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Biology of Reproduction 67, 718-724 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Activation of Mitogen-Activated Protein Kinases and AP-1 Transcription Factor in Ovotoxicity Induced by 4-Vinylcyclohexene Diepoxide in Rats1

Xiaoming Hua, Jodi A. Flawsd, I. Glenn Sipesb,c, and Patricia B. Hoyer2,a,b,c

a Departments of Physiology b Pharmacology and Toxicology, c Southwest Environmental Health Sciences Center, The University of Arizona, Tucson, Arizona 85724 d Department of Epidemiology/Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day—a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days—a time when the earliest signs of impending follicular destruction is seen, 15 days—a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25–100 µm) and large (100–250 µm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 ± 0.14 for 10 days, 1.48 ± 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 ± 0.11 for 10 days, 1.37 ± 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0.29 ± 0.04; 15 days, 0.51 ± 0.04, VCD/control, P < 0.01). VCD did not affect any of these measurements in large preantral follicles or liver. Phosphorylation status of c-Jun protein as measured by Western blotting was increased (1.22 ± 0.1, VCD/control, P < 0.05) after the 15-day daily dosing with VCD, but total c-Jun protein levels were unaffected. Using antibodies against c-Jun or phospho-c-Jun for supershift DNA binding assay, c-Jun and phospho-c-Jun were identified in the AP-1-DNA binding complex, and the binding activity was reduced in tissues with increased phospho-c-Jun protein levels. Taken together, these data provide evidence that accelerated atretic signals induced by VCD is associated with MAPK/AP-1 signaling pathways and phosphorylation of c-Jun plays a significant role in transmitting the apoptotic signals.

apoptosis, follicle, ovary, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the human ovary, only about 400 of the 2 million oocytes contained in primordial follicles at birth are ovulated during the reproductive lifetime of a woman [1]. Thus, throughout her life, greater than 99% of ovarian follicles in a woman undergo degeneration by a process known as atresia. Because the ovary contains a finite number of primordial follicles at birth, human exposure to environmental chemicals that destroy these follicles can result in early menopause [2, 3].

One occupational chemical that has been shown to destroy oocytes contained in primordial and primary follicles of rats and mice is 4-vinyl-1-cyclohexene diepoxide (VCD) [4]. The parent compound, 4-vinyl-1-cyclohexene, and its epoxide metabolites represent a potential health hazard in women because they are released into the environment as by-products in the chemical synthesis of rubber tires, insecticides, flame retardants, and plasticizers [5].

Previous studies have demonstrated that repeated daily dosing of rats with VCD (80 mg/kg i.p.) is required to cause ovotoxicity. The earliest evidence of impending VCD-induced follicle damage is seen following 10 daily doses, and after a 15-day dosing, there is a loss of about 50% of small preantral (primordial and primary) ovarian follicles [6–8]. Following 30 days of daily dosing with VCD, the majority of small preantral follicles in immature as well as in adult rats are destroyed [4]. Our recent reports have indicated that ovotoxicity induced by repeated daily dosing with VCD in rats is associated with activation of intracellular apoptotic events, including the cellular caspase cascade, and redistribution of Bcl-2-related proteins [9, 10]. Additionally, this has been found to involve estrogen receptor-mediated mechanisms [11]. These observations have led to the conclusion that VCD dosing causes follicle loss by accelerating the overall process of ovarian atresia, known to occur via programmed cell death, apoptosis [7, 12]. However, the molecular mechanisms responsible for VCD initiating the apoptotic cascade in ovarian follicles are incompletely understood.

Three major mammalian mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase, are regulated by distinct signal transduction pathways that control many aspects of mammalian cellular physiology, including cell growth, differentiation, and apoptosis [13–15]. In general, the ERK cascade is activated by growth factors and is critical for cell proliferation [16]. Conversely, the JNK and p38 pathways are stimulated by genotoxic agents and cytokines mediating the stress response, growth arrest, and apoptosis [17–20]. However, controversial evidence has indicated that there are more complex roles of these MAPK pathways regulating distinct cellular effects in different cell lineages.

Activation of MAPKs is mediated by upstream phosphorylations via MAPK kinases (MAPKKs) [13–15]. Once activated, MAPKs regulate gene expression via phosphorylation of downstream transcription factors. Activator protein-1 (AP-1) is one of the downstream transcription factors that interact with regulatory DNA sequences known as TPA response elements or AP-1 sites [21]. AP-1 is a collective term referring to a response element regulated by dimeric transcription factors composed of Jun, Fos, or activating transcription factor subunits [22]. Homodimers of c-Jun or heterodimers of c-Jun and c-Fos modulate biological responses by binding to the DNA consensus sequence TGA(C/G)TCA (12-o-tetradecanoylphorbol 13-acetate-responsive element, TRE) found in the promoter region of a number of genes [23]. There is ample evidence that the activation of AP-1, especially that mediated by c-Jun protein, is essential for cell proliferation and differentiation. Recently, it has been suggested that certain components that affect AP-1 activity may also be involved in initiating programmed cell death [22, 23].

Thus, the purpose of this study was to investigate the relationship between VCD-induced ovotoxicity and alterations in MAPKs and AP-1 DNA binding activity in rats. In addition, the possible involvement of c-Jun and phospho-c-Jun protein was also studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Polyclonal primary antibodies for c-Jun and phospho-c-Jun were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), respectively. Anti-rabbit secondary antibody conjugated with horseradish peroxidase was purchased from New England BioLab (Beverly, MA). AP-1 consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3') was purchased from Promega Corporation (Madison, WI). BSA (RNase/DNase free) and poly(dI-dC)·(poly(dI-dC) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). MAPK immunoprecipitation kinase assay kits, p38 assay kits, and JNK kinase assay kits were purchased from Upstate Biotechnology. [{gamma}-32]ATP (~3000 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). Bio-Rad DC protein assay kits were purchased from Bio-Rad Laboratories (Hercules, CA). VCD (purity > 99%) and all other chemicals were reagent grade and purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and Tissue Collection

Immature (21 days old) female Fisher 344 rats were obtained from Harlan Laboratories (Indianapolis, IN), housed in plastic cages, given food and water ad libitum, and maintained on a 12L:12D cycle. Animals were allowed to acclimate for 1 wk before experiments began. All experiments were approved by the University's Institutional Animal Care and Use Committee. Rats (28 days old) were given daily i.p. injections of either sesame oil (2.5 ml/kg, vehicle control) or VCD dissolved in sesame oil (80 mg/kg, 0.57 mmol/kg) for 1, 10, or 15 days, as described previously [6, 7, 24]. Four hours following the final dose, rats were killed by inhalation of CO2. Tissues (liver and ovaries) were excised, and ovarian preantral follicles were isolated.

Follicle Isolation

Small preantral follicles (fraction 1, 25- to 100-µm diameter) and large preantral follicles (fraction 2, 100- to 250-µm diameter) were prepared by gentle enzymatic dissociation of ovaries and sorting with micropipettes as previously described [25]. Pools of follicles were collected from both ovaries of six rats in each treatment (control or VCD) for each observation (N). Whereas fraction 1 follicles are highly enriched in primordial and primary follicles (targeted by VCD), early growing follicles (not targeted by VCD) are also contained. Following isolation, follicles were washed twice with medium 199 and used for preparation of subcellular fractions. The purity and viability of isolated follicles as evaluated microscopically by trypan blue dye exclusion is >99% [25]. Thus, follicles used for measurements are healthy or undergoing apoptosis to some degree but are not yet compromised in membrane integrity.

Preparation of Nuclear and Cytoplasmic Extracts

The nuclear and cytoplasmic extracts from liver or ovarian follicles were prepared as previously described, with some modifications [26]. Briefly, fresh liver pieces or follicles were resuspended and homogenized in ice-cold lysis buffer: 10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM sodium orthovanadate (Na3VO4), 0.5 mM PMSF, 2 µg/ml aproptinin, and 2 µg/ml leupeptin. After incubating the homogenate for 20 min on ice, the samples were centrifuged (at 800 x g) to collect the supernatant as the cytosolic extracts, and the final pellet was incubated in extraction buffer: 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 mM N3VO4, 0.5 mM PMSF, 2 µg/ml aproptinin, and 2 µg/ml leupeptin. After incubating on ice for 30 min with intermittent mixing, the extract was centrifuged for 10 min at 14 000 x g and the supernatant was used as nuclear extracts. The protein concentration of these extracts was determined by using the Bio-Rad protein assay kits as per the manufacturer's directions. All extracts were stored at -70°C and used within 1 mo.

Western Blot Analysis

Twenty micrograms of nuclear extract protein were resolved over a 15% polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was preincubated in blocking buffer (5% nonfat dry milk, 1% Tween 20, in 20 mM TBS, pH 8.0) for 1 h at room temperature, then incubated with appropriate primary antibodies in blocking buffer from 1 h at room temperature to overnight at 4°C, followed by incubation with anti-rabbit secondary antibodies conjugated with horseradish peroxidase and detected by chemiluminescence and autoradiography using x-ray film. Antibodies against c-Jun and phospho-c-Jun demonstrated several bands, with the most intense staining at the predicted molecular weight (both at ~39 kDa). In each sample, quantification was made only on the band displaying the proper molecular weight. Background for each sample was assessed by subtracting the optical density (OD) in a blank region (nonsample loading lane) on the gel from the OD in the appropriate protein band. Densitometric measurements of the bands in Western blot analysis were performed using the Eagle Eye II system with Eaglesight software Version 3.2 (Stratagene, La Jolla, CA).

Electrophoretic Mobility Shift Assay and Supershift Assay

Electrophoretic mobility shift assay (EMSA) was performed using the gel-shift assay system from Promega Corporation as described by the manufacturer for AP-1. Briefly, AP-1 consensus oligonucleotide was end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase to a specific activity of ~2 x 105 cpm min-1 µl-1. To determine the DNA-binding activity of AP-1, the labeled probe (1 µl) and nuclear extract (10 µg protein) were mixed with 30 µg of BSA and 5 µg of poly(dI-dC)·(poly(dI-dC) in a binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol; final volume 20 µl). After a 20-min incubation (room temperature), protein-DNA complexes were resolved by electrophoresis over a 4% native polyacrylamide gel with 0.5% of Tris-borate/EDTA electrophoresis buffer. For unlabeled probe competition, 50-fold excess of unlabeled oligonucleotide was added. The gel was dried and the radioactivity in the bands was quantified using an InstantImager Electronic Autoradiography System from Packard Instrument Company (Meriden, CT). The supershift analysis was performed essentially the same except that antibodies (1 µg/reaction) were incubated with nuclear extracts for 2 h before the probe was added.

MAPK (ERK) Activities

MAPK (ERK) activity in tissue homogenates was determined using MAPK assay kits from Upstate Biotechnology (catalog #17-184) according to the manufacturer's protocol. In brief, ERK in 200 µg of cytoplasmic extract protein was immunoprecipitated by incubating the homogenate with 4 µg of polyclonal anti-MAPKs antibody for 1 h. This was followed by an additional incubation with 50 µl of protein A-Sepharose for 1 h. The protein A-Sepharose beads were washed twice with buffer A (50 mM Tris, pH 7.5; 1 mM EDTA; 1 mM EGTA; 0.5 mM Na3VO4; 0.1% 2-mercaptoethanol; 1% Triton X-100; 50 mM sodium fluoride; 5 mM sodium pyrophosphate; 10 mM sodium ß-glycerophosphate; 0.1 mM PMSF, 1 µg/ml of aprotinin, pepstatin, leupeptin; and 1 µM microcystin) followed by two washes with kinase buffer (20 mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol). The beads were suspended in 40 µl kinase buffer containing 10 µCi [{gamma}-32P]ATP, and the kinase reaction was carried out for 20 min at 30°C using 20 µg of myelin basic protein as substrate. The phosphorylated substrate was then separated from the residual [{gamma}-32P]ATP by using P81 phosphocellulose paper accompanied by extensive washing. Quantification was made using a scintillation counter.

p38 Kinase Activities

The p38 MAPK activity in tissue homogenates was determined using p38 assay kits from Upstate Biotechnology (catalog #17-169) according to the manufacturer's protocol. Briefly, 200 ng inactivated MAPKAP kinase-2 was first activated by incubation with 40 µg cytoplasmic extract protein or 0.06 U purified active p38 for 15 min at 30°C. Then the activated MAPKAP kinase-2 was incubated with 8.6 nM synthetic peptide substrate KKLNRTLSVA in 60 µl kinase buffer containing 10 µCi [{gamma}-32P]ATP for 10 min at 30°C. The phosphorylated substrate was then separated from the residual [{gamma}-32P]ATP using P81 phosphocellulose paper accompanied by extensive washing, followed by quantitation using a scintillation counter.

JNK Kinase Activities

The JNK kinase activity in tissue homogenates was determined using JNK kinase assay kits from Upstate Biotechnology (catalog #17-166) according to the manufacturer's protocol. Briefly, 40 µg cytoplasmic extract protein or 1 µg pure recombinant JNK2 were resuspended in 20 µl of kinase buffer containing 10 µCi [{gamma}-32P]ATP, and the kinase reaction was carried out for 30 min at 30°C using 5 µg of purified c-Jun (1-169)-GST as substrate. The reaction was stopped with an equal volume of Laemmli sample buffer, and the phosphorylated c-Jun-GST was analyzed by 12% SDS-PAGE and quantified using an InstantImager Electronic Autoradiography System from Packard Instrument Company.

Statistical Analysis

All experiments involving follicle isolations were repeated with separate groups of rats (6 rats/group) for independent observations. Quantitative analysis for all experiments were done using the following method. The data were analyzed statistically using individual values (control or VCD), ODs from the Eagle Eye system, and radioactivity from scintillation counting or the Instant-Imager system. Mean differences were analyzed using analysis of variance (ANOVA) and, where appropriate, Fisher probable least-squares difference post hoc test. Statistical significance was assigned at P < 0.05. The individual values were then expressed as a VCD/control ratio to emphasize the relative extent as the effect of VCD above the normal rate of atresia (apoptosis).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of MAPK Members by VCD Dosing in Rats

MAP kinases include ERK, JNK, and p38 kinase. To evaluate the contribution of MAPK members in the VCD-induced ovotoxicity of rats, activities of ERK, JNK, and p38 kinase in cytosolic extracts of rat ovarian follicles were analyzed (Fig. 1). There was a decrease (P < 0.05) in the activity of JNK in small ovarian follicles collected after a single dose of VCD, whereas ERK and p38 kinase activities were unaffected. Following daily dosing with VCD for 10 or 15 days, activities of JNK and p38 kinase in small ovarian follicles were increased (P < 0.01),= whereas there was no effect on ERK. There were no effects of VCD in large preantral follicles and liver at any time (data not shown).



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  		FIG. 1. Effects of VCD on MAPKs activities. Cytoplasmic extracts were prepared from isolated ovarian small follicles collected from F344 female rats following 1, 10, or 15 days of daily dosing with VCD (80 mg/kg i.p.) or vehicle control. Cytoplasmic extracts were used to analyze the activity of ERK, JNK, and p38 kinases as described in Materials and Methods. Means are expressed as mean of VCD treatment versus vehicle control ± SEM; n = 3; *P < 0.05, **P < 0.01 compared with control

Effects of VCD on Follicular Content of c-Jun and Phospho-c-Jun Protein

As one of the substrates of JNK and a prominent member of the AP-1 transcription family, c-Jun has been implicated in the regulation of a wide range of biological processes including development, differentiation, transformation, and apoptosis. The transcriptional activities of c-Jun can be regulated by changes in the level of c-Jun expression as well as posttranslational modifications of the c-Jun protein [27]. In particular, the transactivation potential of c-Jun critically depends on its phosphorylation of amino acid residues Ser-63 and Ser-73 mediated by JNK [28]. Although the growth regulatory functions of c-Jun have been firmly established, its role in regulating responses to stress is more controversial [29]. To understand the role of c-Jun protein in ovarian follicular atresia induced by VCD dosing, total c-Jun and phospho-c-Jun protein levels were detected by Western blot analysis using anti-c-Jun and anti-phospho-c-Jun primary antibodies. Western blot data indicated that a single dose of VCD has no effect on the protein levels of total c-Jun and phospho-c-Jun in target or nontarget tissues (large preantral follicles and liver tissues). Following daily dosing with VCD for 15 days, levels of phospho-c-Jun protein in small preantral follicles were increased (P < 0.05), but total c-Jun protein levels were not (Fig. 2).



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  		FIG. 2. Effect of VCD on c-Jun and phospho-c-Jun protein levels. Nuclear extracts were prepared from isolated small or large preantral ovarian follicles or liver tissue collected from F344 female rats following 15 days of daily dosing with VCD (80 mg/kg i.p.) or vehicle control. Western analysis was performed as described in Materials and Methods. A) A representative gel from three independent experiments. B) Data for results using antibodies against total c-Jun (open bar) or phospho-c-Jun (shaded bar) are expressed as mean of VCD treatment versus vehicle control ± SEM; n = 3; *P < 0.05

Effects of VCD Dosing on the AP-1 DNA Binding Activity in Nuclear Extracts of Rats

Transcription factor AP-1 converts extracellular signals into changes in the expression of specific target genes whose regulatory regions contain AP-1 binding sites. AP-1 activity can be modulated by interactions with other transcriptional regulators and is further controlled by upstream kinases that link AP-1 to various signal transduction pathways [20]. To understand the association between VCD-induced acceleration of atresia in ovarian follicles and VCD-induced alterations in AP-1 binding activity, the effects of VCD dosing on nuclear protein DNA-binding of AP-1 in nuclear extracts of ovarian follicles were examined using EMSA (Fig. 3). A single dose of VCD caused an increase (P < 0.05) in AP-1 binding activity in isolated small preantral follicles. Following repeated daily dosing with VCD for 10 days, AP-1 binding activity was decreased (0.29 + 0.04, VCD/control, P < 0.01). Repeated daily dosing with VCD for 15 days also caused a decrease in AP-1 binding activity in small preantral follicles (0.51 ± 0.04, VCD/control, P < 0.01), but to a less extent than with 10-day dosing (P < 0.05). Unlike small ovarian follicles, no changes in DNA binding activity were seen in nuclear extracts from large follicles or liver at any time. Binding was specific for AP-1 sequences because the radiolabeled complex could be eliminated with a 50-fold excess of unlabeled AP-1 probe (Fig. 3, A and B, lane 2).



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  		FIG. 3. Effects of VCD on AP-1 binding activity. Nuclear extracts were prepared from isolated small or large preantral ovarian follicles or liver tissue collected from F344 rats following 1 day (A) or 10 or 15 days (B) of daily dosing with VCD (80 mg/kg i.p.) or vehicle control. Nuclear extracts were then analyzed by EMSA with 32P-labeled AP-1 probes as described in Materials and Methods. A, B) Representative gels from three independent experiments. Lane 1: no nuclear extracts; lane 2: 50-fold molar excess of unlabeled AP-1 probe; lane 3: liver control; lane 4: liver VCD; lane 5: F1 follicle control; lane 6: F1 follicle VCD; lane 7: F2 follicle control; lane 8: F2 follicle VCD. C) Data are expressed as mean of VCD treatment versus vehicle control ± SEM; *P < 0.05, **P < 0.01 compared with control; n = 3

Involvement of c-Jun and Phospho-c-Jun in VCD-Induced Changes of AP-1 DNA Binding Activity

A growing body of evidence has demonstrated a diversity and complexity of biological responses catalyzed by AP-1 that appear to be related to its interaction with many transcription regulating proteins or to its cross-talk to other transcription factors [30]. To investigate the association between c-Jun or c-Jun phosphorylation and AP-1 activation in VCD-induced ovotoxicity in rats, protein components of the AP-1 complex were detected using specific primary antibodies directed against total c-Jun or phospho-c-Jun proteins (Fig. 4). Preincubation of nuclear extract with c-Jun antibody reduced AP-1 DNA binding activity as detected by EMSA, whereas preincubation of nuclear extract with phospho-c-Jun antibody enhanced AP-1 DNA binding activity. These findings suggest that both c-Jun and phospho-c-Jun proteins are contained in the nuclear AP-1 DNA binding complex in ovarian follicles. Preincubations with unrelated rabbit antiserum did not supershift or disrupt the complex.



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  		FIG. 4. The c-Jun and phospho-c-Jun involvement in AP-1 binding activity. Nuclear extracts were prepared from isolated small preantral ovarian follicles collected from F344 female rats following 1 or 15 days of daily dosing with VCD (80 mg/kg i.p.) or vehicle control. Nuclear extracts were prepared and analyzed by EMSA with 32P-labeled AP-1 probes alone or probes and nuclear extracts that had been preincubated with antibodies against c-Jun or phospho-c-Jun as described in Materials and Methods. A) A gel representative of three independent experiments. Lane 1: with 50-fold molar excess of unlabeled AP-1 probe; lane 2: F1 follicle control; lane 3: F1 follicle control with anti-c-Jun antibody; lane 4: F1 follicle control with anti-phospho-c-Jun antibody; lane 5: F1 follicle VCD; lane 6: F1 follicle VCD with anti-c-Jun antibody; lane 7: F1 follicle VCD with anti-phospho-c-Jun antibody. B) Data are expressed as relative changes of AP-1 DNA binding activity (after preincubation with specific antibodies) compared with AP-1 DNA binding activity (without incubation with specific antibodies). Mean ± SEM; **P < 0.05 compared with control; n = 3


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A variety of intracellular or extracellular signals can activate MAPK cascades and AP-1 transcriptional regulator, and this activation can lead to diverse biological responses, from cell proliferation to apoptosis [20, 29]. Although it is now well established that MAPK/AP-1 pathways play an important role in the induction of apoptosis following exposure to various toxic chemicals [31], little is known about their association with the type of cell death induced by reproductive toxicants, especially in vivo. The present study is the first to determine the association between ovotoxicity induced by VCD and the activation of MAPKs/AP-1 pathways in rats.

Sustained Activations of JNK and p38 Kinase Accompany VCD-Induced Ovotoxicity

MAPKs are important upstream regulators of transcription, and their signaling is critical to the transduction of a wide variety of extracellular stimuli into intracellular events. Differential activation of specific MAPK pathways is believed to play an important role in either the inhibition or the induction of apoptosis. Most often, activation of JNK and p38 kinase pathways is associated with entry into apoptosis, whereas activation of ERK is associated with proliferation [32, 33]. However, in some circumstances, opposite roles for these kinases have been described. In the absence of costimulatory signals, cross-linking of surface IgM in B cell-linkage cell results in the activation of ERK followed by the induction of apoptosis. Furthermore, inhibition of IgM-mediated apoptosis by CD40 ligand involves activation of JNK [34, 35]. These apparent contradictions in the roles that MAPKs play in modulating apoptotic responses underscore the need to more fully understand their activities and the consequences of their activation in specific cell types and under different conditions of stress.

The present studies demonstrated that a single dose of VCD inhibited the activity of JNK in isolated small preantral follicles, with no effects on ERK and p38 kinase activities. Conversely, following 10 or 15 days of repeated daily dosing with VCD, there was an activation of JNK and p38 kinase but not ERK. The data in Figure 1 also indicated that, in follicles collected after 10 days of daily dosing with VCD, there was greater activation of JNK and p38 kinase than from those after 15 days of dosing. Our previous studies have demonstrated that evidence of impending VCD-dependent ovotoxicity is first seen on daily dosing Day 10 in primordial follicles (a time when there is an increase in percentage of unhealthy follicles, although no significant follicle loss has yet occurred). However, relative to control, about 50% loss of primordial follicles [24] has occurred by Day 15. Therefore, the difference between 10 and 15 days likely reflects that, for 10 days, there are more follicles entering an apoptotic pathway, whereas for 15 days, a significant amount of those follicles to be targeted have already been destroyed. Unlike repeated dosing, a single dose of VCD caused an inhibition of JNK activity. This supports a previous study, which determined by morphologic evaluation that a single dose of VCD provides protection against the normal rate of atresia [12].

VCD-Induced Downstream Apoptotic Signals Are Transmitted Through Decreased Activation of Transcription Factor AP-1

AP-1 is a widely distributed consensus sequence for regulation by transcription factors that mediate many aspects of cell physiology in response to environmental changes such as stress, radiation, or growth factor signals, thereby acting as an environmental biosensor [30]. Although much is known about heterodimerization between c-Fos and c-Jun, there is still a significant lack of knowledge about how these factors interact with DNA in the nucleus to activate or repress expression of genes they regulate [30].

Recent data have implicated AP-1 transcription factors in the control of cell death and survival. However, among growth regulators, transcription factors that regulate AP-1 are somewhat unusual because, in addition to being responsive to growth factors, they are also activated by genotoxic stresses, such as UV or alkylating agents, known to cause growth arrest and/or cell death. Furthermore, the consequences of AP-1 activation seem to be cell-type specific. While it may promote apoptosis in some cell types, it is required for survival in others. Given the many different configurations for AP-1 activation, the exact functions are likely to be dependent on the composition of transactivating factors and their state of posttranslational modification [36].

The experiments reported here are the first to show, in vivo, that a single dose of VCD increases the AP-1 DNA binding activity whereas repeated daily dosing represses AP-1 DNA nuclear protein binding activity in rat small ovarian follicles. Comparing these results with VCD-induced MAPK activity and morphologic changes, it can be concluded that VCD dosing alters transcription factors that regulate AP-1 binding in rat small preantral follicles. This event likely plays an important role in transducing signals to the nucleus.

VCD-Induced Ovotoxicity Is Associated with Phosphorylation of c-Jun Protein

The c-Jun protein, a member of the activator protein-1 (AP-1) family of transcription factors, has been implicated in the regulation of many important biological processes, including cell cycle progression, differentiation, and apoptosis [15, 37–39]. Accordingly, its expression and function are up-regulated in response to diverse stimuli, including mitogens and a wide range of stresses. Its transcriptional activities are regulated by changes in the level of c-jun expression as well as posttranslational modifications of c-Jun protein. In particular, the transactivation potential of c-Jun critically depends on its phosphorylation at amino acid residues Ser-63 and Ser-73 mediated by JNK [27, 28].

Consistent with the sustained up-regulation of JNK activity, the results presented here have shown that repeated daily dosing of VCD increases the phosphorylation status of c-Jun protein in ovarian small preantral follicles. It has been well established in other systems that phosphorylation of c-Jun protein enhances transcriptional activation of AP-1 in the nucleus. For example, hypoxia-induced phosphorylation of c-Jun is associated with an increase in AP-1 DNA binding activity in HepG2 cell lines [40]. However, it appears that VCD-induced phosphorylation of c-Jun protein in small preantral follicles results in reduced AP-1 DNA binding activity of nuclear protein, i.e., preincubation of nuclear protein with anti-c-Jun antibody reduced AP-1 DNA binding activity, whereas preincubation with anti-phospho-c-Jun antibody enhanced AP-1 DNA binding activity. Additionally, JNK kinase activity (phosphorylation of c-Jun) was inversely associated with AP-1 DNA binding activity with 1, 10, and 15 days dosing. Taken together, these results support that, in small ovarian preantral follicles, phosphorylation of c-Jun protein reduces its transcriptional regulating capacity. In order to obtain more detailed mechanistic information, future studies will involve utilization of transgenic animal models and development of an in vitro system to evaluate the role of c-jun in the ovarian toxicity induced by VCD.

In summary, the studies reported here demonstrate that repeated daily dosing of rats with VCD induces the activation of MAPK members, JNK and p38 kinase, selectively in small preantral ovarian follicles. Moreover, VCD-mediated signaling for apoptosis appears to involve phosphorylation of c-Jun protein and a reduction in AP-1 transcriptional activation. Future studies will be aimed at identification of the genes that are regulated in these pathways.


   ACKNOWLEDGMENTS
 
We would like to thank Patty Christian for her technical assistance.


   FOOTNOTES
 
First decision: 5 March 2002.

1 Supported by NIH Grant RO1-ESO9246 and Center Grant ESO6694. Back

2 Correspondence: Patricia B. Hoyer, 1501 North Campbell Ave., Dept. of Physiology, The University of Arizona, P.O. Box 245051, Tucson, AZ 85724. FAX: 520 626 2382; hoyer{at}u.arizona.edu Back

Accepted: March 25, 2002.

Received: February 5, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991 124:43-101[Medline]
  2. Mattison DR, Evans MI, Schwimmer WB, White BJ, Jensen B, Schulman JD. Familial premature ovarian failure. Am J Hum Genet 1984 36:1341-1348[Medline]
  3. Mattison DR, Plowchalk BS, Meadows MJ, Miller MM, Malek A, London S. The effect of smoking on oogenesis, fertilization, and implantation. Semin Reprod Endocrinol 1989 7:291
  4. Smith BJ, Mattison DR, Sipes IG. The role of epoxidation in 4-vinylcyclohexene-induced ovarian toxicity. Toxicol Appl Pharmacol 1990 105:372-381[CrossRef][Medline]
  5. Rappaport SM, Fraser DA. Air sampling and analysis in rubber vulcanization area. Am Ind Hyg Assoc J 1977 38:205-209[Medline]
  6. Springer LN, Flaws JA, Sipes IG, Hoyer PB. Follicular mechanisms associated with 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. Reprod Toxicol 1996 10:137-143[CrossRef][Medline]
  7. Springer LN, McAsey ME, Flaws JA, Tilly JL, Sipes IG, Hoyer PB. Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. Toxicol Appl Pharmacol 1996 139:394-401[CrossRef][Medline]
  8. Kao SW, Sipes IG, Hoyer PB. Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice. Reprod Toxicol 1999 13:67-75[CrossRef][Medline]
  9. Hu X, Christian P, Thompson KE, Sipes IG, Hoyer PB. Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades. Biol Reprod 2001 65:87-93[Abstract/Free Full Text]
  10. Hu X, Christian P, Sipes IG, Hoyer PB. Expression and redistribution of cellular Bad, Bax, and Bcl-xL protein is associated with VCD-induced ovotoxicity in rats. Biol Reprod 2001 65:1489-1495[Abstract/Free Full Text]
  11. Thompson KE, Sipes IG, Greenstein Bd, Hoyer PB. 17ß-Estradiol affords protection against 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in Fischer-344 rats. Endocrinology 2002 143:1058-1065.[Abstract/Free Full Text]
  12. Borman SM, VanDePol BJ, Kao SW, Thompson KE, Sipes IG, Hoyer PB. A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide is protective in rat primary ovarian follicles. Toxicol Appl Pharmacol 1999 158:244-252[CrossRef][Medline]
  13. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995 80:179-185[CrossRef][Medline]
  14. Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 1996 74:589-607[CrossRef][Medline]
  15. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development. Curr Opin Cell Biol 1998 10:205-219[CrossRef][Medline]
  16. Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 1995 80:199-211[CrossRef][Medline]
  17. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995 270:1326-1331[Abstract/Free Full Text]
  18. Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase induce cell death. J Biol Chem 1996 271:3229-3237[Abstract/Free Full Text]
  19. Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu FF, Woodgett JR. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Curr Biol 1996 6:606-613[CrossRef][Medline]
  20. Rincon M, Whitmarsh A, Yang DD, Weiss L, Derijard B, Jayaraj P, Davis RJ, Flavell RA. The JNK pathway regulates the in vivo deletion of immature CD4(+)CD8(+) thymocytes. J Exp Med 1998 188::1817-1830[Abstract/Free Full Text]
  21. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 1987 49:729-739[CrossRef][Medline]
  22. Karin M, Liu Zg, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997 9:240-246[CrossRef][Medline]
  23. Foletta VC, Segal DH, Cohen DR. Transcriptional regulation in the immune system: all roads lead to AP-1. J Leukoc Biol 1998 63:139-152[Abstract]
  24. Springer LN, Tilly JL, Sipes IG, Hoyer PB. Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat. Toxicol Appl Pharmacol 1996 139::402-410[CrossRef][Medline]
  25. Flaws JA, Salyers KL, Sipes IG, Hoyer PB. Reduced ability of rat preantral ovarian follicles to metabolize 4-vinyl-1-cyclohexene diepoxide in vitro. Toxicol Appl Pharmacol 1994 126:286-294[CrossRef][Medline]
  26. Raju U, Gumin GJ, Tofilon PJ. Radiation-induced transcription factor activation in the rat cerebral cortex. Int J Radiat Biol 2000 76:1045-1053[CrossRef][Medline]
  27. Binetruy B, Smeal T, Karin M. Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature 1991 351::122-127[CrossRef][Medline]
  28. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 1991 354:494-496[CrossRef][Medline]
  29. Potapova O, Basu S, Mercola D, Holbrook NJ. Protective role for c-Jun in the cellular response to DNA damage. J Biol Chem 2001 276::28546-28553[Abstract/Free Full Text]
  30. Wagner EF. AP-1—introductory remarks. Oncogene 2001 20:2334-2335[CrossRef][Medline]
  31. Corcoran GB, Fix L, Jones DP, Moslen MT, Nicotera P, Oberhammer FA, Buttyan R. Apoptosis: molecular control point in toxicity. Toxicol Appl Pharmacol 1994 128:169-181[CrossRef][Medline]
  32. Chen YR, Wang X, Templeton D, Davis RJ, Tan TH. The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J Biol Chem 1996 271:31929-31936[Abstract/Free Full Text]
  33. Seimiya H, Mashima T, Toho M, Tsuruo T. c-Jun NH2-terminal kinase-mediated activation of interleukin-1beta converting enzyme/CED-3-like protease during anticancer drug-induced apoptosis. J Biol Chem 1997 272:4631-4636[Abstract/Free Full Text]
  34. Sakata N, Patel HR, Terada N, Aruffo A, Johnson GL, Gelfand EW. Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells. J Biol Chem 1995 270:30823-30828[Abstract/Free Full Text]
  35. Hornung M, Lindemann D, Kraus C, Peters A, Berberich I. The CD40 TRAF family member interacting motif carries the information to rescue WEHI 231 cells from anti-IGM-induced growth arrest. Eur J Immunol 1998 28:3812-3823[CrossRef][Medline]
  36. Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene 2001 20:2390-2340[CrossRef][Medline]
  37. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000 103:239-252[CrossRef][Medline]
  38. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1991 1072::129-157[Medline]
  39. Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta 1997 1333:F85-F104[Medline]
  40. Widsom R. AP-1: one switch for many signals. Exp Cell Res 1999 253:180-185[CrossRef][Medline]



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