* Department of Food Science and Human Nutrition,
Department of Microbiology and Molecular Genetics, and
Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824-1224
Received July 18, 2002; accepted November 4, 2002
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ABSTRACT |
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Key Words: transcription factor; mitogen-activated protein kinases; trichothecene; mycotoxin; vomitoxin; immunotoxicity; TNF-; IL-1ß; IL-6.
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INTRODUCTION |
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Leukocytes are primary targets of trichothecenes. Numerous studies of host resistance, mitogen-induced lymphocyte proliferation and humoral immune response have yielded a common theme that trichothecenes can be either immunostimulatory and immunosuppressive, depending on dose, exposure frequency, and timing of functional immune assay (Bondy and Pestka, 2000). Acute high dose trichothecene exposure severely injures actively dividing tissues, including bone marrow, lymph nodes, spleen, thymus, and intestinal mucosa that can result in immunosuppression (Ueno, 1987
). Acute low-dose trichothecene exposure aberrantly affects immune function by initiating a rapid and transient upregulation of proinflammatory cytokines (Bondy and Pestka, 2000
). These latter effects can be exacerbated by concurrent exposure of experimental animals to a trichothecene and an inflammagenic stimulus such as lipopolysaccharide (Islam et al., 2002
; Tai et al., 1988a,b; Taylor et al., 1991
; Zhou et al., 2000
).
The capacity of trichothecenes to upregulate the proinflammatory cytokines TNF-, IL-1ß, and IL-6 appears to drive immunotoxic effects such as shock and IgA nephropathy in mice (Bondy and Pestka 2000
). Trichothecene-induced cytokine mRNA expression may be mediated transcriptionally or post-transcriptionally. Relative to the former, altered transcription factor binding activity in vitro is of particular significance (Li et al., 2000
; Ouyang et al., 1996
; Wong et al., 2002
). Notably, the TNF-
, IL-1ß, and IL-6 promoters all contain binding sites for activating protein (AP-1), CCAAT enhancer-binding protein (C/EBPs), cyclic AMP response element (CRE)-binding protein (CREB, also known as activator transcription factor [ATF]) and nuclear factor-
B (NF-
B) (Godambe, 1994; Haudek et al., 1998
; Hiscott et al., 1993
; Husmann et al., 1996
; Spriggs et al., 1992
; Tanabe et al., 1988
).
The underlying molecular toxic event for trichothecenes in eukaryotic cells is believed to involve high affinity binding to peptidyl transferase site of the 60s ribosomal subunit (Middlebrook 1989; Ueno, 1987). Interestingly, translational inhibitors that bind to ribosomes have been observed to rapidly activate mitogen-activated protein kinases (MAPKs) in vitro (Iordanov et al., 1997
), suggesting that these signal transducers may mediate trichothecene toxicity. The three most widely studied MAPK subfamilies are: (1) p44 and p42 MAPKs, also referred to as extracellular signal regulated protein kinase 1 and 2 (ERK 1/2); (2) p54 and p46 c-Jun N-terminal kinase 1 and 2 (JNK 1/2) also known as stress-activated protein kinases (SAPK 1/2); and (3) p38 MAPK (Cobb et al., 1999
; Widmann et al., 1999
). MAPKs are important intermediates in signaling pathways that have been implicated in many physiological processes, including cell growth, differentiation, and apoptosis. In support of the contention that these kinases mediate trichothecene immunotoxicity, both our laboratory and others have demonstrated that VT and other trichothecenes activate ERK, JNK and p38 in vitro (Anderson, 1999; Shifrin and Moon et al., 2002; Yang et al., 2000
).
A major consequence of MAPK phosphorylation is the activation of transcription factors (Davis, 1995), which serve as immediate or downstream substrates of these kinases. For example, JNK 1/2 phosphorylates c-Jun, which is a component in the AP-1 homodimer or heterodimer (Dong et al., 2002
). Also, p38 (Bhat et al., 2002
) and ERK 1/2 (Hungness et al., 2002
) drive activation of C/EBP. Phosphorylation/activation of CREB/ATF members is mediated by JNK (Dong et al., 2002
), p38 (Bhat et al., 2002
) and ERK (Belmonte et al., 2001
). Finally, all three MAPK signaling pathways have been implicated in nuclear factor
B (NF-
B) activation through phosphorylation of its inhibitor I
B
(Lee et al., 1997
; Schwenger et al., 1998
; Zhao and Lee, 1999
). It is reasonable to suggest that the capacity of trichothecenes to activate MAPKs may contribute to transcriptional activation of cytokine genes.
Elucidation of how environmental stressors affect global regulation of gene expression and, more specifically, immune function at the kinase and transcription factor levels requires that in vitro findings be extended to animal models. Intact animals have been recently used to study the effects of chemical and physiologic stimuli on activation of MAPKs (Hu et al., 1997; Suh, 2001
; Takagi et al., 2000
; Yuan et al., 1999
; Zhong et al., 2001a
,b
) as well as to investigate activation of transcription factors in specific organs (Alam et al., 1992
; Armstead et al., 1999
; Ashida and Matsumura, 1998
; Blackwell et al., 1999
; Blazka et al., 1996
; Zhong et al., 2001a
,b
). These studies suggest that it might be feasible to integrate both strategies to study the molecular effects of trichothecenes in vivo.
Cytokine mRNAs and proteins are typically induced by VT in vivo in rapid (12 h) and transient fashions (48 h) (Azcona et al., 1995; Zhou et al., 1998; Zhou and Pestka, 1997; ). Here, we hypothesized that MAPKs and transcription factors are activated prior to or concurrently with proinflammatory cytokine gene upregulation in vivo. To test this hypothesis, mice were treated orally with VT and their spleens analyzed temporally for MAPK phosphorylation as well as for activation of splenic nuclear proteins binding to four different consensus transcriptional control motifs associated with cytokine promoters. Response elements associated with AP-1, C/EBP, CREB, and NF-
B were selected based on the presence of these DNA sequences in the TNF-
, IL-1ß and/or IL-6 promoters as well as on the recognized capacities of these transcription factors to mediate proinflammatory cytokine gene transactivation in vitro. The results indicated that VT induced the rapid and transient phosphorylation of ERK, p38 and JNK in vivo. These effects were concurrent with or followed by time-dependent increases and decreases in transcription factor binding corresponding to nuclear translocation. Both timing and differential activation of MAPKs and transcription factors by VT were consistent with the profile, magnitude, and duration of proinflammatory cytokine expression described previously and confirmed in this study.
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MATERIALS AND METHODS |
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For MAPK studies, mice were administered VT (1 to 100 mg/kg) by a single oral gavage in 0.25 ml of endotoxin-free water. These doses were selected to bracket VT doses of 5 to 25 mg/kg, which are sufficient to induce strong proinflammatory cytokine responses in this mouse model (Azcona et al., 1995; Zhou et al., 1997, 1998
). Control mice were given 0.25 ml of endotoxin-free water. Mice were killed after 15, 30, 60, and 120 min by cervical dislocation under methoxyflurane (Schering-Plough Animal Health, Union, NJ). Spleens were removed and whole cell lysates analyzed for MAPK phosphorylation.
For transcription-factor studies, mice were gavaged orally with 25 mg/kg of VT. They were then killed at 0.5, 1.5, 4, and 8 h (kinetic studies) or at 0.5, 1.5, or 4 h post-treatment (competition, supershift, and Western blot analyses) and spleens were excised and pooled (23 per group) for preparation of nuclear protein extracts to conduct EMSA.
For cytokine mRNA studies, mice were gavaged orally with 12.5 mg/kg of VT. Spleens were removed at 0, 3, 6, and 9 h and analyzed for proinflammatory cytokine mRNA.
Detection of MAPK phosphorylation.
Phosphorylation of MAPKs was assayed by Western blot, using rabbit polyclonal antibodies specific for phospho-JNK 1/2, phospho-ERK 1/2, and phospho-p38 MAPK (Cell Signaling, Beverly, MA) as described in manufacturers instruction. Spleen cells were dispersed in SDS lysis buffer (1% SDS, 1 mM sodium ortho-vanadate, 10 mM Tris pH [7.4]), transferred to a microcentrifuge tube, boiled for 5 min, and then sonicated briefly. The lysate was centrifuged at 15,000 x g for 15 min at 4°C to pellet insoluble material. Protein concentration of the resultant supernatant was determined using a Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Inc., Melville, NY). Total cellular proteins were resolved by 8% (w/v) SDSPAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham, Arlington Heights, IL). After blocking with 5% (w/v) nonfat dry milk, the immobilized proteins were incubated with phospho-specific antibodies followed by horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Amersham). Bound peroxidase was determined using an ECL chemiluminescence detection kit (Amersham). To assess loading, membranes were stripped and reprobed with specific antibodies that recognize both phosphorylated and unphosphorylated forms (Cell Signaling) of each MAPK.
Preparation of nuclear extracts.
Spleen cells were dissociated and passed through a 100 mesh stainless steel screen. Cells were suspended in Dulbeccos phosphate buffered saline (PBS, Sigma). Erythocytes were lysed for 2 min at 25°C in 0.01 M KHCO3 containing 0.14 M NH4Cl. Nuclear extracts were prepared using the method of Olnes and Kurl (1994) with modifications to prevent protein modification or degradation. Briefly, cells were lysed in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM Mg Cl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM dithiothreitol) with phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride), protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 2 mg/ml leupeptin, 2 mg/ml aprotinin), and 0.6% (w/v) Nonidet P-40. Nuclei were pelleted by centrifugation, suspended in hypertonic buffer containing 20 mM HEPES pH 7.9, 0.4 M KCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 10% (w/v) glycerol, and the phosphatase and protease inhibitors indicated above. After 30 min at 4°C, soluble proteins released were collected by centrifugation at 15,000 x g for 10 min. The supernatant was dialyzed for 2 h at 4°C against 1000 volumes of dialysis buffer (20 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10% [w/v] glycerol supplemented with leuprotinin, aprotinin, and sodium fluoride). Resultant extracts were analyzed for protein, aliquoted, and then stored at 80°C until analysis.
Electrophoretic mobility shift assay (EMSA).
EMSA was used to characterize the binding activity of AP-1, C/EBP, CREB, and NF-B transcription factors in nuclear extracts (Chodosh, 1993
). Double stranded AP-1, C/EBP, CREB, and NF-
B consensus probes (Santa Cruz Biotech, Santa Cruz, CA) (Table 1
) were radiolabeled with [
-32P-ATP] using Ready to GoTM Polynucleotide Kinase Kit (Pharmacia Biotech, Inc., Piscataway, NJ). Nuclear extracts containing 5 to10 µg protein were added to DNA-binding reaction buffer consisting of 20 mM HEPES pH 7.9, 60 mM KCl, 1 mM EDTA, 0.5 mM DTT, 2 µg poly dIdC in a total volume of 20 µl. These were preincubated on ice for 15 min to block nonspecific binding. Following the addition of 1 µl 32P-labeled probe containing 30,000 cpm, the incubation was continued for 30 min at room temperature to promote the formation of nucleoprotein complexes. Resultant nucleoprotein complexes were analyzed by loading samples on a 4% (w/v) native polyacrylamide gel in 0.5 x TBE buffer, dried, and visualized by autoradiography. In each set of experiments, a self-competition was performed by adding excess of the same unlabeled oligonucleotide probe or a corresponding mutant oligonucleotide probe (Table 1
) to the binding reaction at the preincubation step.
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Western blot analysis of nuclear transcription factors.
Electrophoresis of nuclear proteins was performed with 10% (w/v) polyacrylamide gel in the presence of 0.1% SDS in the discontinuous buffer system of Laemmli (1970). The proteins were transferred to a PVDF membrane (Amersham, Piscataway, NJ). Membranes were incubated with polyclonal antibodies for C/EBP, AP-1, CREB, and NF-
B family members, washed and then incubated with horseradish-peroxidase-conjugated donkey anti-rabbit IgG antibody (Amersham). Enzyme activity in bands was visualized using chemiluminescence as described for MAPKs.
RNA isolation and quantitation of cytokine mRNA.
Total RNA from spleen was isolated using an RNAqueous Kit (Ambion Inc., Austin) according to the manufacturers protocol. The resulting RNA was dissolved in 50 µl of elution buffer and stored at 80°C. All PCR reactions for IL-6 and TNF- cytokine mRNA quantification were performed on an ABI PRISM 7700 Sequence Detector System using Taqman One-Step RT-PCR Master Mix Reagents Kit according to the manufacturers protocol (Applied Biosystems). Ct values for Il-6, TNF-
and 18S rRNA were adjusted using the standard curves of known amounts of total RNA (ranging 1.37 to 1000 ng/) and normalized by dividing either IL-6 or TNF-
adjusted amounts by the 18S adjusted amount. IL-1ß cytokine mRNA was quantitated by Quantikine colorometric ELISA method (R&D systems, Inc., Minneapolis) according to the manufacturers protocol.
Statistics.
The data were analyzed by Dunnetts and Student-Neuman-Keuls tests using Sigma-Stat Statistical Analysis System (Jandel Scientific, San Rafael, CA). A p value of <0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Although the exact mechanisms by which VT induces MAPK phosphorylation remain unresolved, trichothecenes (Shifrin and Anderson, 1999; Yang et al., 2000
) and other translational inhibitors (Iordanov et al., 1997
), which bind to eukaryotic ribosomes known to activate MAPKs. Iordanov et al. (1997)
found that anisomycin and antibiotics that bind to 28S rRNA are strong activators of JNK. Notably, two ribotoxic enzymes, ricin A chain, and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA, are strong agonists of JNK1 and of its activator, SEK1/MKK4. Anisomycin and the ribotoxic enzymes apparently mediate signal transduction from altered 28S rRNA to JNK in active ribosomes but not in inactive ribosomes. These investigators described the capacity of the ribosomes to sense cellular stress as a "ribotoxic stress response." The possibility exists that VTs capacity to activate MAPKs in vitro and in vivo is also reflective of a ribotoxic stress response, and that it drives subsequent events related to transcription factor activation and ultimately, cytokine expression. The molecular transducer linking ribosomal binding of trichothecene and the almost immediate activation of MAPKs is not known and represents a fertile area for future investigation.
JNK 1/2 and p38 are generally believed to be stress responsive MAPKs and to be susceptible to chemicals, while ERK is thought activated by receptor-mediated mechanisms. In contrast, our results suggest that ERK can be rapidly activated by VT and confirm findings that have been made in vitro with VT (Moon et al., 2002) and other trichothecene mycotoxins (Yang et al., 2000). It is possible that the prolonged ERK response, as compared to JNK and p38, may be receptor-driven via the cytokines that are initially induced by VT (Fig. 12
).
AP-1 regulates many genes associated with proinflammatory cytokine production, monocytic differentiation, B- and T-cell activation, Th1/Th2 differentiation of immunoglobulin production, and apoptosis (Pennypacker, 1998). AP-1 binds to the palindromic TRE sequence TGA(C/G)TCA (Liebermann et al., 1998
). Members of the AP-1 family include four Fos proteins (c-Fos, Fos B, Fra-1, and Fra-2) and three Jun proteins (c-Jun, JunB, and JunD) (Pennypacker, 1998
). These can interact as either a Jun homodimeric complex or as a Fos/Jun heterodimeric complex. The seven subunits are subject to regulation via phosphorylation and by chemical oxidation of specific cysteine residues mapping within the DNA binding domains of Fos and Jun. Our results suggest that VT exclusively affected Jun homodimers including a JunB homodimer as well as dimers involving differential association with c-Jun, JunB, and/or JunD. Based on our results, AP-1 is likely to play a prominent role in transactivation of proinflammatory cytokines by VT.
IL-1ß, IL-6, IL-8, TNF-, M-CSF, and IP-10 expression are regulated, in part, by C/EBP (Akira and Kishimoto, 1997
; Koj, 1996
; Ohmori and Hamilton, 1994
; Pope et al., 1994
). C/EBP family members are derived from myeloid lineage and impact monocyte/macrophage differentiation (Valledor et al., 1998
). The C/EBP family contains several family members (Akira et al., 1992
). These include C/EBP
, which is found in undifferentiated myeloid cells, as well as C/EBPß (nuclear factor IL-6 [NF-IL6]) and C/EBP
that are mainly expressed in macrophages (Hanson, 1998
). C/EBP proteins dimerize with other C/EBP proteins in a cell differentiation state- and tissue-specific manner, thus facilitating the differential regulation of hematopoiesis target genes (Yamanaka et al., 1998
). C/EBPß was specifically found to be affected by VT. This protein translocates into the nucleus following phosphorylation. The capacity of VT to sequentially upregulate and downregulate formation of at least two complexes associated with C/EBPß might mediate initial increases in proinflammatory cytokines and other inflammatory genes, whereas disappearance of these complexes might facilitate the eventual downregulation of their expression.
CREB/ATF transcription factors act through binding to the cAMP responsive element (CRE) palindromic octanucleotide, TGACGTCA (Mayr and Montminy, 2001). CREB-1, CREB-2, ATF-1, and ATF-2 are the best-characterized members of this family. Although the CREB/ATF family shares highly related COOH terminal leucine zipper dimerization and basic DNA binding domains, they are highly divergent in their amino terminal domains (Meyer and Habener, 1993
). It was notable that unlike AP-1 and C/EBP, the CREB binding response to VT was multiphasic, with a marked depletion at 0.5 h followed by upregulation at 1.5 and 4 h. This observation may relate to differential regulation by MAPKs. Even though CREB/ATF proteins bind CREs in their homodimeric form, they sometimes also bind as heterodimers, both within the CREB/ATF family and with members of the AP-1 and C/EBP families. These latter hybrid complexes exhibit different specificities for AP-1, C/EBP, and CREB motifs. The interaction of CREB with these other factors may also explain the existence of at least four CREB bands as well as multiple bands EMSAs for AP-1 in this current study. Further investigation into identification of these multiple bands is warranted, using gel conditions that provide higher band resolution as well as use of multiple antibodies during supershifts.
The NF-B transcriptional activator family responds to numerous stimulatory agents that include LPS, phorbol esters, IL-1, IL-2, TNF-
. NF-
B regulates many genes involved in acute phase responses and inflammation, such as GM-CSF, G-CSF, TNF-
, IL-1ß, IL-6, IL-2, IL-8, and NO synthase (Akira and Kishimoto, 1997
; Baldwin, 1996
). This factor was first identified as a protein complex consisting of a 65 kDa DNA (p65 or RelA) binding subunit and an associated 50 kDa protein (p50). p65 is functionally related to c-Rel (p75). Heterodimers or homodimers of these proteins are normally inactive in the cytoplasm, and are associated with an inhibitory factor, I
B. After stimulation, I
B is phosphorylated, ubiquinated, and degraded releasing the NF-
B dimer which translocates into the nucleus as an active form and can activate (via p65, c-Rel, and Rel B heterodimers with p50) or suppress (via p50 homodimer) transcription of various responsive genes (Baldwin, 1996
). The results found here suggest that both a c-Rel-p50 heterodimer and p50 homodimer may be upregulated in spleen late in the time period tested. This latter protein may have attenuating properties that contributes to cytokine downregulation by 6 h.
EMSA is based on the principal that proteins of differing size, molecular weight, and charge will exhibit different electrophoretic mobility in non-denaturing gels (Laniel et al., 2001). Interaction with DNA-binding proteins will cause DNA to characteristically migrate more slowly than free DNA. Clearly, EMSA is an in vitro assay and regulation of genes in vivo within the context of chromatin is undoubtedly much more complex, involving interactions of multiple factors. Our results represent the first step in elucidating this complex system in vivo that can be used as a basis for further detailed analyses. Any interpretation of the EMSA results or design of future follow-up studies must recognize that formation and electrophoretic mobility of DNA protein complexes are modulated by DNA sequence, molecular weights of the protein and the DNA, ionic strength and the pH of the binding and electrophoresis buffers, concentration of gel matrix, and temperatures used for binding reaction and electrophoresis (Taylor et al., 1994
). For the purposes of simplicity, a uniform buffer concentration and assay condition for all four transcription-factor families was employed in the current study. Also, consensus sequences were used here because no single gene was targeted. Another consideration is that EMSA was applied to crude nuclear extracts from the spleen. A possible complication with using animal tissues can result from the presence of numerous enzymes that can degrade or modify the transcription factors (Laniel et al., 2001
). We dealt with this issue by including both protease and phosphatase inhibitors in the extraction buffer.
Taken together, the patterns observed here for MAPK phosphorylation and differential activation/inhibition of binding activity within each transcription factor family are consistent with the known half-life of this toxin in tissue and the transient nature of VT-induced proinflammatory cytokine and other inflammation-associated genes. The data presented herein suggest the feasibility of examining the in vivo effects of VT and other toxicants on kinase signaling pathways and on activation of individual transcription factors within the context of other factors. One limitation of this study is that, for some endpoints, earlier submaximal responses were not observed at time points chosen. Thus future studies should examine additional time points to more precisely pinpoint peak MAPK and transcription factor responses. Although use of crude spleen homogenates precluded identification of specific cell phenotypes in which MAPK, transcription factor and cytokine effects occurred, future studies can address this issue by using immunohistochemistry to detect phosphorylated MAPKs, transcription factor activation/ sequestration, and cytokine mRNA expression in the context of cellular phenotype. These data are significant to human and animal health, because the induction of cytokines by VT and inflammation-associated genes may play a role in acute gastroenteritis as well as in chronic illness resulting from immune dysfunction. Furthermore, the results offer a venue for understanding how upstream events such as MAPK activation relate to regulation of gene expression in intact mice. The precise linkages might be explored in vivo using pharmacologic inhibitors for specific MAPKs in conjunction with the new in vivo imaging strategy of Carlson et al. (2002) that employs transgenic mice, which express luciferase under control of transcription factors.
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ACKNOWLEDGMENTS |
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NOTES |
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