EGR1 Is a Novel Target for AhR Agonists in Human Lung Epithelial Cells

Jeanelle M. Martinez*,1, Seung Joon Baek{dagger}, Donna M. Mays*, Patricia K. Tithof{dagger}, Thomas E. Eling{ddagger} and Nigel J. Walker*

* Laboratory of Computational Biology and Risk Analysis, NIEHS, Research Triangle Park, North Carolina, 27709; {dagger} College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996; and {ddagger} Laboratory of Molecular Carcinogenesis, NIEHS, Research Triangle Park, North Carolina, 27709

Received July 7, 2004; accepted August 30, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor early growth response 1 (EGR1) was previously identified as a potential novel target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human lung epithelial cells by toxicogenomic analysis. EGR1 has been implicated in the pathogenesis of vascular disease and is altered by a number of factors that include stress, inflammation, and hypoxia. Depending on its downstream targets or protein interactions, EGR1 regulates important biological processes that include cell growth, apoptosis, and differentiation. The following experiments were conducted to determine if EGR1 is indeed a target of TCDD and polycyclic aromatic hydrocarbons (PAHs) that can act through a similar mechanism. Pulmonary epithelial cells were exposed to TCDD for 24 h and an increase in EGR1 mRNA was measured. In addition, EGR1 protein was increased by TCDD and PAHs that have binding affinity to the aryl hydrocarbon receptor. The transcriptional activity of the EGR1 promoter was measured with a luciferase construct; however, no increases in luciferase activity were detected in TCDD or PAH-treated cells. Using actinomycin to inhibit RNA synthesis, we found that TCDD increased the half-life of EGR1 mRNA from 13 to 22 min. Thus, the increase in EGR1 expression appears to be mediated through a post-transcriptional mechanism that leads to the higher EGR1 protein levels in TCDD and PAH treated cells, compared to vehicle treated cells. Increased expression of a transcription factor EGR1 with tumorigenic and other biological activities could contribute to the deleterious pulmonary effects of exposure to these environmental agents.

Key Words: early growth response 1; mRNA stabilization; cell transformation; 2,3,7,8-tetrachlorodibenzo-p-dioxin; lung epithelial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of chemical contaminants highly prevalent in our environment elicit some or all of their toxicity through the cytosolic aryl hydrocarbon receptor (AhR). AhR is a basic helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS)-containing transcription factor (Hankinson, 1995Go). Upon ligand binding, the AhR is transferred into the nucleus, where it dissociates from a heat shock protein-chaperone complex, and forms a heterodimer with another bHLH, the aryl hydrocarbon nuclear translocator (ARNT) (Heid et al., 2000Go; Kazlauskas et al., 2001Go; McGuire et al., 1994Go). The AhR/ARNT heterodimer binds to a DNA xenobiotic responsive element (XRE, also known as the AhRE or DRE) in the promoter region of a large number of AhR responsive genes. Many AhR ligands are considered carcinogenic either individually or as part of a mixture. A well characterized AhR agonist is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a classified known human carcinogen by the National Toxicology Program (NTP, 2002Go). A number of PAHs have been evaluated for carcinogenicity in chronic rodent bioassays. The most studied PAH is benzo(a)pyrene (BaP), and it is considered a probable human carcinogen (IARC, 1987Go). In humans, accidental exposure to TCDD is associated with increases for risk of lung cancer and the development of chronic obstructive pulmonary disease (Pesatori et al., 1998Go; Steenland et al., 1999Go). Personal exposure to PAHs is also associated with an increased risk for lung cancer (Zmirou et al., 2000Go). The mechanism of how TCDD and PAHs target the lung to cause disease is largely unexplained. One approach to develop a better understanding of the biochemical events that lead to pulmonary toxicity is to determine how these chemicals alter gene expression. We have found that TCDD alters the expression of a plethora of genes in lung cells by toxicogenomic analysis (Martinez et al., 2002Go).

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., 1996Go; Svaren et al., 1996Go). EGR1 is reported to possess pro-tumorigenic activity in some cases, and is also considered a tumor suppressor gene (Calogero et al., 2001Go; Liu et al., 1998Go). 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., 2001Go; Virolle et al., 2001Go), 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., 2000Go). EGR1 is down-regulated in several types of neoplasia, as well as in an array of tumor cell lines (Huang et al., 1995Go, 1997Go). 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., 2001Go). EGR1 expression supports FGF-dependent angiogenesis during tumor growth (Fahmy et al., 2003Go), 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and chemical treatment. All cell culture reagents, unless otherwise stated, were purchased from Life Technologies (Life Technologies, Rockville, MD). HPL1A cells were grown as described previously (Masuda et al., 1997Go). A549 cells (ATCC No. CCL-185) were purchased from American Type Culture Collection (Manassas, VA) grown in F12 nutrient mixture (HAM) with glutamine and supplemented with 10% defined FBS (HyClone, Logan, UT), 100 units/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, 25 µg/ml amphotericin B, and 1 mM HEPES. Near confluent adherent cells were washed with PBS and further treated with trypsin/EDTA. The cells were pelleted and resuspended in medium to a final concentration of 100,000 cells/ml. Cells were fed every 3–4 days in media plus or minus TCDD. A 756 nM stock solution of TCDD was prepared in fetal bovine serum as reported previously (Spencer et al., 1999Go). TCDD, dissolved in DMSO at 100 µM, was purchased from Research Triangle Institute (Research Triangle Park, NC). 1-Methyl anthracene, anthracene, benzo[a]pyrene; benzo[b]fluoranthene, biphenylene, chrysene, dibenzothiophene, fluoranthene, naphthalene, and phenanthrene were purchased from Sigma-Aldrich (Milwaukee, WI).

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 2{Delta}{Delta}CT, where {Delta}CT = target gene CT – actin CT, and {Delta}{Delta}CT is based on the mean {Delta}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 4–12% 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., 2003Go). 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 60–80% 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EGR1 Expression after TCDD Treatment
Our previous results with cDNA microarray analysis using HPL1A and A549 cells indicated that TCDD increased the expression of EGR1. After 24 h of exposure to TCDD, EGR1 and the positive control gene CYP1A1 was induced in A549 and HPL1A cells. To confirm the microarray results, we measured the expression of EGR1 mRNA by real-time RT-PCR. HPL1A cells were incubated for 24 h with 0, 0.1, 1, or 10 nM TCDD, the RNA was isolated and the EGR1 expression measured. As shown in Figure 1, treatment of HPL1A cells with TCDD for 24 h resulted in an increased expression of EGR1 by approximately two-fold. Similar results were seen with A549 cell line (data not shown). An increase in mRNA over vehicle treatment was observed at all concentrations tested. Thus, TCDD increased the expression of EGR1 mRNA and confirmed the previous results from microarray analysis. Thus, these findings indicate that TCDD exposure of these human pulmonary cells increased EGR1 expression and the increase is comparable to changes in TCDD induced protein expression reported by other investigators (Tamaki et al., 2004Go).



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FIG. 1. Induction of EGR1 in HPL1A and A549 cells following treatment with TCDD. Increased EGR1 mRNA expression in HPL1A cell line treated with a concentration of 10 nM TCDD using serum (0.1%) as a vehicle. Samples were analyzed using SYBR Green based real-time RT-PCR; experiments were repeated three times and each sample analyzed in triplicate.

 
Effect of PAHs on EGR1 Expression
To determine if the results are restricted to TCDD, PAHs were incubated with A549 cells and the expression of EGR1 was measured by Western blot analysis. We selected six environmentally relevant compounds with variable AhR binding affinity. Anthracene (AN), dibenzothiophene (DBT), and naphthalene (NA) are poor to non-AhR agonists; whereas benzo(a) pyrene (BaP) is a strong ligand, and fluoranthene (FA) and phenanthrene (PHE) are weak AhR ligands (Machala et al., 2001Go; Piskorska-Pliszczynska et al., 1986Go). As shown in Figure 2, several PAHs (5 µM) increased the expression of EGR1 as observed for TCDD. BaP, FA, and PHE effectively caused an increase in EGR1 protein and there was no increase observed with AN, DBT, or NA. Thus, both TCDD and certain PAHs modulate the expression of the EGR1 protein.



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FIG. 2. Western analysis of EGR-1 protein after PAH treatment. A549 cells were grown to 60% confluency and then treated in serum free media with the following chemicals: AN, anthracene; NIB, dibenzothiophene; NA, naphthalene; BaP, benzo[a]pyrene; FA, fluoranthene; PHE, phenanthrene (5 µM) for 24 h. The cell lysates were isolated and analyzed by Western blotting as described in Materials and Methods. Equal amounts of protein (20 µg) were loaded onto each lane. Protein levels of EGR1 were measured using specific antibody obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

 
EGR1 Gene Is Not Transcriptionally Activated by TCDD or PAHs
To investigate the transcriptional activity of the EGR1 promoter, A549 and HPL1A cells were transfected with a plasmid containing the human EGR1 promoter region (1.2 Kb) in a luciferase construct (Fig. 3A) (Baek et al., 2003Go). Cells were treated for 24 h with a concentration of sulindac sulfide (30 µM) that is known to increase the EGR1 promoter activity of this luciferase reporter construct (Beak et al., 2004Go). However, no increase in luciferase activity was detected in cells treated with TCDD for 24 h (Fig. 3B). In addition to TCDD, we also tested if PAHs would alter the EGR1 promoter activity using this same luciferase assay. As shown in Figure 3C, none of the PAHs tested increased the luciferase activity in the A549 cells, while sulindac sulfide, the positive control, greatly increased the promoter activity. Thus, neither TCDD nor PAHs appear to induce transcriptional activation of EGR1.



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FIG. 3. Effects of TCDD and PAHs on transcriptional activity using a 1.2 Kb EGR1 promoter. (A) The human EGR1 luciferase construct was prepared as described previously (Baek et al., 2003Go). (B) HPL1A and A549 cells were transfected with pEgr1260/LUC for 5 h. Cells were then treated with TCDD (10 nM) or vehicle for 24 h, and luciferase activity was measured. EGR1 promoter activity in A549 cells treated with sulindac sulfide (30 µM) was used as a positive control for transcriptional activation of the EGR1 construct. (C) A549 cells were transfected with pEgr1260/LUC for 5 h, treated with indicated chemicals at (5 µM) for 24 h and luciferase activity was measured. For these experiments, the internal control vector (pRL-null) was used to normalize for transfection efficiency. 1-MA, 1-methyl anthracene; Abbreviations used: Veh, vehicle, SS, sulindac sulfide; BaP, benzo[a]pyrene; B(b) F, benzo[b]fluoranthene, CHR, chrysene; 1-MA, 1-methyl anthracene; NA, naphthalene, PHE, phenanthrene, SS, sulindac sulfide; BP, biphenylene. The data represent means ± SD from three different experiments.

 
TCDD Effect on Protein Expression
HPL1A cells were grown to 60% confluency, treated with 10 nM TCDD in vehicle (0.1% serum) for the indicated times, and the cells lysed. Because EGR1 has a known serum responsive element in its promoter, the control cells were also treated with 0.1% serum. The expression of EGR1 protein was estimated by Western blot analysis (Fig. 4A). In the TCDD-treated cells, a modestly higher expression of EGR1 was observed but the induction of EGR1 protein appeared to be prolonged by treatment with TCDD. At the longer times after treatment with TCDD a higher expression of EGR1 was seen in the TCDD treated samples as compared to controls. (Fig. 4B). Thus, not only did TCDD induce a modest increase in the induction of EGR1 protein, but the major response was a prolongation of the protein expression.



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FIG. 4. Prolonged induction of EGR1 protein by TCDD in HPL1A cells. HPL1A cells were treated with vehicle (serum) or TCDD for the indicated times (h). Western blot analysis of EGR1 expression was performed as indicated in the Materials and Methods. Actin antibody staining was used as a loading control. STD is a protein lysate from troglitazone-treated HCT116 cells with known expression of EGR1 (Baek et al., 2003Go).

 
TCDD Regulates EGR1 at the Post-Transcriptional Level
TCDD did not transcriptionally activate the EGR1 promoter as shown in Figure 3, but EGR1 protein was induced in HPL1A cells, thus it is possible that TCDD may regulate expression at the post-transcriptional level by increasing EGR1 mRNA stability. To test this hypothesis, HPL1A cells were treated with either 10 nM TCDD or vehicle for 24 h, then 5 µg/ml of actinomycin D was added and the cells were harvested for RNA analysis after various time-points. The levels of EGR1 mRNA were measured by real-time RT-PCR. TCDD treatment increased the half-life of EGR1 mRNA from 13 to 22 min (Fig. 5) using nonlinear regression. This increase in half-life is similar to the stabilization of TGF{alpha} mRNA as reported previously (Gaido et al., 1992Go). The real-time RT-PCR data was confirmed by Northern blot analysis (data not shown). This result suggests that the stabilization of EGR1 RNA stability is a major factor for the induction and prolonged expression of EGR1 protein by TCDD.



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FIG. 5. Increased stability of EGR1 mRNA as measured by real-time RT-PCR. HPL1A cells were treated with TCDD (10 nM) or vehicle (serum) for 24 h and subsequently with actinomycin D (5 µg/ml), referred to in Figure 5 as Time Zero. At the indicated times after treatment with actinomycin D, total RNA was isolated and examined using real-time RT-PCR with primers for human EGR1 or actin. The relative level of EGR1 mRNA (relative to the level of actin) was calculated, and the results were plotted as the percentage of the mRNA level present at time zero of actinomycin D treatment. Values are the mean ± SE. Statistical analysis was performed by non-linear regression (curve fit) with GraphPad Prism4 software (GraphPad Software Inc., San Diego, CA). To test if the curves were different a two-tailed t-test resulted in a p value < 0.001.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The EGR1 transcription factor was identified as a novel target in human peripheral lung epithelial cells from a TCDD toxicogenomic analysis (Martinez et al., 2002Go). In this study, we report for the first time that the ubiquitous environmental contaminants TCDD and PAHs alter the protein expression of the transcriptional regulator-EGR1. We found higher levels of EGR1 mRNA and protein in both the HPL1A and A549 cell lines treated with either TCDD or PAHs. Evidence is presented to support the conclusion that the up-regulation of EGR1 occurs at a post-transcriptional level and appears to be responsible for the increase in the level of EGR1 protein. Neither TCDD nor PAHs, alter the transcriptional activity of EGR1 as measured by promoter assays, but the presence of TCDD increases mRNA stability of EGR1 suggesting it is responsible for the higher levels of EGR1 protein observed. Thus, EGR1 is identified as a novel target for TCDD and PAHs whereby the mechanism for induction of this gene belongs to a growing list of genes that are regulated via post-transcriptional events.

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, 1995Go; Gaido et al., 1992Go; Tamaki et al., 2004Go). 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., 1986Go; Monk et al., 2003Go; Pasco et al., 1988Go). Transforming growth factor (TGF{alpha}) 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, 1995Go; Gaido et al., 1992Go). 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., 2000Go). 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., 2004Go; Tamaki et al., 2004Go). Another important AhR regulated growth factor is TNF-{alpha}. 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., 2002Go; Vogel and Abel, 1995Go). This study indicates that in addition to TCDD-regulation of uPA, IL-1ß, and TGF{alpha} 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., 1996Go). 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., 1997Go). 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., 2002Go; Winzen et al., 1999Go). TCDD is known to activate these pathways (Henley et al., 2004Go; Tan et al., 2002Go), and data we obtained from TCDD-toxicogenomic studies highly suggests there are signal transduction cascades initiated (Martinez et al., 2002Go). 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, 2002Go). 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, 2003Go). 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)Go 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., 2003Go). 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., 2003Go). Experimental evidence also links EGR1 to vascular and inflammatory stress (Harja et al., 2004Go), and in human emphysematous lung, EGR1 was up-regulated compared to control lung (Zhang et al., 2000Go).

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.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Jong Sik Kim and Dr. Mari Ida for their critical review of this manuscript, and Dr. Jeong-Ho Kim for his expert technical assistance with the luciferase assays.


    NOTES
 

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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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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