by activation of the phosphatase and tensin homolog (PTEN) tumor suppressor gene during UV irradiation (Ferrigno et al., 2001
; Virolle et al., 2001
), and re-expression of EGR1 causes a suppression in growth of transformed cells both in soft agar and in athymic nude mice (Liu et al., 2000
). EGR1 is down-regulated in several types of neoplasia, as well as in an array of tumor cell lines (Huang et al., 1995
, 1997
). However, up-regulation of EGR1 is also indicative of neoplastic progression and is regulated by peroxisomal proliferator-activated receptor gamma ligands that are pro-apoptotic (Virolle et al., 2001
). EGR1 expression supports FGF-dependent angiogenesis during tumor growth (Fahmy et al., 2003
), and is involved in the pathogenesis of vascular disease. Thus, EGR1 appears to regulate the expression of numerous proteins linked to several biological functions that are related to the pathogenesis of disease. An increase in the expression of EGR1 may play a contributing role to toxic effects of these ubiquitous environmental contaminants. The aim for the present study was to determine if EGR1 gene expression corresponds to an increase in EGR1 protein, and then to understand how environmental chemicals alter EGR1 expression.
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MATERIALS AND METHODS |
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RNA extraction and reverse transcription. Total RNA was prepared using Qiagen RNeasy midiprep columns (Qiagen, Valencia, CA) according to the manufacturer's recommendations, RNA was quantitated by UV spectroscopy at 260 nM and stored in RNase-free H2O at 70°C. Reverse transcription reactions were carried out at 37°C for 1 h using 50 ng RNA that was heat denatured at 70°C for 10 min in 10 µl volume containing: MgCl2 (5.5 mM), 1X PCR buffer, dNTP (0.5 mM), RNAsin (0.4 units), oligo d(t) (2.5 µM), Molony Murine Leukemia Virus reverse transcriptase (MMLV-RT) (1.25 units) (PE Applied Biosystems, Foster City, CA).
SYBR green detection. Real-time fluorescence PCR detection was carried out using an ABI Prism 7700 Sequence Detection System. PCR reactions were carried out in microAmp 96 well reaction plates; SYBR Green PCR buffer 1X, MgCl2 (5 mM), dATP, dCTP, dGTP, dUTP (0.2 mM each), Taq Polymerase (0.25 units/µl) (PE Applied Biosystems, Foster City, CA), forward and reverse primers (0.2 µM each) (Research Genetics, Huntsville, AL), and cDNA (10 µl) in a final PCR reaction volume of 50 µl. Amplification parameters were: denaturation at 94°C 10 min, followed by 40 cycles of 95°C, 15 s; 60°C, 60 s. Primers and probes were designed using Primer Express Software (PE Applied Biosystems, Foster City, CA). Samples were analyzed in triplicate, and beta-actin was used as an endogenous control. Fold induction was calculated using the formula 2CT, where
CT = target gene CT actin CT, and
CT is based on the mean
CT of respective control (non-TCDD treated).
Western blot analysis. Cells were washed twice with PBS, and then lysed using ice-cold solubilization buffer (50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate and protease inhibitors (complete protease inhibitor cocktail tablets were purchased from Roche Molecular Biochemicals, Mannheim, Germany) for 30 min on ice. Plates were scraped and samples were spun in a microcentrifuge for 5 min at 12,000 rpm, and supernatant containing cell lysate was removed. Quantitation of protein was done using the bicinchoninic acid (BCA) protein-assay kit from Pierce (Rockford, IL). A Molecular Devices Thermomax microplate reader (Molecular Devices Corp., Menlo Park, CA) was used to measure the absorbance of 96 well plates at a wavelength of 562 nm. Protein extracts were separated by electrophoresis on a 412% Tris-Glycine gel (Invitrogen Life Technologies, Carlsbad, CA) and transferred to 0.45 µm pore size polyvinylidene difluoride (PVDF) membrane (Invitrogen Life Technologies, Carlsbad, CA). The filters were preincubated for 60 min in 1x phosphate buffered saline and 5% dry milk and subsequently sealed for overnight incubation at 4°C with the antibody EGR1, (SC-110) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), diluted 1:500 in 1x PBS and 2% dry milk. After washing 3x 10 min in 1x PBS, the membranes were incubated with a secondary sheep anti-mouse IgG for EGR1 that was conjugated to peroxidase and diluted 1:2500 in 1x PBS 1% dry milk. Detection was carried out using the ECL-Plus detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The blot were then striped and re-blotted with actin antibody (Santa Cruz, CA).
Transfection and luciferase assay. HPL1A and A549 cells were plated in six-well plates at 200,000 cells/well in HAMS F12 complete media. A 1260 kb fragment of the EGR1 promoter (pEgr1260/LUC) was isolated and ligated into pGLBasic3 luciferase vector as previously described (Baek et al., 2003). After growth for 24 h, plasmid mixtures containing 1 µg pEgr1260/LUC, and 0.1 µg of pRL-null (Promega, WI) were transfected by lipofectamine (Life Technologies, MD) according to the manufacturer's protocol. After 5 h of transfection, the cells were treated with vehicle (0.1% serum) plus or minus TCDD (10 nM) for 24 h and then harvested in 1X luciferase lysis buffer. Luciferase activity was determined and normalized to the pRL-null luciferase activity using Dual Luciferase Assay Kit (Promega, WI). pRL-null vector was used to adjust the transfection efficiency.
RNA stability. When reaching 6080% confluence in 60 mm plates, the cells were treated with vehicle (0.1% serum) plus or minus TCDD (10 nM). After 24 h of TCDD treatment incubation, the transcription inhibitor, actinomycin D (5 µg/ml), was added and samples were harvested at various time-points. RNA was isolated as above and levels of EGR1 mRNA were analyzed by real-time RT-PCR. The values presented are percent of the time-zero values for treated and control samples. Statistical analysis was performed by non-linear regression (curve fit), Top to zero equation (Y = Top*exp(-K*X), and the two curves were compared by a paired t-test using GraphPad Prism4 software (GraphPad Software Inc., San Diego, CA).
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RESULTS |
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DISCUSSION |
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This study illustrates that both transcriptional and post-transcriptional processes are responsible for the alterations of gene expression detected by microarray analysis. Furthermore, a rather modest induction of RNA does result in a measurable change in protein expression and thus low level changes (less than two-fold) in gene expression deserve further consideration. The fold-change in EGR1 protein by TCDD is similar to the fold-changes reported for other growth factors that are regulated by TCDD through stabilization of RNA (Gaido and Maness, 1995; Gaido et al., 1992
; Tamaki et al., 2004
). In this report we show that the PAHs with strongest ligand binding to the AhR: BaP, FA, and PHE increased the expression of EGR1 protein while those PAHs with minimal AhR binding ability (AN, DBT, and NA) did not. This data suggests that the AhR mediates the increase in EGR1 expression; however, additional evidence is required to support this conclusion.
Detection of genes that are altered by TCDD because of changes in RNA stability is increasing. For example, the increased expression of CYP1A1, CYP1A2 by TCDD occurs though both transcription and post-transcriptional levels (Kimura et al., 1986; Monk et al., 2003
; Pasco et al., 1988
). Transforming growth factor (TGF
) and urokinase plasminogen activator (uPA) are up-regulated post-transcriptionally in human keratinocytes by TCDD due to a change in mRNA stability (Gaido and Maness, 1995
; Gaido et al., 1992
). For uPA, TCDD causes activation of a 50 kD cytoplasmic protein that binds to the 3' untranslated region (UTR) of uPA mRNA via a phosphorylation event resulting in increased mRNA stability (Shimba et al., 2000
). More recently, an important inflammatory cytokine interleukin-1B (IL-1ß) is also reported to be upregulated by both TCDD and PAHs by a post-transcriptional mechanism that is dependent on the AhR (Henley et al., 2004
; Tamaki et al., 2004
). Another important AhR regulated growth factor is TNF-
. While the regulation has not been shown to be via increased RNA stability, the TCDD induced fold increase for this growth factor is relatively low (Kern et al., 2002
; Vogel and Abel, 1995
). This study indicates that in addition to TCDD-regulation of uPA, IL-1ß, and TGF
via an increase in RNA stability we can now add another important transcription factor-EGR1.
Stability of mRNA can be mediated by a number of different mechanisms. The control of mRNA stability can involve A/U-rich elements (ARE) in the 3' UTR or specific RNA stem loop motifs (Peng et al., 1996). Increased stability might be due to a reduction in the level of proteins that destabilize EGR1 mRNA or specific mRNA binding proteins, including AUF1 (Sirenko et al., 1997
). Interacting with ARE may protect EGR1 mRNA from mRNA binding proteins, including AUF1 or from degradation by endo- and exonuclease, thereby increasing RNA stability. Signaling transduction pathways that include p38 and ERK are also known to be involved in mRNA stabilization (Andoh et al., 2002
; Winzen et al., 1999
). TCDD is known to activate these pathways (Henley et al., 2004
; Tan et al., 2002
), and data we obtained from TCDD-toxicogenomic studies highly suggests there are signal transduction cascades initiated (Martinez et al., 2002
). The molecular mechanisms by which TCDD, and presumably PAHs that bind to the AhR, increase the stability of the mRNA message is not clear but an understanding of this molecular mechanism would provide critical insight into the diversity of TCDD biological activity EGR1 can regulate both positively and negatively, the expression of a wide range of genes, many of which are involved in diverse biological processes. For example, EGR1 is associated with genes that play a role in inflammation, wound healing, angiogenesis, and vascular disease (Adamson and Mercola, 2002
). It is possible that the prolonged expression of EGR1 protein could be involved with these biological processes in the lung. Injury to airway epithelium involves changes in biological processes including cell migration, proliferation, apoptosis, and an immune response (Tesfaigzi, 2003
). The detection for genes that are regulated by EGR1 is increasing and many of these genes are involved with the responses mentioned above, for example Liu et al. (2002)
show human fibrosarcoma cells transfected with EGR1 lead to an alteration in expression of 25 genes that are associated with defense and immunity proteins. Genes altered in over-expression of EGR1 in TRAMP C2 prostate cancer cells are involved in cellular growth, cell cycle progression, and apoptosis (Virolle et al., 2003
). There is also a set of genes that are specifically related to cardiovascular function altered in adenoviral over-expression of EGR1 in human endothelial cells (Fu et al., 2003
). Experimental evidence also links EGR1 to vascular and inflammatory stress (Harja et al., 2004
), and in human emphysematous lung, EGR1 was up-regulated compared to control lung (Zhang et al., 2000
).
We have shown that environmentally relevant compounds that are AhR agonists increase the expression of the transcription factor EGR1 through post-transcriptional mechanisms. It is not clear what specific molecular effects TCDD or PAHs have on biological effects that are mediated by EGR1 or EGR1-regulation of downstream genes. The results presented here suggest that a number of environmental chemicals can alter EGR1. A better understanding of EGR1 regulated genes may provide clues to how EGR1 contributes to the deleterious effects of these environmental chemicals in human lung.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Laboratory of Computational Biology and Risk Analysis, NIEHS, 111 Alexander Dr., PO Box 12233, MD C4-05, Research Triangle Park, NC 27709. Fax: (919) 541-4704. E-mail: martine2{at}niehs.nih.gov.
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