ToxSci Advance Access originally published online on July 16, 2007
Toxicological Sciences 2007 99(2):502-511; doi:10.1093/toxsci/kfm182
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Transcriptional Regulation of Deoxynivalenol-Induced IL-8 Expression in Human Monocytes
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,1
* Department of Microbiology and Molecular Genetics
Center for Integrative Toxicology
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824-1224
1 To whom correspondence should be addressed at 234 G.M. Trout Building, Michigan State University, East Lansing, MI 48824-1224. Fax: (517) 353-8963. E-mail: pestka{at}msu.edu.
Received June 5, 2007; accepted July 3, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The trichothecene mycotoxin deoxynivalenol (DON), commonly present in contaminated grains worldwide, induces expression of the chemokine interleukin (IL)-8 in human monocytes. The purpose of this study was to test the hypothesis that DON modulates transcriptional and posttranscriptional regulation of IL-8 expression in the U937 human monocyte model. When U937 cells were transfected with a wild-type IL-8 promoter luciferase construct (–162/+44 IL-8 LUC) and incubated with DON (1 µg/ml) or the positive control, lipopolysaccharide (LPS) (1 µg/ml), there was a significant increase in luciferase expression. Mutation of the nuclear factor-
B (NF-B) binding site significantly impaired both DON- and LPS-induced luciferase expression. In contrast, mutating the activator protein-1 binding site resulted in significantly increased DON- and LPS-induced luciferase expression. CCAAT/enhancer-binding protein ß, octamer-1, or NF-B repressing factor binding site mutations did not affect DON-induced luciferase activity. Consistent with reporter studies, the NF-B inhibitor caffeic acid phenethyl ester completely ablated both DON-induced IL-8 mRNA and protein expression. When NF-B subunit binding to a specific IL-8 promoter probe was evaluated by enzyme-linked immunosorbent assay (ELISA), DON was observed to increase p65 binding by 21-fold, have no effect on p50 binding and decrease p52 binding. DON was not found to stabilize IL-8 mRNA in U937 cells. Taken together, these data suggest that DON-induced IL-8 expression is likely to be mediated at the transcriptional level by NF-B, specifically p65, but does not appear to involve mRNA stabilization. Key Words: mycotoxin; trichothecene; chemokine; transcription; immunotoxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Deoxynivalenol (DON, vomitoxin) is a trichothecene mycotoxin that contaminates cereal grain crops and foods worldwide (Pestka and Smolinski 2005
). Besides its emetic, growth and anorectic effects, DON can either stimulate or suppress immune function, depending on dose and duration of exposure (Pestka et al., 2004). Low-dose DON exposure evokes gene expression of a variety of immune- and inflammation-related genes that could potentially contribute to its diverse toxic effects. These genes include interleukin (IL)-6 (Sugita-Konishi and Pestka 2001; Wong et al., 2001), IL-2 (Li et al., 1997, 2000), tumor necrosis factor (TNF)-
(Chung et al., 2003b; Sugita-Konishi and Pestka, 2001), cyclooxygenase-2 (COX-2) (Moon et al., 2003), and macrophage inhibitory protein-2 (Chung et al., 2003a).
There are at least two mechanisms by which DON can upregulate gene expression. DON increases binding of several transcription factors, including nuclear factor-B (NF-B) (Ouyang et al., 1996), activator protein-1 (AP-1) (Li et al., 2000), and cAMP response element-binding protein (Jia et al., 2006) in a number of leukocyte models. These findings have been confirmed in vivo in the spleens of mice orally exposed to DON (Zhou et al., 2003). Measurement of heteronuclear RNAs (hRNAs) and expression of reporters under control of selected promoters have further revealed that DON-induced increases in IL-6 (Jia et al., 2006; Wong et al., 2001), TNF- (Chung et al., 2003b), and COX-2 (Moon et al., 2003) result, in part, from increased transcription. Other investigations suggest that mRNA stabilization also contributes to DON upregulation of TNF-, IL-6, COX-2, and IL-2 (Chung et al., 2003b; Li et al., 1997; Moon et al., 2003). Thus, DON can potentiate gene expression both by increasing transcription and by stabilizing mRNA transcripts.
Mitogen-activated protein kinases (MAPKs) have been shown to drive transcription factor activation and mRNA stability (Liu et al., 2000; Neininger et al., 2002; Schmeck et al., 2004; Vanden Berghe et al., 1998; Winzen et al., 1999; Yu et al., 2003). Notably, DON-induced p38, extracellular signal–regulated kinase (ERK), and c-jun N-terminal kinase (JNK) phosphorylation typically precede upregulation of immune gene expression in both in vitro and in vivo models, and furthermore, DON-induced gene transcription and mRNA stabilization are often MAPK dependent (Chung et al., 2003b; Islam et al., 2006; Moon et al., 2003; Pestka et al., 2005).
Recently, DON-induced expression of the CXC chemokine IL-8, a neutrophil chemoattractant, was observed in human peripheral blood mononuclear cell cultures (Islam et al., 2006). Flow cytometry revealed that human monocytes were the effector population in these cultures. Since IL-8 has been implicated in many chronic diseases ranging from inflammatory bowel disease (Alzoghaibi et al., 2003; Banks et al., 2003) to rheumatoid arthritis (Georganas et al., 2000; Suzuki et al., 2000), understanding the mechanisms by which DON upregulates this chemokine is of potential toxicological significance. However, the mechanisms immediately preceding IL-8 expression remain unclear.
IL-8 upregulation has been, in other models, linked to elevated transcription (Bhattacharyya et al., 2002; Buss et al., 2004), increased mRNA stability (Bosco et al., 1994; Thorpe et al., 2001), or a combination of these two mechanisms (Holtmann et al., 1999; Li et al., 2002). The purpose of this study was to test the hypothesis that DON-induced IL-8 in monocytes is mediated transcriptionally, through activation of multiple transcription factors, and posttranscriptionally, by increasing IL-8 mRNA stability. Human U937 cells originating from the pleural effusion of an individual with diffuse histiocytic lymphoma (Sundstrom and Nilsson, 1976) were employed to test this hypothesis. These cells markedly express IL-8 in response to DON and, thus, closely mimic primary monocytes (Islam et al., 2006; Sugita-Konishi and Pestka, 2001). The results presented here indicate that DON-induced IL-8 expression in monocytes was driven, in part, by NF-B–mediated transcription but did not involve mRNA stabilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemicals and cell culture.
DON and Salmonella typhimurium lipopolysaccharide (LPS) (1.5 EU/ng) were purchased from Sigma Chemical Co (St Louis, MO), as were other chemicals unless noted. U937 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were grown at 37°C with 6% CO2 in RPMI-1640 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco BRL, Rockville, MD). DON or LPS were dissolved in RPMI-1640 without FBS or antibiotics and incubated with the cells for various time intervals before RNA isolation (15 min to 3 h), nuclear protein isolation (3 h), and supernatant fraction collection (12 h) for ELISA.
For NF-B inhibition, U937 cells (1 x 106 cells/ml) were treated with 100 µg/ml of caffeic acid phenethyl ester (CAPE; Calbiochem, San Diego, CA), dissolved in ethanol, or vehicle 2 h prior to addition of DON or LPS where indicated. This CAPE concentration was selected based on the manufacturer's recommendations and verified to be nontoxic by the 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide viability assay (data not shown).
Promoter constructs.
To study IL-8 promoter activity, a previously described IL-8 promoter-firefly luciferase reporter construct (162/+44 IL-8 LUC) (Casola et al., 2002) was kindly provided for this investigation by Dr A. Casola (University of Texas, Galveston, TX). The site-directed mutagenesis protocol was based on Strategene's QuikChange Kit and primers were created with the web-based program at http://www.stratagene.com/tradeshows/feature.aspx?fpld=118 using portions of sequence from the published IL-8 promoter sequence (Mukaida et al., 1989) (Table 1). Briefly, reactions consisted of 1x Pfx reaction buffer (Invitrogen, Carlsbad, CA), 50 ng of 162/+44 IL-8 LUC, 0.35µM of each primer, 10mM dNTP, and RNase- and DNase-free water to a total volume of 50 µl. Pfx (2.5 U; Invitrogen) was added, and the reactions were run at 95°C for 2 min with 15 repetitions of 95°C for 30 s, 55°C for 1 min, and 68°C for 12 min. After cooling, 20 U of DpnI (New England Biolabs, Beverly, MA) was added, and the reactions were incubated for 2 h at 37°C. The product (2 µl) was used to transform JM109 Escherichia coli cells, clones were selected, and plasmids isolated (Wizard MiniPrep from Promega, Madison, WI) for sequencing at Michigan State University's Research Technology and Support Facility (RTSF). Following confirmation of specific mutation, plasmids were amplified in JM109 E. coli cells and isolated using the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA).
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Potential effects of selected mutations of the IL-8 promoter were estimated using two bioinformatics programs that predict transcription factor binding, PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) (Farre et al., 2003
; Messeguer et al., 2002) and TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) (Heinemeyer et al., 1998). Using the available data, one or both programs predicted that the mutations for AP-1, NF-B, CCAAT/enhancer-binding protein ß (C/EBPß), and octamer-1 (Oct-1) would prevent binding of their respective transcription factors; comparable information for NF-B repressing factor (NRF) was unavailable on these sites.
Transient transfection of U937 cells.
U937 cells (107 cells/210 µl of RPMI-1640 without FBS or antibiotics) were electroporated with 15 µg of plasmid –162/+44 IL-8 LUC or one of the five mutated promoter constructs containing a firefly luciferase reporter using a Bio-Rad (Hercules, CA) Gene Pulser at 280 V and 960 µF in a 0.4-cm gap cuvette (Bio-Rad). For normalization, electroporations were conducted simultaneously with 0.5 µg pRL SV40 (Promega), a Renilla luciferase reporter gene under control of the constitutive SV40 promoter. Following electroporation, cells were diluted in 12 ml growth media; incubated at 37°C for 1 h; then treated with vehicle, 1 µg/ml DON, or 1 µg/ml LPS, and plated at 1 ml/well. Since DON induces IL-8 mRNA and IL-8 hRNA in a concentration-dependent fashion from 250 to 1000 ng/ml (Islam et al., 2006), the highest concentration was used to maximize resolution of promoter studies. A similar rationale was used for selecting LPS concentration. After 11 h, both firefly and Renilla luciferase levels were measured with a Dual-Luciferase Reporter Assay (Promega) according to manufacturer's instructions. A Turner 20e Luminometer (Turner Designs, Sunnyvale, CA) was used for luminometry measurements. Firefly luciferase levels were normalized against Renilla luciferase levels measured in the same sample. All normalized treatment data were expressed relative to vehicle control for each IL-8 promoter construct.
Assessment of NF-B binding.
Nuclear proteins were isolated with the Nuclear Extraction Kit (Active Motif, Carlsbad, CA) according to manufacturer's instructions, quantitated using a DC Protein Quantitation Kit (Bio-Rad), and diluted to 2.5 µg/µl. Binding of NF-B family members was assessed with the TransAM NF-B Family Flexi Kit (Active Motif) using IL-8 promoter-specific probe sequence (5'-GATCCATCAGTTGCAAATCGTGGAATTTCCTCTA-3'). The probe was biotin-labeled with the Biotin 3' End DNA Labeling Kit (Pierce, Rockford, IL) per manufacturer's instructions and duplexed by incubating 1 pmol/µl of each oligonucleotide together at 95°C for 5 min and ramping back to 4°C by decreasing 1°C per minute. Nuclear protein (20 µg) was incubated with a duplex, biotin-labeled IL-8–specific probe, then added to a strepavidin-coated 96-well plate. Following washing, wells were incubated sequentially with primary antibodies for each NF-B subunit followed by anti-rabbit horseradish peroxidase (HRP)-conjugated antibody. Bound HRP was determined by addition of substrate, and plates were read at 450 nm on a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA).
RNA isolation and reverse transcription real-time PCR.
Treated cultures were centrifuged for 10 min at 300 x g, supernatant fraction was removed, and RNA was isolated with an RNaqeous kit (Ambion, Austin, TX). Briefly, cells were lysed, nucleic acids precipitated with ethanol, RNA trapped on a glass fiber filter, and then eluted from the filter. Samples were treated with Turbo DNA free (Ambion) to remove gross DNA contamination. Eluted RNA was incubated with Turbo DNase and the enzyme precipitated with the proprietary inactivation reagent.
Reverse transcription real-time PCR was performed using One-Step PCR Master Mix (Applied Biosystems; Foster City, CA) and IL-8 Pre-Developed Assay Reagents (PDAR) (Applied Biosystems) multiplexed with the 18S PDAR (Applied Biosystems). Fold change was determined using a relative quantitation method (Smolinski and Pestka, 2005). PCR was conducted according to manufacturer's instructions using an ABI 7900HT (384 wells), at the Michigan State University's RTSF. First, standard curves are created using dilutions of total RNA from LPS-treated U937 cells. An equation for the trend line of the standard values was used to convert the Ct values obtained in the assay to nanogram amounts of target. The amounts were normalized by dividing the IL-8 value by the 18S (endogenous control) value. Relative expression was determined by dividing all normalized values by the average of the control normalized value.
IL-8 protein determination.
Treated cultures were centrifuged for 10 min at 300 x g and supernatant fractions collected and stored at – 20°C. OptELISA IL-8 Kits (Pharmingen, San Diego, CA) were used according to manufacturer's instructions with two modifications. First, the highest standard utilized was 1600 pg/ml, instead of 400 pg/ml. Second, to economize on reagents, 50 µl of antibody dilutions and samples were used per well instead of 100 µl. Samples were read at 450 nm in a Vmax Kinetic Microplate Reader (Molecular Devices).
Statistics.
Data were analyzed with SigmaStat v 3.1 (Jandel Scientific, San Rafael, CA). IL-8 protein and RNA data were analyzed using ANOVA with Student-Newman-Keuls Method for pairwise comparisons unless otherwise noted. Samples treated with inhibitor were compared to the vehicle control using a t-test. A t-test was used for the luciferase experiments by comparing the mutated promoter construct to the IL-8 wild-type promoter in the same treatment group and in the NF-B binding assay by comparing DON and LPS treated to the vehicle control group. A p value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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DON Induces IL-8 Transcription
DON has been previously shown to increase IL-8 hRNA expression in U937 monocytes, which is strongly indicative of an accelerated transcription rate for this chemokine gene (Islam et al., 2006
). To further confirm the action of DON on IL-8 transcription, U937 cells were transfected with a wild-type IL-8 promoter luciferase (–162/+44 IL-8 LUC) construct, incubated with DON (1 µg/ml) or the positive control, LPS (1 µg/ml), and luciferase expression was assessed. Incubation with either DON or LPS (as positive control) was found to significantly induce IL-8 promoter-driven luciferase reporter expression as compared to cells treated with vehicle only (Fig. 1). These results provide further evidence that increased IL-8 transcription results from DON exposure.
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12). Bars with different letters are significantly different (p 
0.05).DON Induction of IL-8 Transcription Requires NF-
BIL-8 transcription is potentially controlled by several transcription factors (Sharma et al., 1998
; Wu et al., 1997). To identify the transcription factors important for DON-induced IL-8 transcription, the –162/+44 IL-8 LUC plasmid was mutated using site-directed mutagenesis at binding sites of five transcription factors reported previously to regulate IL-8 transcription (AP-1, C/EBPß, Oct-1, NF-B, and NRF) (Fig. 2). Relative luciferase levels for U937 cells transfected with each mutated IL-8 promoter plasmid were compared to cells transfected with the wild-type IL-8 promoter.
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Mutation of the NF-
B site was found to significantly reduce both DON- and LPS-induced luciferase as compared to the wild-type promoter (Fig. 3A). In contrast, mutation of the AP-1 site significantly increased both DON- and LPS-induced IL-8 promoter-driven luciferase compared to luciferase expression from the wild-type IL-8 promoter (Fig. 3B).
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Mutations in the C/EBPß (Fig. 4A), Oct-1 (Fig. 4B), or NRF (Fig. 4C) sites did not alter the DON-induced luciferase expression as compared to the wild-type IL-8 promoter construct. While mutation in the C/EBPß site similarly did not alter LPS-induced luciferase as compared to luciferase expression from the IL-8 wild-type promoter (Fig. 4A), LPS-induced luciferase expression was potentiated by the Oct-1 mutation (Fig. 4B) and attenuated for the NRF mutation (Fig. 4C).
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NF-
B Inhibition Ablates DON-Induced IL-8 mRNA and Protein ExpressionTo further confirm the role of NF-
B, CAPE, a specific inhibitor for this transcription factor was employed. DON- and LPS-induced IL-8 mRNA (Fig. 5) and IL-8 protein (Fig. 6) were completely inhibited following CAPE pretreatment in U937 cells. CAPE was also similarly able to suppress LPS-induced IL-8 protein (data not shown).
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DON Increases Binding Activity of p65
Increased transcription can result, in part, from increased DNA binding of transcription factors to promoter regions. Based on the observation that the NF-
B site mutation specifically reduced DON-induced luciferase, the effects of the toxin on binding of five NF-B subunits (p65/RelA, c-Rel, RelB, p50/NF-B1, and p52/NF-B2) were assessed using an ELISA-based binding assay. Two of the five subunits, RelB and c-Rel, were undetectable in all U937 cell samples tested (data not shown) while detectable levels of p65, p50, and p52 were observed. DON and LPS were found to significantly (p < 0.05) increase p65 binding by 21- and 40-fold, respectively (Fig. 7). Binding to the biotin-labeled IL-8 wild-type probe was not altered with competition from an unlabeled probe with a mutation in the NF-B site, identical to the one used in the IL-8 promoter-driven luciferase studies, while binding was reduced by approximately 80% or greater, with an unlabeled wild-type probe (data not shown).
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Although p50 binding was equivalent in untreated and DON-treated samples, there was a small, but significant (p < 0.05), increase in p50 binding in LPS-treated U937 cells (Fig. 7). While in untreated and LPS-treated samples p52 binding was equivalent, there was a trend (p = 0.056) toward reduced p52 binding in the DON-treated samples (Fig. 7). Accordingly, DON treatment could potentially affect NF-
B binding by increasing p65 binding and, to a lesser extent, by decreasing p52 binding.
DON Does Not Affect IL-8 mRNA Stability
In addition to transcriptional effects, DON could possibly upregulate IL-8 mRNA expression by increasing the mRNA stability as observed for IL-6 (Jia et al., 2006; Wong et al., 2001), TNF- (Chung et al., 2003b; Wong et al., 2001), and COX-2 (Moon et al., 2003). Based on its aforementioned efficacy (Fig. 8), CAPE was used here to block IL-8 transcription and enable assessment of DON's effects on IL-8 mRNA half-life. U937 cells were pretreated with CAPE for 2 h. After addition of DON or LPS, RNA was isolated every 15 min for 2 h. The half-life of the IL-8 transcript was approximately 40 min in all treatment groups (Fig. 8). Neither the inclusion of DON nor LPS altered the stability of IL-8 mRNA as compared to the control group. Thus, DON did not appear to affect IL-8 mRNA stability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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IL-8 transcription can potentially be regulated at the promoter level by several different transcription factors. While some cell models require multiple factors, working together, for optimal IL-8 expression (Jijon et al., 2002
; Sharma et al., 1998; Wu et al., 1997), NF-B is the only transcription factor required for LPS-induced IL-8 in THP-1 cells (Makarov et al., 1997; Haas et al., 1998) and in T98G glioblastoma cells (Mukaida et al., 1994). Analogous observations have been made in other cell culture models (Ameixa and Friedland, 2002; Dragneva et al., 2001; Hippenstiel et al., 2000; Li et al., 2002). Consistent with these latter studies, the data presented here indicate that DON-induced IL-8 expression was highly NF-B dependent. The observed role for NF-B in DON-induced IL-8 expression correlates with our prior observations that NF-B binding increases in DON-treated murine macrophages and T cells (Ouyang et al., 1996; Wong et al., 2002).
CAPE prevents NF-B nuclear translocation without altering binding of two other transcription factors with binding sites in the IL-8 promoter, AP-1 and Oct-1, or the basal transcription factor TFIID in U937 cells (Natarajan et al., 1996). The complete inhibition of DON-induced IL-8 protein and mRNA by CAPE supports the contention for a proposed role for NF-B in DON-induced IL-8 expression.
The transcription factor ELISA employed here offers two levels of specificity as determined by specific antibodies and site-specific promoter binding. Our observations are consistent with previous investigations implicating the p65 (RelA) subunit of NF-B in IL-8 expression (Buss et al., 2004; Kunsch et al., 1994; Lakshminarayanan et al., 1998; Vlahopoulos et al., 1999). Of the three NF-B subunits detectable here in U937 cells, p65 is the only one to contain a transactivation domain (Liang et al., 2004). The other two NF-B subunits, p50 and p52, only contain a DNA-binding and dimerization domain and cannot activate transcription. Dimers of NF-B consisting of p50 and p52 have been found in several cell types including HepG2 (Roman et al., 2000), C33A epithelial (Miller et al., 1998), and JB6 epithelial (Hsu et al., 2001) cells. Based upon the observation that, following DON exposure, p65 binding significantly increases, p52 binding decreases, and p50 remains equivalent to the control, a shift in the composition of NF-B might be predicted. Accordingly, DON-treated cells are more likely to contain a lower proportion of dimers composed of p50 and p52 and a higher proportion of heterodimers composed of p65 and p50 than vehicle-treated cells. These latter heterodimers would likely contribute to DON-induced IL-8 transcription. An additional complexity of the NF-B system is that dimers are sequestered in the cytoplasm by either IB or by the protein precursors of p50 or p52. The investigation of the upstream inhibitory proteins should be examined in the future to further elucidate how DON activates NF-B.
Comparison of DON-induced luciferase and IL-8 responses revealed that relative increases of wild-type IL-8 promoter-driven luciferase (two- to threefold) were much lower than relative increases in IL-8 protein (46-fold). Similarly, small increases in relative luciferase expression were also observed for the LPS positive control. It should be noted that the studies described here specifically focused on promoter-driven transcription and identification of transcription factors necessary for DON-induced IL-8 expression. However, other mechanisms for IL-8 transcriptional regulation are likely to be involved. Notably, the IL-8 promoter-driven luciferase construct lacks the structure and regulation of chromatin. Histone alteration has been found in several studies to contribute to IL-8 expression with histone acetylation mediating accessibility to the IL-8 promoter site. In the case of oxidative stress within human aveolar epithelial cells, histone acetylation results in chromatin remodeling leading to increased transcription of the IL-8 gene triggered by activation of NF-B and AP-1 (Rahman et al., 2002). Tomita et al. (2003) attributed H2O2- and trichostatin A–induced IL-8 expression to hyperacetylation of histone H4 in BEAS-2S epithelial cells. Yamamoto et al. (2003) found, using HeLa epithelial cells, that TNF-–induced IL-8 production required phosphorylation and acetylation of histone H3. Thus, to obtain a more complete understanding of the effects of DON on transcription of IL-8 and other chemokines, it will be necessary in the future to assess the contributions of histone acetylation and chromatin remodeling.
The concentrations of DON necessary to induce IL-8 expression in monocytes range from 250 to 1000 ng/ml (Islam et al., 2006). Concentrations slightly higher than these can be seen in mouse serum up to 4 h after following oral exposure to 5 mg/kg body weight of DON (Azcona-Olivera et al., 1995), thus suggesting the relevance of the in vitro data described herein. It should be noted that, given the modest reporter responses, we employed to highest concentration in this range (1000 ng/ml) to enhance resolution for comparison of promoters.
It was surprising that mutation of the AP-1 site significantly increased IL-8 promoter-driven luciferase expression in both DON- and LPS-treated cultures. Using the PROMO web-based transcription factor binding site recognition program, an increase in the percent similarity of the binding site in the IL-8 promoter to a consensus binding site for a basal transcription factor, TFIIB, was predicted for the mutated AP-1 IL-8 promoter as compared to the wild-type promoter. This increase in similarity provides a plausible explanation for the increase in luciferase seen with the mutant AP-1 construct as compared to the wild type.
Differences observed between LPS and DON responses with other mutated IL-8 promoters possibly reflect DON's inability to act through TLR receptors (Islam et al., 2006). While there was no difference between the wild-type IL-8 promoter and the mutant Oct-1 or mutant NRF with DON exposure, the Oct-1 mutation increased the LPS-induced luciferase and the NRF mutation decreased the LPS-induced luciferase. Both Oct-1 and NRF have been reported to act as negative regulatory elements (Nourbakhsh et al., 2001; Wu et al., 1997) in the IL-8 promoter. The increase in luciferase seen with the Oct-1 mutation might be explained if the Oct-1 site acts as a standard negative regulatory element in LPS-treated U937 cells but is not involved in regulation of DON-induced IL-8. Previously, the function of NRF has varied, being observed to be a negative regulatory element for one stimulus but a positive element necessary for transcription with a different stimulus (Nourbakhsh et al., 2001). The results here suggest that NRF was not necessary for DON-induced transcription but did contribute to LPS-induced transcription.
DON has been shown to increase stability of mRNAs for TNF-, IL-6, and COX-2 in RAW 264.7 (Moon et al., 2003), as well as IL-2 in EL-4 thymoma cells (Li et al., 1997). Clearly, IL-8 expression in U937 cells does not share this common DON-induced posttranscriptional mechanism with the aforementioned genes. Other studies suggest that stabilization of IL-8 mRNA, indeed, depends on stimulus and cell type. For example, THP-1 monocytes exposed to LPS (Frevel et al., 2003) exhibit increased IL-8 mRNA stability, whereas those treated with urate crystals (Liu et al., 2000) or Staphylococcus toxin (Dragneva et al., 2001) do not. Similarly, transfection of HEK-293 cells with constitutively active MEKK1 and MKK6 increases IL-8 mRNA stability (Holtmann et al., 1999), while treatment with neutrophil elastase does not (Devaney et al., 2003).
While U937 cells behave similarly to primary human monocytes relative to IL-8 expression (Islam et al., 2006), their employment in reporter studies such as those described herein were challenging because only relatively low transfection efficiency was achieved, regardless of the method used. Difficulties with expression of reporter constructs have been described in U937 cells (Duvoix et al., 2004; Malone et al., 1989), and several other investigations have confirmed that transfection efficiency for U937 cells is inherently poor (Kusumawati et al., 1999; Shimokawa et al., 2000). We overcame this obstacle by using firefly and Renilla luciferases as reporter and transfection control, respectively, which ensure accurate and reproducible normalization. Another consideration is that U937 cells contain CYP450s (Nagai et al., 2002; Wang et al., 2004), phase II drug metabolizing enzymes (Duvoix et al., 2004), and even a multidrug resistance transporter (Belloc et al., 1997), suggesting that this cell line can effectively metabolize or export some xenobiotics. These characteristics provide a possible explanation for observations in preliminary mRNA stability studies that conventional transcriptional inhibitors actinomycin D and DRB were ineffective at arresting IL-8 transcription. This problem was overcome by using the highly specific inhibitor CAPE (Natarajan et al., 1996).
The capacity of DON to induce IL-8 has possible clinical significance because this cytokine is associated with several chronic diseases (Alzoghaibi et al., 2003; Banks et al., 2003; Georganas et al., 2000; Suzuki et al., 2000), including primary IgA nephropathy, the most common glomerulonephritis in the world (Huang et al., 2001; Lim et al., 2003). Interestingly, chronic DON ingestion can evoke aberrant IgA production and kidney deposition in the mouse, thus mimicking this human disease (Pestka, 2003). It is further notable that two of the principal effects of chronic DON consumption in nonruminant mammalian species are anorexia and reduced weight gain (Pestka and Smolinski, 2005). IL-8 has also been found to play a role in anorexia, with elevated IL-8 levels observed cerebrospinal fluid of human patients exhibiting anorexia after a trauma (Chuang et al., 2005) and confirmed by a study infusing the brains of rats with IL-8, among other cytokines (Sonti et al., 1996). Rats given IL-8 had a significantly reduced total feed intake due to a smaller meal size. Thus, it might be speculated that IL-8, along with other anorectic cytokines such as TNF- and IL-1ß, could mediate reduction in food intake and weight gain in DON-exposed animals and humans.
In conclusion, DON-induced IL-8 expression was highly NF-B dependent. These data suggest that this transcription factor might be a possible target for reducing the toxicity of DON or other trichothecenes in a prophylactic or therapeutic manner. A critical question that remains to be answered relates to how DON mediates NF-B activation in the monocyte. DON and other trichothecenes are known to rapidly induce MAPK activation in leukocytes via a mechanism known as the ribotoxic stress response (Pestka et al., 2004). DON induces phosphorylation p38, ERK, and JNK in concentration-dependent fashion in human monocytes (Islam et al., 2006). p38 inhibition completely represses DON induction of IL-8 expression in monocytes (Islam et al., 2006). Both double-stranded RNA-associated protein kinase (PKR) and hematopoietic cell kinase function upstream of p38, ERK, and JNK (Zhou et al., 2003, 2005). Interestingly, PKR triggers the release of NF-B, through activation of IB kinase complex (IB) (Zamanian-Daryoush et al., 2000) or phosphorylation of IB (Kumar et al., 1994), whereas p38 can increase the activation of NF-B (Schmeck et al., 2004; Vanden Berghe et al., 1998). Given the general importance of NF-B, future research should focus on how PKR, p38, and other kinases communicate the initial ribosome-binding event to activation of NF-B and other transcription factors.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The National Institute for Environmental Health Sciences (ES 3358 to J.J.P.).
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ACKNOWLEDGMENTS |
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We would like to thank Sarah Godbehere and Mary Rosner for technical assistance and Drs R. Schwartz, L. Mansfield, and N. Kaminski for scientific advice during this project.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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