Peroxisome Proliferators Do Not Activate the Transcription Factors AP-1, Early Growth Response-1, or Heat Shock Factors 1 and 2 in Rats or Hamsters

Michelle L. O'Brien*, Michael L. Cunningham{dagger}, Brett T. Spear*,{ddagger} and Howard P. Glauert*,§,1

* Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40506; {dagger} Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; {ddagger} Department of Microbiology and Immunology and Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, Kentucky 40506; and § Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40506

Received December 3, 2001; accepted May 1, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice, whereas hamsters are less responsive to these compounds. PPs increase peroxisomal ß-oxidation and P4504A subfamily activity, which have been hypothesized to result in oxidative stress. Work in our laboratory indicated that differential modulation of the redox-sensitive transcription factor NF-{kappa}B may contribute to the resulting difference in species susceptibility following PP administration. Therefore, we hypothesized that other redox-sensitive transcription factors such as AP-1, early growth response gene 1 (Egr-1), and heat-shock factors 1 and 2 (HSF1/2) may also be alternatively activated in differentially susceptible species. Accordingly, we measured the activation of these transcription factors using gel mobility shift assays, with hepatic nuclear extracts derived from rats and Syrian hamsters fed two doses of three peroxisome proliferators (dibutyl-phthalate [DBP], gemfibrozil and Wy-14,643) for 6, 34, or 90 days. Although changes were observed at various time points, no consistent, dose-responsive changes were observed in the DNA binding activities of these transcription factors following PP treatment. The lack of increased binding of AP-1, Egr-1, and HSFs suggests that these factors are not involved in increased cell proliferation following PP administration, although we cannot rule out that these factors are activated at earlier time points than those examined in this study.

Key Words: peroxisome proliferators; hamsters; rats; Egr-1; HSF; AP-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferators (PPs) are a diverse group of compounds that cause hepatocarcinogenesis in rats and mice (Rao and Reddy, 1991Go). Central to the mechanism of PP induced tumorigenesis is their ability to activate a ligand-activated transcription factor, the peroxisome proliferator-activated receptor alpha (PPAR{alpha}), a member of the nuclear receptor superfamily (Issemann and Green, 1990Go). PPAR{alpha} activation results in increased transcription of several target genes including acyl-CoA oxidase (AOX) and P4504A1 and 4A6, whose increased enzyme activities have been hypothesized to increase O2- • and H2O2 production (Desvergne and Wahli, 1999Go; Osumi et al., 1991Go; Sundseth and Waxman, 1992Go). In contrast, the peroxisomal H2O2 detoxification enzyme catalase is only modestly increased following PP treatment, and other cellular antioxidants (including glutathione peroxidase and vitamin E) are decreased (Lock et al., 1989Go). These intracellular changes in the production and detoxification of H2O2 may lead to increases in reactive oxygen species (ROS) in the liver.

Increased reactive oxygen species (ROS) have been implicated in both cell proliferation and apoptosis (Gamaley and Klyubin, 1999Go; Hasmall and Roberts, 1999Go). Moreover, increased ROS in the liver following PP treatment may result in alterations in transcription factors involved in apoptosis and cell proliferation, including c-fos and c-jun, subunits of the transcription factor AP-1, early growth response gene 1 (Egr-1), and heat shock transcription factors (HSF1, HSF2). c-fos, c-jun, and Egr-1 are early response genes whose transcription is increased with entry into the cell cycle, indicating that their gene products may function in proliferation and/or apoptosis (de Belle et al., 1999Go; Fausto, 2000Go; Gius et al., 1999Go; Huang et al., 1994Go; Karin, 1991Go; Nair et al., 1997Go). Redox regulation of AP-1 involves changes in the oxidation status of critical cysteine residues as well as an associating protein Ref-1. AP-1 is highly activated under hypoxic conditions and by antioxidants (Gius et al., 1999Go). In addition, AP-1 can be slightly activated by H2O2, but H2O2 decreases the activity of AP-1 in the presence of phorbol esters (Meyer et al., 1994Go). Redox regulation of Egr-1 mimics that of AP-1, requiring a reduced state for binding and the ability of the oxidized protein to be rescued in vitro by the protein Ref-1 (Huang and Adamson, 1993Go). Much like AP-1, Egr-1 binding activity can be slightly increased by H2O2 (Nose and Ohba, 1996Go). Increased transcription of the hsp70 gene occurs during G1 -> S phase transition (Morano and Thiele, 1999Go). The hsp70 gene contains a heat shock element (HSE) that binds HSF1 and HSF2, and the Hsp70 protein functions to chaperone steroid receptors, as well as other proteins, assisting in their proper folding and function (Morano and Thiele, 1999Go). Interestingly, oxidative stress has been shown to increase HSF binding to HSEs, but without concomitant increases in hsp70 transcription (Bruce et al., 1993Go; Jornot et al., 1997Go).

Species-specific differences present a challenging problem in determining the risk of continual human exposure to PPs. As previously mentioned, PPs increase peroxisomal ß-oxidation in hamsters that is similar to that seen in rats and mice (James and Roberts, 1996Go; Lake et al., 1993Go; Styles et al., 1988Go). In addition, PP treatment of hamsters results in slight but significant decreases in catalase activity (Durnford et al., 1998Go; Lake et al., 2000Go). However, hamsters do not develop hepatocellular carcinomas after PP administration, nor do they demonstrate increased cell proliferation. Moreover, NF-{kappa}B, which is activated by oxidative stress and PPs in rats and mice, was refractory to activation in hamsters (Nilakantan et al., 1998Go; Li et al., 2000Go; Tharappel et al., 2001Go). Increased NF-{kappa}B activity was positively correlated with cell proliferation (Durnford et al., 1998Go; Tharappel et al., 2001Go; Cunningham, unpublished results), suggesting that NF-{kappa}B activation may facilitate the association between PPs, ROS, and cell proliferation. We therefore hypothesized that the DNA binding activity of other transcription factors sensitive to oxidative stress (AP-1, Egr-1, HSFs) may be differently modulated in rats and hamsters following treatment with PPs. The objective of this study was to determine if the DNA binding activities of these redox-sensitive transcription factors are differentially regulated in rats and hamsters following exposure to three known PPs for 6, 34, and 90 days.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Gemfibrozil and all reagents for nuclear protein extraction were purchased from Sigma Chemical Company (St. Louis, MO.) and were molecular biology grade. Antibodies to c-fos (4–10G), c-jun/AP-1(D)-G, HSF2(C-20) and Egr-1(588) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A rat monoclonal HSF1 (Ab-4) antibody was obtained from NeoMarkers, Inc. (Union City, CA). Poly(dI-dC) • poly(dI-dC) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ) and suspended at a concentration of 5 µg/µl in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA (TE). Wy-14,643 ([4-chloro-6-(2,3-xylindino)-2-pyrimidinylthio]-acetate) was obtained from Chemsyn Science Laboratories (Lenexa, KS). Dibutyl-phthalate (DBP) was obtained from Research Triangle Institute (Research Triangle Park, NC).

Experimental design and animal treatments.
The National Toxicology Program at Research Triangle Park, NC performed all animal treatments. Male Sprague-Dawley rats (4–6 weeks old, Harlan Sprague-Dawley, Inc., Indianapolis, IN) and male Syrian hamsters (4–6 weeks old, Frederick Cancer Research and Development Center, Frederick, MD) were treated with PPs in an NTP-2000 unrefined diet (Ziegler Brothers Inc, Gardners, PA). Animals (n = 3) were administered a control diet or the following amounts of PPs: Wy-14,643 at 50 and 500 ppm, DBP at 5000 and 20,000 ppm in both rats and hamsters, and gemfibrozil at 1000 and 16,000 ppm in rats, 6000 and 24,000 ppm in hamsters. Animals were treated for either 6, 34, or 90 days, euthanized by CO2 exposure, and livers frozen in liquid nitrogen with subsequent storage at –80°C.

Nuclear extract preparation.
Nuclear extracts were prepared from frozen liver, using a modification of previously published methods to account for small sample sizes (Fei et al., 1995Go; Hattori et al., 1990Go; Stuempfle et al., 1996Go). For nuclear extracts, all stock solutions were prepared in advance and stored either at 4°C or –20°C. Samples were homogenized on ice using a motor-driven Teflon pestle homogenizer (Glas-Col) in ice-cold buffer A (10 mM HEPES–KOH, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, pH 8.0, 2 M sucrose, 10% glycerol, 1 mM DTT, 0.2% NP-40, 0.1 mM PMSF, 2 µg/ml each aprotinin, leupeptin, pepstatin A, and 2 mM benzamidine HCl). Following homogenization, 1 ml of Buffer A was overlaid with 3 ml of tissue solution in a 5-ml centrifuge tube (Beckman SW55Ti) that had been precooled on ice. This solution was centrifuged in a Beckman Coulter Optima L-90 K-Ultra Centrifuge using a swinging bucket rotor (SW 55Ti) at 80,000 x g (33.6 K rpm for 55Ti), 4°C, for 30 min. Following centrifugation, the supernatant was removed by aspiration and the nuclear pellet was transferred to a 1.5-ml Eppendorf tube using 1 ml Buffer B (10 mM HEPES–KOH, pH 7.9, 1 mM EDTA, pH 8.0, 0.6% NP-40, 0.5 mM PMSF, 150 mM NaCl). The extract was centrifuged at 3000 x g (6000 rpm) for 4 min in a refrigerated microfuge. After removing the supernatant, 0.5 volumes of Buffer C (20 mM HEPES–KOH, pH 7.9, 420 mM NaCl, 0.2 mM EDTA, pH 8.0, 1.2 mM MgCl2, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 2 mM benzamidine HCl) were added to the sample and samples were incubated on a rotating platform at 4°C for 60 min. Following incubation, samples were vortexed for approximately 10 s followed by centrifuging at max speed (16,000 x g, 14,000 rpm), 10 min at 4°C. Supernatants were aliquoted, snap frozen in a dry ice-ethanol bath, and stored at –80°C.

Protein assay of nuclear extracts.
Protein concentrations of nuclear extracts were determined using the bicinchoninic acid (BCA) method (Pierce Chemical Company, Rockford, IL), using IgG as standard.

Electrophoretic mobility shift assays (EMSA).
EMSAs were used to determine the DNA binding activity of AP-1, Egr-1, or HSF1 and HSF2.

Probe synthesis.
A double-stranded oligonucleotide containing the AP-1 consensus binding sequence (TRE) was obtained from Santa Cruz Biotechnology, Inc. The sequence of this oligonucleotide was 5` cgcttgaTGACTCAgccggaa 3` (the consensus TRE site in capitol letters). Radioactive labeling was carried out by combining 100 ng of the TRE with 2.5 µl [{gamma}-32P]ATP (6000 Ci/mmol, NEN, Boston, MA), 5 µl reaction buffer (5X) and 10 u T4 polynucleotide kinase (PNK, New England BioLabs) in a reaction totaling 25 µl. The reaction was incubated at 37°C for 10 min and unincorporated 32P was removed by filtration using a Sephadex G-25 column. The eluted reaction was brought to 200 µl (0.5 ng/µl) using TE and 1 µl was counted in a Packard Tricarb 2100TR Liquid Scintillation Analyzer to determine specific activity. Average cpm for 0.5 ng (1 µl) was ~50,000.

Egr-1 and HSE binding sites were synthesized as single-stranded oligonucleotides purchased from Operon Technologies, Inc. (Alameda, CA). For Egr-1, sequences described by Nair et al.(1997), which corresponds to the EBS1 Egr-1 binding site of the p53 promoter (–770 to –762), were used: sense 5`-agctGCGCCTACGCTC-3`, antisense 5`-agctGAGCGTAGGCGC-3` (binding sequence in capitols). For HSFs, an ideal HSE binding sequence described by Sarge et al.(1993), which contains an optimal HSF binding site, was used: sense 5`-agctcTAGAAGCTTCTAGAAGCTTCTag-3` and antisense, 5`-agctcTAGAAGCTTCTAGAAGCTTCTag-3` (capital letters designate NGAAN inverted repeat). Oligonucleotides were checked for integrity before use by {gamma}-32P end labeling using PNK (as described above for the TRE sequence) and examination on a 15% polyacrylamide, 7 M Urea, 1 x TBE gel electrophoresed using 1 x TBE running buffer. After electrophoresis, gels were exposed to Kodak XOMAT-AR film and confirmed to contain a single band. (+) Strands were end-labeled using [{gamma}-32P]ATP and annealed to unlabeled (–) strand in a reaction consisting of 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.5, and 0.001 M EDTA, in a volume of 200 µl. The reaction was incubated at 95°C for 5 min in a hot block. After 5 min the block was switched to a setting of 37°C, and was allowed to slowly cool overnight to anneal the strands. The annealed oligonucleotides were counted to determine radioactive incorporation, and average cpm for 1 µl were ~50,000.

Other oligonucleotides used in analysis included a TRE mutant oligonucleotide obtained from Operon Technologies, Inc. and annealed as described previously. This oligonucleotide contained the sequence: (+) 5`-agctcgcttgaTGACTTGgccggaa-3` and (–) 5`-agctttccggcCAAGTCAtcaagcg-3` (mutated nucleotides underlined). Additionally, for verification of AP-1 binding, a TRE site described in the collagenase gene promoter (cTRE) was used for comparison (Schonthal et al., 1988Go). The sequence of this oligonucleotide, also synthesized by Operon Technologies, Inc., is (+) 5` tcgagagtcaTGAGTCAgacacctctggcc 3` and (–) 5` tcgaggccagaggtgtcTGACTCAtgactc 3` (TRE in capitol letters). Labeling of this oligonucleotide was essentially as described for HSE and Egr1.

AP-1 EMSA.
For analysis of TRE binding proteins in nuclear extracts from PP-treated rats and hamsters, the following binding reaction was assembled: 4 µg protein, 1 µg poly(dI-dC) • poly(dI-dC), 10 mM HEPES–KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 15% glycerol, 0.1% NP-40, 0.5 ng TRE in a total volume of 20 µl. For consistency, all samples were brought to the same protein concentration using Buffer C. Samples were incubated for 20 min at room temperature. Included in each gel were a negative control reaction (no protein) and a positive control (HeLa cell extract, Santa Cruz). Extracts were loaded onto a 5% polyacrylamide, 0.5x TBE gel that was electrophoresed for 90 min at 180 volts using 0.5x TBE as running buffer. Binding specificity was determined by adding 4 µg (2 µl) of antibodies raised against c-jun, c-fos, or preimmune serum (IgG) to the reaction prior to probe addition and incubating the reaction for 15 min at room temperature. For competition experiments, 100x cold TRE or mTRE were added to the protein binding buffer reactions 10 min prior to probe addition. For analysis of cTRE binding, the exact same reaction was carried out as with TRE, with the exception of 0.5 ng cTRE as probe.

Egr-1 EMSA.
Egr1 binding was analyzed by using a modified reaction buffer based on the findings of previously published Egr-1 reactions (Hamilton et al., 1998Go; Liu et al., 1999Go; Minc et al., 1999Go; Peng et al., 1999Go). Briefly, 6 µg of nuclear extracts were incubated in a reaction containing 10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol, and 0.2 µg poly(dI-dC) • poly(dI-dC) in a total volume of 30 µl for 15 min at 4°C. One-half ng Egr-1 probe was added to the reaction following incubation and the reaction was incubated for an additional 20 min at 4°C. Binding proteins were separated on a 6% polyacrylamide, 0.5x TBE gel using 0.5x TBE running buffer run at 250 volts for 87 min. Binding specificity was examined by adding 2 µg anti-Egr-1 or IgG to the buffered reaction and incubating the reaction at 4°C for 60 min prior to probe addition (Peng et al., 1999Go). In addition, competition reactions were performed by adding 100x Egr-1 oligonucleotide to the buffered reaction at 15 min prior to probe addition. As with AP-1, a negative control (no protein) reaction was included with every EMSA run, and reactions were brought to the same concentration using Buffer C, while cocktails were used to add reaction components (except probe) to avoid inconsistencies.

HSF EMSA.
Analysis of HSF binding was conducted on nuclear extracts from PP-treated animals, essentially as previously described (Sarge et al., 1993Go). Briefly, 6 µg of nuclear extracts were incubated in a reaction containing 10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM DTT, 10 µg bovine serum albumin, 0.5 µg poly(dI-dC) • poly(dI-dC), and 0.5 ng HSE. Reactions were incubated for 20 min at room temperature and separated on a 4% polyacrylamide, 0.5 x TBE gel using 0.5 x TBE running buffer electrophoresed at 180 volts for 75 min. Binding specificity was examined using 2 µg anti-HSF1, anti-HSF2, or IgG incubated with protein for 20 min at 4°C prior to probe addition. Cold competition experiments were performed by adding 100 x HSE to the reaction at the time of probe addition. Furthermore, to confirm HSF binding, the comigration of a bacterially expressed HSF1 construct (generously provided by Dr. Kevin Sarge, University of Kentucky) was used as a positive control.

EMSA analysis.
All gels were dried under vacuum and exposed overnight at –80°C to Kodak XOMAT-AR film. Gels were then reexposed to a phosphorimaging screen and analyzed using the STORM Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant 5.0 software (Molecular Dynamics). Specific bands were counted as well as two separate areas of background per lane. The ratio of the specific band to background was considered the arbitrary numeric value for specific binding and was used for statistical analysis.

Statistical analysis.
Sample counts were analyzed using ANOVA followed by Bonferroni, for examination of differences in treatment means using the statistical software package Systat V.8 (SPSS, Inc.). Data are reported as mean ± SEM for n = 3 samples per group.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is unknown whether PP-induced proliferation activates immediate-early genes, as has been previously demonstrated with liver regeneration following chemical injury or partial hepatectomy (Brenner, 1998Go; Fausto, 2000Go; Peng et al., 1999Go; Scearce et al., 1996Go). Furthermore, whether the activities of these transcription factors are critical to species susceptibility to PPs is unknown. To investigate immediate early gene involvement in PP induced proliferation, we examined AP-1 and Egr-1 DNA binding as well as HSF binding in rats and hamsters administered three known PPs for 6, 34, and 90 days.

EMSAs of rat hepatic nuclear extracts using a consensus AP-1 binding site (TRE) revealed several bands (Fig. 1Go). The slower-migrating band contained AP-1, based on its ability to compete with cold consensus TRE but not with a mutated TRE, and by its ability to be displaced and shifted by an anti-c-jun antiserum but not by nonspecific antiserum (Fig. 1AGo). Furthermore, using a labeled probe of the collagenase gene AP-1 binding site (cTRE) (Schonthal et al., 1988Go), we observed a band which comigrated with the consensus AP-1 binding band, and in both cases the same band was displaced by incubation with the c-jun antiserum (Fig. 1CGo). A shifted band was not observed using antibodies to c-fos and may be explained as either a technical problem or the recent finding that jun family dimers make up shiftable AP-1 binding complexes in untreated rat hepatocytes (Rahmani et al., 1999Go). The same binding pattern and c-jun antibody-shiftable complex was observed in hamster nuclear extracts (data not shown). Using this same consensus TRE oligonucleotide, we examined AP-1 in nuclear extracts from rats or hamsters treated with DBP, gemfibrozil, or Wy-14,643. As shown in Table 1Go, both rats and hamsters contained similar levels of constitutive AP-1 binding activity. Furthermore, increased AP-1 binding activity was observed in hamsters at 6 days with all three compounds, although only the 50 ppm Wy-14,643 dose was significant, and a dose response was not observed in any case examined. Extracts from hamsters treated for 34 days with Wy-14,643 demonstrated a dose response increase in AP-1 binding, although only the 500 ppm dose was significant. Both DBP and Wy-14,643, however, had no effect on AP-1 binding in rats at any of the time points examined. Although no differences were observed in the earlier time points with gemfibrozil, a dose response increase in AP-1 binding activity was observed in 90-day gemfibrozil treated rats.



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FIG. 1. Determination of AP-1-specific binding. EMSA, using a consensus TRE oligonucleotide (A, B, C) or the collagenase TRE site (C) and nuclear extracts derived from rat liver. (–) Indicates probe and buffer while (+) indicates probe and buffer incubated with nuclear extracts. Competitions with 100-fold molar excess of self (100x TRE) or mutant oligonucleotides (100x mTRE) as well as incubation with antibodies specific for AP-1 (anti-c-fos, anti-c-jun) or nonspecific preimmune serum (IgG) were used to determine specificity of binding. Arrows indicate AP-1 complex while the anchored arrow indicates supershifted (ss) AP-1 with antibody to c-jun.

 

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TABLE 1 Effects of Peroxisome Proliferators on Ap-1 DNA Binding Activity in Male Sprague-Dawley Rats and Male Syrian Hamsters
 
To determine whether PPs affected Egr-1 DNA binding activity, we examined nuclear extracts for binding activity to an oligonucleotide containing the Egr-1 binding site found within the p53 promoter (Nair et al., 1997Go). Rat hepatic nuclear extracts contained proteins that formed complexes with this Egr-1 site containing oligonucleotide (Fig. 2Go). Complex II contained Egr-1, an observation based on cold competition experiments and its ability to be supershifted with an Egr-1 antiserum, while control IgG had no effect. The same shiftable complex was observed using nuclear extracts derived from hamsters (data not shown). Higher levels of constitutive Egr1 binding were observed in rats as compared to hamsters (2.7-fold averaged across all time points; Table 2Go). As with AP-1 binding, no consensus effect was seen between compounds in either of the two species. Interestingly, gemfibrozil treatment of rats significantly decreased Egr-1 binding activity at both 6 days (59 and 29% of control for low and high dose, respectively) and 90 days (35 and 62% of control for low and high dose, respectively); however, neither DBP nor Wy-14,643 had any significant effect on Egr-1 binding. No significant changes in Egr-1 binding were observed in hamsters with any of the three compounds at the time points examined.



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FIG. 2. Determination of Egr-1-specific binding. Gel mobility-shift assay using an oligonucleotide coding for the Egr-1 binding site in the p53 promoter and nuclear extracts derived from rat liver. (–) Indicates buffer and probe while (+) indicates buffer and probe incubated with nuclear extracts. Competitions with 100-fold molar excess of self (100x Egr-1) as well as incubation with antibodies to Egr-1 (anti-Egr-1) or nonspecific preimmune serum (IgG) were used to determine specificity of binding. Complex II was determined to contain Egr-1, and SS indicates supershifted protein in the presence of anti-Egr-1. Gel (B) was run longer than (A) to determine complex specificity.

 

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TABLE 2 Effects of Peroxisome Proliferators on Egr-1 DNA Binding Activity in Male Sprague-Dawley Rats and Male Syrian Hamsters
 
To determine if HSF1 and HSF2 are activated in the liver following PP exposure, we performed EMSAs with nuclear extracts from PP-treated rats and hamsters. A band was observed to bind to the optimized HSE oligonucleotide in rat nuclear extracts (Fig. 3Go). This band was specific since it could be competed by a molar excess of cold HSE oligonucleotide and its ability to be supershifted by either anti-HSF1 or -HSF2 antisera, but not by control antiserum. This same band and shiftable complex were observed in hamster nuclear extracts (data not shown). In addition, the shifted band containing HSFs in liver extracts was observed to comigrate with a bacteria-produced HSF1 bound to the HSE probe (data not shown). Furthermore, more HSF1 appears to be present in this complex, based on the more intense band observed with the antibody complex. In rats treated with DBP for 6 days, a dose-response increase in HSE binding was observed, although only significant for the highest dose, 20000 ppm (Table 3Go). Conversely, no changes were observed in hamsters with 6-day DBP treatment. Neither the 34- nor 90-day DBP treatments had an effect on HSF binding in either animal. No significant changes in HSF binding were observed following gemfibrozil treatment at any of the three time points examined, although a near significant (p = 0.054 in Bonferroni's test) decrease was observed in rats following 90 days of dosing at 1000 ppm. Wy-14,643 treatment of rats increased HSF binding in the latter time points (34 and 90 days) while decreased activity was observed in hamsters, with varied significance.



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FIG. 3. Determination of HSF-specific binding. Gel mobility-shift assay using an HSE oligonucleotide and nuclear extracts derived from rat liver. (–) Indicates buffer and probe alone, while (+) indicates buffer and probe incubated with nuclear extracts. Competitions with 100-fold molar excess of self (HSE) as well as incubation with antibodies to HSF1, HSF2, or nonspecific preimmune serum (IgG) were used to determine specificity of binding. HSF was determined to contain HSF1 and HSF2, while SS indicates supershifted protein in the presence of anti-HSF1 and anti-HSF2.

 

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TABLE 3 Effects of Peroxisome Proliferators on HSF DNA Binding Activity in Male Sprague-Dawley Rats and Male Syrian Hamsters
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPs are a diverse group of chemicals that increase cell proliferation while decreasing apoptosis, events that could contribute to the hepatocarcinogenic properties of these chemicals. Species susceptibility to PPs may be mediated though an alteration in cell-cycle kinetics, since induction of S-phase shows a strong correlation with tumorigenesis across species (James and Roberts, 1996Go). Increased levels of several early-response proteins (c-fos, c-jun, c-myc, Ha-ras) are correlated with the proliferation of the liver following partial hepatectomy or chemical insult (i.e., CCl4), suggesting the possibility of a role for these proteins in the mitogenic response of the liver (Brenner, 1998Go; Coni et al., 1990Go; Fausto, 2000Go). The objective of this study was to determine if the gene products of early response (the AP-1 family of transcription factors and Egr-1) or stress responsive (HSFs) genes are activated following PP administration. More importantly, both a PP responsive (rat) and less responsive (hamster) species were used in this study to compare the relevance of the activity of these transcription factors to increased cell proliferation. The potential role of these transcription factors, which can be modulated by ROS, makes them of great interest in growth and proliferation as well as in the transcription of phase II enzymes—important in the defense against ROS.

One mechanism that has been proposed for the carcinogenic and promoting activity of peroxisome proliferators is that they increase oxidative stress. Peroxisome proliferators greatly increase the number and volume of peroxisomes in the cell as well as several enzymes of lipid metabolism (Reddy and Lalwani, 1983Go). Specifically, the enzymes of the peroxisomal ß-oxidation pathway, which produces hydrogen peroxide as a by-product, are highly induced. On the other hand, catalase, the peroxisomal enzyme that detoxifies hydrogen peroxide, is only slightly induced. It has therefore been proposed that toxicity and carcinogenesis by peroxisome proliferators is due, at least in part, to overproduction of hydrogen peroxide or other active oxygen species (Reddy and Lalwani, 1983Go). Peroxisome proliferators have also been found to decrease the levels of several cellular antioxidants and antioxidant enzymes, including vitamins C and E, DT-diaphorase, and glutathione peroxidase (Glauert et al., 1992Go; O'Brien et al., 2001aGo,bGo). Possible adverse effects include lipid peroxidation, oxidative DNA damage, or changes in gene expression. Despite much work on this oxidative damage hypothesis, definitive answers have not been found. Several studies have found that the administration of peroxisome proliferators leads to lipid peroxidation and oxidative DNA damage, but others have not (Antonenkov et al., 1988Go; Cattley and Glover, 1993Go; Conway et al., 1989Go; Elliott and Elcombe, 1987Go; Glauert et al., 1992Go; Goel et al., 1986Go; Hegi et al., 1990Go; Huang et al., 1994Go; Huber et al., 1991Go; Kasai et al., 1989Go; Lake et al., 1987Go; Reddy et al., 1982Go; Srinivasan and Glauert, 1990Go; Takagi et al., 1990aGo,bGo). These studies, in summary, do not provide convincing evidence that lipid peroxidation or oxidative DNA damage from hydrogen peroxide overproduction are critical factors in peroxisome proliferator-induced carcinogenesis.

Another possible mechanism by which oxidative stress from peroxisome proliferators could influence carcinogenesis is the activation of oxidative stress-sensitive transcription factors. In the present study, we did not observe changes in the activation of three redox sensitive transcription factors: AP-1, Egr-1, and HSF1/2. We have observed, however, that the oxidative stress-sensitive transcription factor NF-{kappa}B can be activated by peroxisome proliferators, and that the activation is related to oxidative stress induced by peroxisome proliferators. The peroxisome proliferators Wy-14,643, ciprofibrate, gemfibrozil, and dibutylphthalate increase the DNA-binding activity of NF-{kappa}B in rats, mice, and hepatoma cell lines (Li et al., 2000a, 1996Go; Nilakantan et al., 1998Go; Tharappel et al., 2001Go). In addition, ciprofibrate activates stably transfected NF-{kappa}B-regulated reporter genes in H4IIEC3 rat hepatoma cells (Li et al., 2000a). Several lines of evidence support the hypothesis that this activation is mediated by oxidative stress produced by peroxisome proliferators. First, the antioxidant, vitamin E, inhibits ciprofibrate-induced NF-{kappa}B activation, both in vivo and in vitro (Calfee et al., 1998Go; Li et al., 2000a). Second, overexpression of the hydrogen peroxide-producing enzyme that is induced by peroxisome proliferators, fatty acyl CoA oxidase, is sufficient to activate NF-{kappa}B in Cos1 cells (Li et al., 2000b). Third, overexpression of the hydrogen peroxide-detoxifying enzyme catalase in the livers of transgenic mice inhibits the activation of NF-{kappa}B by ciprofibrate (Nilakantan et al., 1998Go). In addition, the ciprofibrate-induced increase in hepatocyte proliferation is decreased by catalase overexpression, indicating a possible role for NF-{kappa}B in cell proliferation by peroxisome proliferators. Finally, NF-{kappa}B is not activated by peroxisome proliferators in hamsters, which have much higher levels of the antioxidant enzymes glutathione peroxidase, glutathione S-transferase, glutathione reductase, and DT-diaphorase (O’Brien et al., 2001aGo,bGo; Tharappel et al., 2001Go). Overall, the results of the present study and previous studies imply that the administration of peroxisome proliferators does not induce a generalized activation of all transcription factors that are sensitive to oxidative stress. It is possible that other signal transduction pathways are inhibiting the activation of the transcription factors examined in the present study.

Oxidative stress has been implicated in the induction of cell proliferation by peroxisome proliferators. Cell proliferation is induced shortly after the administration of peroxisome proliferators, with a peak of DNA synthesis occurring 2–3 days after beginning administration (Reddy and Lalwani, 1983Go). Cell proliferation then decreases, but with some peroxisome proliferators, such as Wy-14,643, a low level of cell proliferation continues throughout administration (Chen et al., 1994Go; Eacho et al., 1991Go; Marsman et al., 1988Go; Yeldandi et al., 1989Go). The initial increase in cell proliferation is associated with the transient activation of NF-{kappa}B, primarily in Kupffer cells, with the subsequent release of cytokines that stimulate cell proliferation in hepatocytes (Rose et al., 1999Go; Rusyn et al., 1998Go). The production of active oxygen by peroxisomal enzymes is almost certainly not involved, because peroxisome proliferation has not taken place at this early time after the beginning of its administration. The long-term increase in cell proliferation, however, correlates well with the induction of peroxisomal ß-oxidation enzymes and the activation of NF-{kappa}B, which has increased DNA-binding activity through at least 90 days of administration (Durnford et al., 1998Go; Tharappel et al., 2001Go). The transcription factors AP-1, Egr-1, and HSF1/2 were not activated after 6, 34, or 90 days of peroxisome proliferator administration, and therefore are not likely to be involved in the induction of cell proliferation seen at these times. It cannot be determined from the results of the present study if these transcription factors could play a role in the increase in cell proliferation seen early after the administration of peroxisome proliferators.

Increased activity of AP-1 or Egr-1 with H2O2, which is presumed to be the causative by-product involved in signaling following PP treatment, is quite controversial. Meyer et al.(1993, 1994) have shown that unlike NF-{kappa}B, which is activated by H2O2, AP-1 is strongly activated by antioxidants. A proposed explanation for the observed slight increases in AP-1 activity observed following oxidative stress may be due to an overcompensation by antioxidant defense systems (Meyer et al., 1993Go, 1994Go). In addition, although AP-1 binding to a TRE was examined in this study, cAMP response elements (CRE) and EpRE binding, both of which involve jun, were not studied. Egr-1 is slightly activated by H2O2, but only at a limited concentration and not to the extent observed with the activator phorbol esters (Nose and Ohba, 1996Go). Contrasting with these findings are those of Huang et al., who demonstrated that Egr-1 requires reducing equivalents for maximal binding and that oxidized Egr-1 does not bind to DNA (Huang and Adamson 1993Go).

The lack of increased hepatic AP-1 DNA binding activity in response to peroxisome proliferators observed in this study agrees with the findings of Menegazzi et al.(1997), although it contradicts the findings of Nilakantan et al.(1998). Interestingly, both studies revealing a lack of AP-1 activity were conducted in male Wistar rats treated with nafenopin (Menegazzi et al., 1997Go) or SD rats treated with DBP, Wy-14,643, or gemfibrozil (this study), whereas increased AP-1 binding was observed in female mice treated with ciprofibrate. This contradiction may be due to species differences, as has been observed with cyclins and PPs in rats and mice (Peters et al., 1998Go) or sex differences. Increased NF-{kappa}B and AP-1 activities observed in the mouse study were correlative with cell proliferation, while overexpression of catalase in these animals resulted in reduced binding for NF-{kappa}B, but AP-1 was not as affected (Nilakantan et al., 1998Go).

Increased production of H2O2 via increased activity of AOX or P450 could serve to activate HSF1, which is activated by stress, resulting in oligomerization and HSE binding (Bruce et al., 1993Go; Morano and Thiele, 1999Go). For example, neuroblastoma cells incubated with H2O2 demonstrate a rapid increase in HSF1 binding that is blocked by supplementation with glutathione (Bijur et al., 1999Go). As with AP-1 and Egr-1, alterations in HSF binding in this study neither correlated with species susceptibility nor the potency of the PP examined. This lack of HSF activation agrees with a lack of HSF activation in the livers of CoCl2-treated rats, which is known to induce oxidative stress (Tacchini et al., 1995Go).

In summary, the administration of the peroxisome proliferators Wy-14,643, gemfibrozil, or dibutyl phthalate to rats or hamsters did not increase the DNA binding activity of the transcription factors AP-1, Egr-1, or HSFs. The lack of increased binding of AP-1, Egr-1, and HSFs following PP exposure indicates that mechanisms that do not rely on these factors may be involved in long-term increases in cell proliferation following PP treatment.


    ACKNOWLEDGMENTS
 
We thank Mr. Nicolas Larmonier for contributions to the AP-1 EMSAs. This research was funded by NIH grant ES09771 and by the Kentucky Agricultural Experiment Station. M.L.O. was supported by NIEHS Training Grant ES07266 and a Dissertation Year Fellowship awarded by the Graduate School, University of Kentucky.


    NOTES
 
1 To whom correspondence should be addressed at Graduate Center for Nutritional Sciences, 204 Funkhouser Building, University of Kentucky, Lexington, KY 40506. Fax: (859) 323-0061. E-mail: hglauert{at}uky.edu. Back


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