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BOR - Papers in Press, published online ahead of print July 6, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043505
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BIOLOGY OF REPRODUCTION 73, 974–982 (2005)
DOI: 10.1095/biolreprod.105.043505
© 2005 by the Society for the Study of Reproduction, Inc.

2,2'-Dichlorobiphenyl Decreases Amplitude and Synchronization of Uterine Contractions Through MAPK1-Mediated Phosphorylation of GJA1 (Connexin43) and Inhibition of Myometrial Gap Junctions1

Daesuk Chung , and Rita Loch Caruso 2 

Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study examined the hypothesis that inhibition of myometrial gap junctions through MAPK1-induced phosphorylation of GJA1 (connexin43) leads to inhibition of spontaneous phasic uterine contractions by 2,2'-dichlorobiphenyl (2,2'-DCB). Uterine strips from Gestation Day 10-pregnant rats exposed in muscle baths to 2,2'-DCB exhibited increased oscillatory frequency and decreased amplitude and synchronization of contractions. To assess effects on gap junctions, Lucifer yellow was injected into myometrial cells and transfer to adjacent cells was scored. After a 1-h treatment, 100 µM 2,2'-DCB decreased Lucifer yellow intercellular transfer in a concentration-dependent manner. The MAP2K1 inhibitor PD98059 increased percentage of dye transfer to adjacent myometrial cells from 18% in cultures exposed for 1 h to 100 µM 2,2'-DCB alone to 48% in cultures cotreated with 50 µM PD98059 and 100 µM 2,2'-DCB. In contrast, the conventional PRKC inhibitor Gö6976 (10 µM) had no significant effect on 2,2'-DCB-induced inhibition of dye transfer. Western blotting showed about a 4.5-fold increase in phosphorylation of GJA1 at S255, a MAPK1 site, after exposure to 100 µM 2,2'-DCB compared to untreated and solvent controls. However, there was no difference in phosphorylation of GJA1 at S368, a PRKC site. Cells treated with 2,2'-DCB increased phosphorylated MAPK1, implicating the increase of activation of MAPK1. Cotreatment with 100 µM 2,2'-DCB and 5 µM PD98059 reversed 2,2'-DCB-induced modification of uterine contractions and increase of pGJA1(S255) in uterine strips. Therefore, this study suggests that 2,2'-DCB decreases amplitude and synchronization of uterine contractions mediated through MAPK1-mediated phosphorylation of GJA1 and subsequent inhibition of myometrial gap junctions.

kinases, parturition, signal transduction, toxicology, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are abundant environmental contaminants despite an ongoing ban on their production in the United States and other countries. Their widespread use, resistance to environmental degradation, and lipophilicity promote their bioaccumulation, especially in fatty tissues. Even though there has been an abundance of research on various adverse effects of PCBs, there are few reports on the effect of PCBs on uterine contractions. Loch-Caruso emphasized uterine muscle as a potential target of PCBs during pregnancy because of the increased lipid concentrations of PCBs in uterine muscle during pregnancy [1]. In a comparison of acute actions on uterine contractions for different PCB congeners [2], the congeners 2,2',4,4'-tetrachlorobiphenyl and 2,4,6-trichlorobiphenyl elicited increased frequency, 3,3',4,4'-tetrachlorobiphenyl and 3,3',4,4',5'-pentachlorobiphenyl showed no acute activity, and 4-hydroxy-2',4',6'-tetrachlorobiphenyl inhibited contractions of midgestation rat uteri.

Serving as intercellular channels, gap junctions play an important role in uterine contraction and parturition [3]. The increased formation of gap junctions between myometrial cells in the uterus is associated with the onset of parturition, allowing proper coordination of smooth muscle contraction and the generation of sufficient contraction force essential for labor [4]. Structurally, gap junctions are composed of a hexameric assembly of integral membrane proteins (connexins) arranged symmetrically around a central aqueous pore [5]. Among the members of the family of connexins, the appearance of GJA1 (also known as Connexin43) in the myometrium coincides with the onset of labor in several species [6]. GJA1 is a phosphoprotein, and one mechanism to regulate gap junctions is through the phosphorylation of GJA1. There are six known phosphorylation sites on the C-terminal of GJA1 proteins: S368 by protein kinase C (PRKC, also known as PKC), S255/S279/ S282 by mitogen-activated protein kinase1 (MAPK1, also known as MAPK), and S247/S265 by v-src [7].

Previous studies demonstrated that PCBs inhibit gap junctions in vivo [8] and in vitro [9]. However, the mechanism by which PCBs inhibit gap junctions is not known. In contrast, the mechanism for the inhibition of gap junctions by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) has been studied in detail. Epidermal growth factor-induced inhibition of gap junctions is associated with a signal transduction pathway involving sequential activation of EGF receptor (EGFR), HRAS1, RAF1, MAP2K1 (also known as MEK), and MAPK3/MAPK1 (also known as ERK1/2) [10, 11]. TPA has been associated with the activation of PRKC followed by activation of MAPK3/1 [12].

In the present study, 2,2'-DCB was selected as a representative ortho-substituted noncoplanar PCB congener to inhibit spontaneous phasic uterine contractions. Although 2,2'-DCB is easily biodegraded, there have been reports that 2,2'-DCB was found in soil and drinking water samples [13]. The present study characterizes the in vitro effects of 2,2'-DCB on spontaneous oscillatory uterine contractions in Gestation Day (GD) 10 rat uteri and examines the hypothesis that inhibition of myometrial gap junctions through phosphorylation of GJA1 leads to 2,2'-DCB-induced desynchronization and decreased amplitude of uterine contractions. By examining the mechanism for 2,2'-DCB action on uterine contraction, this study will show signaling pathways by which ortho-substituted noncoplanar PCBs induce alteration of uterine contractions.


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

Time-pregnant Sprague-Dawley rats were obtained from Harlan Laboratory (Indianapolis, IN) at GD 7–10. The gestational age at time of acquisition did not affect observed uterine contraction or myometrial responses. All animals used in this study were treated humanely according to guidelines of the National Research Council and the University of Michigan, with due consideration to the alleviation of distress and discomfort.

Chemicals

The 2,2'-DCB was purchased from Ultra Scientific (North Kingstown, RI). Type II collagenase, type III trypsin, deoxyribonuclease I, and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture media (RPMI 1640), calcium/magnesium-free PBS (CMF-PBS), and PBS were purchased from Invitrogen (Carlsbad, CA), and bovine calf serum (BCS) was purchased from HyClone (Logan, UT). Lucifer yellow (lithium salt) was obtained from Molecular Probes (Eugene, OR), and PD98059 and Gö6976 were purchased from Calbiochem (La Jolla, CA). Protease inhibitor cocktail stock solution was purchased from Roche (Indianapolis, IN).

Uterine Contractility

Spontaneous isometric oscillatory contractions were evaluated in uterine muscle strips isolated from pregnant rats and suspended in standard muscle baths. Because contractions of the longitudinal muscle layer are primarily responsible for fetal delivery in rats, longitudinal muscle contractions were assessed by monitoring contractions along the longitudinal axis of the uterus. Uteri from GD 10 rats were used because previous studies in our laboratory found that PCBs modify contractions of GD 10 uteri [2, 14]. Sprague-Dawley rats were anesthetized with ether and the uteri were removed. Longitudinal uterine strips 1 mm wide x 20 mm long were excised from the midsection of each uterine horn and suspended in 50-ml muscle baths filled with 37°C prewarmed physiologic salt solution (116 mM NaCl, 4.6 mM KCl, 1.16 mM NaH2PO4•H2O, 1.16 mM MgSO4•7H2O, 21.9 mM NaHCO3, 1.8 mM CaCl•2H2O, 11.6 mM dextrose, and 0.03 mM CaNa2EDTA at pH 7.4). One end of each uterine strip was tied to a stationary post and the other end was tied to a force transducer. The changes of contraction were recorded by computer using the PowerLab system (ADInstruments, Australia). All strips were subjected to a 1.0 g preload tension, allowed to equilibrate for 45 min, and then depolarized with a 10-min exposure to 60 mM KCl to determine maximal contractile force. Strips were rinsed free of KCl and allowed to equilibrate for an additional 3–7 h until spontaneous oscillatory contractions were established. Contraction amplitude and frequency were calculated, respectively, as the average peak force of contraction and average number of peaks in the last 10 min of each 30- or 60-min exposure interval. Contraction completion was calculated as the number of peak force displacements that returned to baseline divided by the number of total contraction peaks in the last 10 min of each 30- or 60-min exposure interval.

Myometrial Cell Isolation and Culture

Myometrial smooth muscle cells were isolated on GD 10 from pregnant Sprague-Dawley rats that had been anesthetized with ether and killed by cardiac puncture. Upon removal, uteri were immediately placed in ice-cold CMF-PBS. After embryos, cervix, ovaries, and adipose tissue were removed, uteri were diced and digested in an enzyme solution containing 300 µg/ml type II collagenase, 300 µg/ml type III trypsin, and 200 µg/ ml deoxyribonuclease I. The digest was filtered through wire mesh with 1.5-mm openings, then through standard cheesecloth to remove large tissue clumps. The filtrate containing isolated cells was centrifuged at 200 x g to pellet the cells. After washing of the cells with CMF-PBS three times, cells were seeded into flasks containing RPMI 1640 medium supplemented with 10% BCS. Cultured cells were incubated at 37°C in a 5% CO2 atmospheric condition. Medium was changed every 2 days and cells were subcultured using 0.25% crude trypsin to remove cells from flasks after 6–7 days, just before confluence. All cells were used at Passage 2. The smooth muscle character of the cultured cells was verified using indirect immunofluorescence labeling with mouse smooth muscle-specific {alpha}-actin antibodies as previously described [15], and the purity of the cell cultures was 99–100%.

Microinjection

Passage 1 cultured cells were removed from flasks by a 5-min exposure to 0.25% crude trypsin in CMF-PBS at 37°C and were seeded into Corning polystyrene dishes at densities of 50,000 cells per dish with RPMI 1640 medium supplemented with 10% BCS. Cells were incubated for 24 h at 37°C in a 5% CO2 atmosphere, during which time cell attachment and growth occurred. After exposure to 2,2'-DCB for 1 h, cells were microinjected with a mixed dye solution of 0.8% (w/v) Lucifer yellow CH and 0.02% propidium iodide. An injection pressure of 6.5 psi for 200 msec was used. Propidium iodide served as a marker of the injected cells by binding to nuclear DNA. The presence of Lucifer yellow fluorescence in neighboring cells was used as a measure of gap junction intercellular communication. Lucifer yellow dye transfer from an injected cell to cells in direct contact with an injected cell was scored by epifluorescence microscopy and expressed as percent dye transfer by dividing the number of adjacent cells that exhibited Lucifer yellow fluorescence by the total number of cells touching the injected cell and multiplying by 100. Cells were injected in RPMI 1640 medium over a 4-min period followed by rinsing with prewarmed CMF-PBS. Cells were scored in the order that they were injected over a 5-min observation period. For experiments with kinase inhibitors, cells were cotreated with PD98059 or Gö6976 in addition to 100 µM 2,2'-DCB. Concentrations of inhibitors were selected according to effective concentrations derived from previously published data [16, 17].

Western Blotting Analysis for GJA1 Phosphorylation

Confluent cells in 75-cm2 plates were washed twice with cold CMF-PBS and lysed with 375 µl of cold lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 10 mM NaF, pH 7.5) containing 100 µl of protease inhibitor cocktail stock solution (1 tablet/10 ml H2O; Roche). Cells were harvested by scrapping and collected in precooled Eppendorf tubes. The collected cells were treated with 550 µl of 40 mM NaOH added to each Eppendorf tube, sonicated on ice for 5 sec, and centrifuged at 16,600 x g for 30 min at 4°C. The pellets were resuspended in 100 µl of running buffer (62.5 mM Tris pH 6.8, 2% SDS, 10% glycerol), sonicated for 5 sec, and centrifuged at 16,600 x g for 5 min at 4°C. Protein concentration was determined by the method of Bradford using a Bio-Rad DC protein kit (Bio-Rad, Hercules, CA), and 20 µg of protein was loaded into the gel for each sample. Equal protein loading was verified by probing blots with anti-rabbit glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotech Inc., Santa Cruz, CA). Proteins were separated by SDS-PAGE using 15% acrylamide. Following electrophoresis, proteins were transferred electrophoretically onto membranes and reacted for 1 h with rabbit polyclonal antibody for GJA1 phosphorylated at Ser368 [pGJA1(S368)] at 1:100 dilution (Cell Signaling, Beverly, MA), rabbit polyclonal antibody for GJA1 phosphorylated at Ser255 [pGJA1(S255)] (Santa Cruz Biotech Inc.) at 1:100 dilution, or rabbit polyclonal antibody for phosphorylated MAPK3/1 proteins (Santa Cruz Biotech Inc.) at 1:100 dilution. The protein-primary complexes were probed with a 1:1000 dilution of AP conjugated anti-rabbit antibodies (Amersham Life Sciences Products, Arlington Heights, IL) as appropriate for 1 h.

Statistical Analysis

Data are reported as the mean ± SEM. Data analysis was conducted using SigmaStat (Jandel Scientific Software, San Rafael, CA). Dye transfer data and Western blot data were analyzed by one-way ANOVA. Contractility data were analyzed by two-way repeated measures ANOVA. Post hoc comparison of means was by a Dunn pairwise multiple comparison tests. A P value of ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of 2,2'-DCB on Uterine Contractions

When uterine strips were exposed continuously to 100 µM 2,2'-DCB, contraction frequency initially increased for 5 min, but then initiated contractions started to diverge to two, three, or more peaks before returning to baseline, beginning about 5 min after exposure (Fig. 1). Subsequent to development of desynchronized contractions, contraction amplitude decreased in the 2,2'-DCB-treated uterine strips. Control strips maintained consistent frequency, amplitude, and completion, so that the pattern of contraction established before treatment was sustained in solvent control strips for the entire 150-min observation period (not shown). Changes of contraction in response to 2,2'-DCB were quantified (Fig. 2). Time-dependent and concentration-dependent changes of contraction amplitude (Fig. 2A), frequency (Fig. 2B), and completion (Fig. 2C) were significant in uterine strips exposed to 2,2'-DCB (two-way repeated measures ANOVA, P ≤ 0.05). Maximal responses were observed for up to 7–9 h (data not shown).



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  		FIG. 1. Representative tracings showing time-dependent changes of contraction patterns of uterine strips exposed continuously to 100 µM 2,2'-DCB. The before treatment tracing was taken from the 30-min period immediately preceding addition of 2,2'-DCB. The pattern of contraction established before treatment was maintained in solvent control strips for the entire 150-min observation period (not shown)



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  		FIG. 2. Effects of 2,2'-DCB on rat uterine contraction. Uterine strips from Gestation Day 10 pregnant rats were suspended in isometric muscle baths and exposed to DMSO (solvent control) or to 30, 60, or 100 µM 2,2'-DCB. A) Average peak amplitude per contraction. B) Frequency of oscillation. C) Contraction completion measured as the percent of contractions that returned to baseline during 10-min periods over 150 min duration of exposure. Bars represent mean ± SEM. n = 7–10 strips per treatment. *Statistically different (P ≤ 0.05) from control within each time point. {dagger}Statistically different from 0 min within each treatment

Treatment with 100 µM 2,2'-DCB decreased contraction amplitude in a time-dependent manner such that amplitude was suppressed to less than 25% of control 1 h after initiating exposure, and this inhibition was sustained for up to 150 min of exposure (P ≤ 0.05). Treatment with 60 µM 2,2'-DCB decreased amplitude to 50% of control 1 h after exposure and showed a U-shaped, time-dependent curve (P ≤ 0.05). Decreases in contraction amplitude by 30 µM 2,2'-DCB were not significantly different at any exposure time point compared to 0 h.

Compared with patterns observed for amplitude of uterine contraction, changes of contraction frequency followed an inverse time-dependent response pattern (Fig. 2B). The increases in contraction frequency by 30 µM 2,2'-DCB were not significantly different at any exposure time point compared to 0 min. Exposure to 100 µM 2,2'-DCB increased contraction frequency to over 1000% of control by 1 h, and this increase was sustained for the entire 150 min exposure period (P ≤ 0.05). In contrast, uterine strips exposed to 60 µM 2,2'-DCB exhibited an inverted U-shaped time-dependence, significantly increased from 0 min to 30 min and sustained for 120 min of exposure (P ≤ 0.05), but returning to control levels 150 min after initiation of exposure to 2,2'-DCB.

Contraction completion, used as an index of contraction synchronization, followed a similar pattern to that seen for contraction amplitude (Fig. 2C), except that decreases in contraction completion by 30 µM 2,2'-DCB were significantly different after 30, 60, and 90 min of exposure compared to 0 min (P ≤ 0.05). Also, decreases in contraction completion by 60 µM 2,2'-DCB were significantly different at 60 and 90 min after exposure compared to solvent control (P ≤ 0.05).

Effect of 2,2'-DCB on Myometrial Gap Junctions

To examine whether 2,2'-DCB inhibited gap junction intercellular communication between myometrial cells, the fluorescent dye Lucifer yellow was microinjected into myometrial cells that had been treated with 2,2'-DCB for 1 h and the transfer of dye to adjacent cells was assessed. As shown in the examples in Figure 3A, extensive transfer of Lucifer yellow dye occurred among untreated control cells and among cells exposed to 25 µM 2,2'-DCB, whereas reduced Lucifer yellow dye transfer was observed in cell cultures exposed to 50 or 100 µM 2,2'-DCB. Exposure to 2,2'-DCB decreased dye transfer in a concentration-dependent manner (one-way ANOVA, P ≤ 0.05). Compared with 98% dye transfer in untreated control cells and 94% dye transfer in solvent controls exposed to 0.1% DMSO, dye transfer decreased to 37, 18, and 18% after a 1-h treatment with 50, 75 or 100 µM 2,2'-DCB (P ≤ 0.05; Fig. 3B).



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  		FIG. 3. Concentration-dependent effect of 2,2'-DCB on gap junction communication in myometrial cells. Cells were untreated (negative control) or exposed to 0.1% DMSO (solvent control) or to 25, 50, 75, or 100 µM 2,2'-DCB for 1 h, and then assessed for transfer of Lucifer yellow dye from microinjected cells to adjacent cells as a measure of gap junction communication. A) Examples of dye transfer: top row, phase contrast; middle row, epifluorescence image showing propidium iodide fluorescence; bottom row, epifluorescence image showing Lucifer yellow fluorescence. Photos taken with 20x objective. B) Summarization of effects of 2,2'-DCB on dye transfer. Bars represent mean ± SEM. n = 12–16 injections (3–4 dishes) per treatment. Columns with different letters denote treatment means that are statistically significantly different from each other (P ≤ 0.05)

Effect of Kinase Inhibitors on 2,2'-DCB-Induced Inhibition of Gap Junctions

To investigate the role of phosphorylation of GJA1 on 2,2'-DCB-induced inhibition of gap junctions, myometrial cells in culture were cotreated for 1 h with 100 µM 2,2'-DCB and either PD98059 or Gö6976, inhibitors for the MAP2K1 and conventional PRKCs ({alpha}, ß, and {gamma} isoforms), respectively, and then assessed for Lucifer yellow dye transfer. The percentage of dye transfer recovered from 18% in cultures treated with 2,2'-DCB alone to 48% in cultures cotreated with 50 µM PD98059 (P ≤ 0.05; Fig. 4). However, dye transfer remained significantly depressed compared with controls in cultures cotreated with 10 µM Gö6976 (P ≤ 0.05). Cotreatment with 100 µM Gö6976 did not change 2,2'-DCB-induced inhibition of gap junctions (data not shown). This suggests that 2,2'-DCB-induced partial inhibition of myometrial gap junctions is mediated through the activation of MAPK.



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  		FIG. 4. The effect of kinase inhibitors on 2,2'-DCB-induced inhibition of gap junction communication in myometrial cells. Cells were untreated (negative control) or exposed for 1 h to 0.1% DMSO (solvent control), 50 µM PD98059 (MAP2K1 inhibitor), 10 µM Gö6976 (conventional PRKC inhibitor), 100 µM 2,2'-DCB, 100 µM 2,2'-DCB plus 50 µM PD98059, or 100 µM 2,2'-DCB plus 10 µM Gö6976 before assessing gap junction communication by Lucifer yellow dye transfer. Bars represent the mean ± SEM. n = 7–16 injections (3–4 dishes) per treatment. Columns with different letters denote means that are statistically significantly different from each other (P ≤ 0.05)

Effect of 2,2'-DCB on Phosphorylation of GJA1

To examine further whether 2,2'-DCB-induced partial inhibition of myometrial gap junctions is mediated by MAPK1, as opposed to PRKC, Western blotting was performed with two phospho-GJA1 antibodies, pGJA1(S368) and pGJA1(S255). The pGJA1(S255) and pGJA1(S368) antibodies recognize GJA1 phosphorylated at ser255 and at ser368 by MAPK1 and PRKC, respectively. Densities of Western blot bands recognized by the two phospho-GJA1 antibodies were normalized to the housekeeping gene GAPDH. Densities of Western blot for GAPDH were not significantly different between treatments. Densitometric analysis showed an almost 6-fold increase of pGJA1(S255) in cells exposed to 2,2'-DCB for 1 h compared to untreated and solvent (0.1% DMSO) control cells (P ≤ 0.05; Fig. 5A). However, there was no statistically significant difference in the abundance of pGJA1(S368) compared with untreated and solvent (0.1% DMSO) controls (Fig. 5B). The Western blot data showed increased phosphorylation of GJA1 at a MAPK1 site but not a PRKC site. Although not included in the present report, TPA was included as a positive control in a previous study by us to show detection of phosphorylation of GJA1 at Ser368 using identical procedures [18].



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  		FIG. 5. The effect of 2,2'-DCB on site-specific phosphorylation of GJA1 in myometrial cells as detected by Western blot analysis. Cultured myometrial cells were untreated, exposed to 0.1% DMSO (solvent control), or exposed to 100 µM 2,2'-DCB for 1 h. A) Immunoreactivity to an antibody for GJA1 phosphorylated at Ser255 [pGJA1(S255)]. B) Immunoreactivity to an antibody for GJA1 phosphorylated at Ser368 (pGJA1(S368)). Bars represent the mean ± SEM. n = 5–12 dishes per treatment. *Significantly different from untreated and solvent controls (P ≤ 0.05)

Effect of 2,2'-DCB on Phosphorylation of MAPK1

To confirm whether MAPK3/1 is activated by 2,2'-DCB in myometrial cells, Western blotting was performed with phospho-MAPK3/1 (pMAPK3/1) antibodies and normalized to GAPDH expression. Although the antibody for phosphorylated MAPK3/1 recognizes both MAPK3 and MAPK1, the only immunoreactive band observed was MAPK1, identified by comparison with molecular weight standards. Densitometric analysis of the ratio of pMAPK1 to GAPDH showed that myometrial cells treated for 1 h with 2,2'-DCB increased immunoreactivity to pMAPK1 compared to untreated and solvent (0.1% DMSO) control cells (P ≤ 0.05)(Fig. 6). Because phosphorylation of MAPK1 has been shown to be increased by 2,2'-DCB, this experiment suggests that 2,2'-DCB activates MAPK1.



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  		FIG. 6. The effect of 2,2'-DCB on phosphorylation of MAPK1 detected by Western blot analysis with rabbit polyclonal antibody for phosphorylated MAPK3/1. Cultured myometrial cells were untreated (negative control) or exposed for 1 h to 0.1% DMSO (solvent control) or 100 µM 2,2'-DCB. Bars represent mean ± SEM. n = 4–8 dishes per treatment. *Significantly different from untreated and solvent controls (P ≤ 0.05)

Effect of PD98059 on 2,2'-DCB-Induced Modification of Uterine Contraction

The MAPK3/1s are activated via phosphorylation of tyrosine by MAP2K1s. To examine further the role of MAPK1-dependent inhibition of gap junctions in 2,2'-DCB-induced modification of uterine contractions, uterine strips suspended in muscle baths were cotreated with 100 µM 2,2'-DCB and 5 µM PD98059. The MAP2K1 inhibitor PD98059 reversed 2,2'-DCB-induced inhibition of uterine contraction amplitude (Fig. 7A) and completion (Fig. 7B) in a time-dependent manner (two-way repeated measures ANOVA, P ≤ 0.05). Compared to strips treated with 2,2'-DCB alone (Fig. 7), the pattern of uterine contraction did not show significant difference until 1 h after treatment. However, the decreases in amplitude and synchronization of contractions reversed significantly 2 h after treatment (P ≤ 0.05). The reversal reached up to 65% of control in amplitude and up to 85% of control in completion of uterine contraction 4 h after treatment. This suggests that 2,2'-DCB-induced modification of uterine contractions is dependent on MAPK1 activity.



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  		FIG. 7. The effect of MAP2K1 inhibitor PD98059 on 2,2'-DCB-induced modification of uterine contractions. Uterine strips were cotreated with 5 µM PD98059 and 100 µM 2,2'-DCB. The average peak amplitude per contraction (A) and the contraction completion (B) as the percentage of contractions that returned to baseline were measured during 10-min periods over the 240-min duration of exposure. Bars represent mean ± SEM. n = 5–10 strips per treatment. *Significantly different from the 2,2'-DCB within each time point. {dagger}Significantly different from 0 min within each treatment. (P ≤ 0.05)

Effect of PD98059 on 2,2'-DCB-Induced Phosphorylation of GJA1

To see whether MAPK1-induced phosphorylation of GJA1 occurs in uterine tissue, Western blots of GJA1 phosphorylated at the MAPK1 site (ser255) were compared for uterine strips that were untreated or exposed to 0.1% DMSO (solvent control), 100 µM 2,2'-DCB, or 100 µM 2,2'-DCB and 5 µM PD98059 for 1 h. Densitometric analysis showed about a 2-fold increase of GJA1 phosphorylated at ser255 relative to GAPDH after exposure to 100 µM 2,2'-DCB. However, pGJA1(S255) was reduced to untreated and solvent control levels in uterine strips exposed to 100 µM 2,2'-DCB and 5 µM PD98059 (P ≤ 0.05; Fig. 8). Therefore, these data suggest that 2,2'-DCB phosphorylates GJA1 through MAPK1 in uterine strips.



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  		FIG. 8. Effect of the MAP2K1 inhibitor PD98059 on phosphorylation of GJA1 at Ser255 (pGJA1(S255)) in uterine strips as detected by Western blot analysis. Uterine tissues were untreated (negative control), or exposed for 1 h to 0.1% DMSO (solvent control), 100 µM 2,2'-DCB, or 100 µM 2,2'-DCB plus 5 µM PD98059 for 1 h. Bars represent mean ± SEM. n = 5–8 dishes per treatment. *Significantly different from all other treatments (P ≤ 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first report to show that 2,2'-DCB decreases amplitude and synchronization of uterine contractions. After in vitro exposure of longitudinal uterine strips from midgestation rats, the most dramatic effects were an increase of contraction frequency to 1288.5% at 110 min, a decrease in the amplitude to 10.5% at 140 min, and a decrease in completed contractions (a measure of synchronization) to 10.9% at 140 min after 100 µM 2,2'-DCB exposure. Other stimulatory PCB congeners, such as 2,4,6-trichlorobiphenyl, elicit an increase of frequency without changing the synchronization [2]. The 2,2'-DCB pattern is also different from that of another environmental contaminant, lindane, which decreases contractile force without significant change of frequency and synchronization [19].

The observation that 2,2'-DCB partially inhibited gap junction communication between myometrial cells suggests that inhibition of gap junctions may be a mechanism by which 2,2'-DCB modifies uterine contractions. Previously, inhibition of gap junction communication by toxicants, including TPA and lindane, has been demonstrated in myometrial cells [18]. Although an earlier study showed that inhibition of gap junctions by PCBs correlated with inhibition of uterine contraction [20], the present study is the first to provide direct evidence that disruption of gap junction communication between myometrial cells is a mechanism for PCB-induced modulation of uterine contraction.

Of the connexins identified in the uterus, it is the increased expression of GJA1 that is associated with parturition [4]. Gap junctions are regulated in various ways, including connexin phosphorylation. Phosphorylation of GJA1 has been associated with down-regulation of gap junction communication [21]. Several phosphorylation sites have been identified on GJA1, including Ser368 phosphorylated by PRKC [22] and Ser279/282 phosphorylated by MAPK1 [23]. In prior studies, PRKC or MAPK1 has been implicated in the phosphorylation of GJA1 and the inhibition of gap junctions [24, 25]. EGF-induced inhibition of gap junctions is associated with activation of MAPK1 because pretreatment of cells with MAPK1 inhibitors prevents the EGF-induced inhibition of gap junctions [26].

To identify the kinases involved in 2,2'-DCB-induced inhibition of gap junctions, we used PD98059 or Gö6976, inhibitors for MAP2K1 and cPRKC, respectively. Because MAPK1s are activated via phosphorylation of tyrosine by MAP2K1s, the MAP2K1 inhibitor PD98059 has been used to investigate the role of the MAPK1 pathway in inhibition through phosphorylation of GJA1 [10, 27]. The MAP2K1 inhibitor PD98059 only partially reversed 2,2'-DCB-induced inhibition of myometrial gap junctions in the present study. However, the same concentration of PD98059 used in the present study was shown previously to protect nonmyometrial cells almost completely from inhibition of gap junctions by other treatments [10, 28]. This discrepancy of PD98059 effectiveness indicates cell- and toxicant-specific activity of the MAP2K1 inhibitor PD98059 for prevention of inhibition of gap junctions. Also, it should be noted that 5 µM PD98059 reversed 2,2'-DCB-induced modification of uterine contractions, whereas 2,2'-DCB-induced inhibition of gap junction communication was only partially prevented at the higher concentration of 50 µM PD98059. The concentration of 5 µM PD98059 was used for the muscle bath contractility experiment because higher concentrations blocked spontaneous uterine contractions. Moreover, the effect of PD98059 was not significant until 2 h of exposure in uterine tissue, but cells in culture showed significant effects after only 1 h of exposure. The discrepancy between results in cells and tissues suggests that there is a difference in efficacy of the MAP2K1 inhibitor between myometrial cells and uterine tissue.

The present study showed that 2,2'-DCB increased phosphorylation of GJA1 at Ser255, which is phosphorylated by MAPK1, but not at Ser368, which is phosphorylated by PRKC. Lampe et al. [29] demonstrated by site-directed mutagenesis that inhibition of gap junction communication by TPA is dependent on the phosphorylation of GJA1 at Ser368. Because we used only Gö6976, an inhibitor for conventional PRKCs ({alpha}, ß, and {gamma} isoforms), the present study does not exclude the possibility that other isotypes of PRKC might be involved in 2,2'-DCB-induced inhibition of myometrial gap junctions through phosphorylation of GJA1.

Moreover, exposure of myometrial cells to 2,2'-DCB increased phosphorylation of GJA1 at Ser255 but not at S279/S282, which is also a MAPK1 phosphorylation site (data not shown). Abdelmohsen et al. showed that quinone-induced inhibition of gap junctions in WB-F344 rat liver epithelial cells is mediated through phosphorylation of GJA1 at S279/S282 by MAPK1 [30]. The different results of the present study and Abdelmohsen et al.'s study suggest that the phosphorylation site of GJA1 by MAPK1 may be cell-specific and toxicant-specific. Understanding the actual sites of phosphorylation and the consequences of phosphorylation at different sites will be necessary to determine the exact mechanisms for effects of 2,2'-DCB on inhibition of gap junction communication.

A band shift of GJA1 has been used previously as a measure of the status of GJA1 phosphorylation in Western blot analysis [12, 22]. However, Western blot analysis of 2,2'-DCB-exposed myometrial cells did not show a band shift of GJA1, even though immunoblotting with antibodies that recognizes pGJA1(S255) indicated phosphorylation of GJA1. Our finding of phosphorylation of GJA1 in the absence of a band shift on Western blot is consistent with other reports [18, 31].

Data showing that myometrial cells exposed to 2,2'-DCB have increased pMAPK1 support our hypothesis that 2,2'-DCB-induced phosphorylation of GJA1 is mediated by MAPK1. Furthermore, reversal of 2,2'-DCB-induced modification of uterine contraction and reversal of 2,2'-DCB-induced increase in pGJA1(S255) by PD98059 in uterine strips supports the conclusion that 2,2'-DCB modifies uterine contractions by a mechanism involving MAPK1-mediated phosphorylation of GJA1 and inhibition of myometrial gap junctions. On the other hand, the mechanism by which 2,2'-DCB activates MAPK1 is unknown and requires additional experiments beyond the scope of the present study.

It has been shown that 2,2'-DCB increased intercellular calcium ion concentration in rat cerebellar granule cells [32] and human granulocytes [33]. Because increased intracellular calcium concentration fundamentally drives uterine muscle contraction through the activation of myosin light chain kinase, an increase in intercellular calcium induced by 2,2'-DCB may contribute to an increase in uterine contraction frequency. Also, 2,2'-DCB-induced changes in membrane fluidity may contribute to modification of uterine contractions. By measuring steady-state fluorescence polarization, Tan et al. showed that an ortho-substitued PCB, 2,2',5,5'-tetrachlorobiphenyl, disrupts membrane structure in cerebellar granule cells and thymocytes [34]. Because gap junction channel formation can be disrupted because of change of membrane structure, 2,2'-DCB-induced desynchronization of uterine contractions may result from the change of membrane structure.

Although longitudinal muscle contraction was evaluated in the present study, uterine contractility involves both circular and longitudinal layers of the uterine muscle. However, it is the contractions of the longitudinal muscle that underlie fetal delivery in the rat, with contractions that move the fetuses along the uterine tubes toward the cervix. Nonetheless, GJA1 is expressed in gap junctions in the circular as well as the longitudinal layer of the uterus, and it is not known whether 2,2'-DCB activity in the uterus is influenced by differential regulation of GJA1 between the circular and longitudinal muscle layers, as seen in bovine myometrium [35]. Such investigations are beyond the scope of the current study, however.

2,2'-DCB has been found in soil and drinking water samples [13], even though it is relatively easily biodegraded by bacteria. In an occupational study, up to 10 µM of 2,2'-DCB was detected in plasma of workers engaged in the manufacture of capacitors [36]. There have been limited reports on altered parturition in animals exposed to PCBs. The congeners 3,3',4,4'-tetrachlorobiphenyl [37, 38] and 2,2'-DCB [39], and the PCB mixture Aroclor 1254 [40], increase gestation length. Although the present study used uterine strips from midgestation (GD 10) rats, uterine strips (longitudinal and circular) from GD 10 rats generate spontaneous contractile activity in muscle baths that is indistinguishable from GD 20 uterine strips [41]. Regardless, because the number and area of gap junctions dramatically increase at or near term [42], additional studies of uteri from late-gestation rats would provide further insight into 2,2'-DCB-induced modification of uterine contractions.

The present study contributes new information to understanding signaling pathways by which ortho-substituted noncoplanar PCBs induce alteration of uterine contractions. Increased understanding of how 2,2'-DCB alters uterine contraction may contribute to improved assessment of the potential risks of 2,2'-DCB and possibly other PCBs to pregnant women and their offspring.


   FOOTNOTES
 
1 Supported by grant P42ES04911 from the National Institute of Environmental Health Science, NIH, issued to R.L.C. Portions of this research were presented at the 37th Society for the Study of Reproduction Annual Meeting, August 2004, Vancouver, British Columbia, Canada. Back

2 Correspondence: Rita Loch Caruso, Department of Environmental Health Sciences, University of Michigan, 1420 Washington Heights, Ann Arbor, MI 48109-2029. FAX: 734 763 8095; rlc{at}umich.edu Back

Received: 5 May 2005.

First decision: 25 May 2005.

Accepted: 5 July 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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