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Am J Physiol Endocrinol Metab 291: E922-E928, 2006. First published June 6, 2006; doi:10.1152/ajpendo.00602.2005
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Phorbol ester treatment of human myometrial cells suppresses expression of oxytocin receptor through a mechanism that does not involve activator protein-1

Allison Ball,1 Jing Wei Wang,1 Susan Wong,1 Barbara Zielnik,1 Jana Mitchell,1 Nanping Wang,2 Michael B. Stemerman,2 and B. F. Mitchell1

1Department of Obstetrics and Gynecology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada; and 2Division of Biomedical Sciences, University of California, Riverside, California

Submitted 1 December 2005 ; accepted in final form 24 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxytocin (OT) is a potent uterine agonist. Its receptor (OTR) is a G protein-coupled receptor that is downregulated by prolonged exposure to OT. We hypothesized that activation of PKC mediated this OT-induced decrease in OTR expression. Diminished PKC activity in late pregnancy could underlie the increased expression of uterine OTR preceding labor onset. Using cell cultures of transformed human uterine myocytes, we determined the effects of PKC agonists and antagonists on the expression of OTR. We also explored the effects of overexpression of activator protein-1 (AP-1, a mediator of many PKC- and phorbol ester-induced effects) using adenoviral expression vectors for the AP-1 subunits c-Jun and c-Fos. Stimulation of PKC using the phorbol ester 12-O-tetradecanoylphorbol 13-acetate caused a rapid, significant (P ≤ 0.05) increase in c-Jun and c-Fos concentrations but a significant decrease in mRNA for OTR within 6 h followed by a significant decrease in OT binding by 24 h. Adenoviral infection of the cells with expression vectors for c-Jun and c-Fos increased the AP-1 subunits but had no effect on OTR expression. Furthermore, there were no changes in c-Fos or c-Jun levels in human intrauterine tissues around the time of labor onset, as measured by Western analyses. We conclude that phorbol ester treatment decreases OTR expression, likely through a mechanism that does not involve AP-1.

parturition; G protein-coupled receptor; preterm labor; protein kinase C

THE MOST POTENT UTERINE CONTRACTILE STIMULANTS, including oxytocin (OT), are ligands for G protein-coupled receptors (GPCRs) that are linked through G{alpha}q/11 subunits to the phospholipase C (PLC) signaling system (7). Activation of PLC causes hydrolysis of phosphatidylinositol bisphosphate, giving rise to two further signaling molecules, inositol trisphosphate (IP3) and diacylglycerol (DAG). The IP3-induced immediate increase in intracellular Ca2+ is well studied in myometrial smooth muscle. However, the role of the DAG-stimulated protein kinase C (PKC) pathway is less clearly defined.

Several isoforms of PKC are present in the pregnant human uterus (11, 14, 21). The commonest pharmacological agonists used to study PKC activity are the group of growth-promoting carcinogens, phorbol esters. The principal mechanism that mediates the effects of such PKC stimulation involves activation of a transcription factor called activator protein-1 (AP-1), which consists of homo- and heterodimers of the c-Jun and c-Fos families (3). Thus regulation of transcription of the jun and fos genes and/or regulation of activity of Jun and Fos proteins may regulate the downstream effects of phorbol ester-induced PKC activation. Myometrial expression of c-jun appears to increase in human pregnancy and is further enhanced at the time of labor onset (8, 25).

The literature is confusing regarding the role of PKC in uterine contractile function. Depending on the experimental approach, both positive and negative effects mediated through a variety of mechanisms have been observed. Myographic techniques using uterine muscle strips have shown that PKC stimulants may either increase or decrease myometrial contractility (13, 22, 26). The effects may be dependent on the dose of the PKC agonist and the species from which the uterine strips were obtained. Similar to most GPCRs, prolonged exposure to OT causes downregulation of OTR (20). The mechanism mediating this downregulation is poorly defined but appears to be complex. Phosphorylation of OTR may target the receptor for internalization and destruction, and it is possible that PKC recognition sites on OTR may play a role in this phosphorylation (24). However, the downregulation also appears to be accompanied by a decrease in mRNA for OTR (20). In keeping with this, our earlier studies using serial deletion constructs of the OTR promoter suggested that the AP-1 response elements were present in a negative regulatory region (27). Conversely, activation of AP-1 subunits appears to increase OTR expression in breast cancer cells (10). Finally, activated PKC may directly inhibit PLC activity (2, 33), thus diminishing the magnitude of agonist stimulation.

The present experiments were undertaken to directly examine the role of PKC in expression of OTR in human myometrial cells, with particular emphasis on the role of AP-1 in mediating these effects. We have used the transformed human myometrial cell line ULTR (18), which has been shown to maintain a smooth-muscle phenotype in culture and which expresses receptors for physiological uterine agonists. On the basis of the literature for uterine myocytes (30), we chose to manipulate PKC activity using the PKC agonist phorbol ester [12-O-tetradecanoylphorbol 13-acetate (TPA)] or the antagonists calphostin C and bisindolylmaleimide hydrochloride (BIM). We explored the role of AP-1 by infecting cells with adenovirus expression vectors for c-Jun and/or c-Fos. We assessed OTR expression by using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to measure mRNA for OTR and using a radioligand-binding assay to assess OTR protein.

There is strong evidence that the mechanism regulating the timing of parturition is regulated by paracrine interactions between the maternal decidua and fetal membranes with the adjacent myometrium (15). Because we cannot obtain samples of human myometrium from the fundal portion of the uterus, we reasoned that if changes in myometrial AP-1 activity were involved in the mechanism regulating parturition, there may be a corresponding change in expression of AP-1 subunits in the choriodecidual tissues. Thus we have compared c-Fos and c-Jun levels assessed using Western blots in choriodecidual tissues obtained from term pregnancies (38–41 wk gestation) prior to labor onset with those obtained after spontaneous labor had occurred.

The results demonstrate an inhibitory action of phorbol ester on OTR expression, although this was not altered by overexpression of AP-1 subunits. No major changes were measured in Fos or Jun expression in choriodecidual tissues obtained before and after labor onset.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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Cell cultures and tissues. An immortalized cell line (ULTR) (18) originating from human myometrium was provided by Dr. J. MacDougall (Fred Hutchinson Cancer Research Center, Seattle, WA). We have determined that these cells express smooth muscle actin, GPCRs for OT and prostaglandin F2{alpha}, and the nuclear receptors for estrogen and progesterone. They also respond to OT with an immediate increase in cytosolic Ca2+ (unpublished observations). ULTR cells were grown to 80% confluence in Dulbecco's Modified Eagle's Medium (GIBCO, Rockville, MD) supplemented with 10% fetal bovine serum (Invitrogen, Eugene, OR) and 1% antimycotic (Invitrogen) at 37°C and 5% CO2-95% air. The PKC agonist TPA and antagonists BIM and calphostin C were obtained from Sigma-Aldrich (Oakville, ON, Canada).

With informed consent and the approval of the Human Research Ethics Board of the University of Alberta, human choriodecidual tissues were obtained immediately after delivery from healthy women having uncomplicated pregnancies at term delivering prior to labor onset (elective repeat cesarean section) or after spontaneous onset of labor without need for labor induction or augmentation. The amnion was separated from the choriodecidua, which was rinsed in cold normal saline and kept on ice until delivery to the laboratory for further use.

RNA extraction. After treatment, the cells were incubated with TRIzol (Invitrogen) for 5 min at room temperature. Chloroform (200 µl/ml TRIzol) was added, and after vigorous agitation followed by incubation on ice for 2–3 min, the samples were centrifuged at 14,000 g for 15 min at 4°C. The aqueous phase was removed, and 0.5 ml isopropyl alcohol/ml TRIzol was added. The samples were then incubated overnight at –20°C, thawed, and centrifuged at 14,000 g for 10 min at 4°C. The supernatant was removed and the pellet washed with 1 ml of 75% ethanol per milliliter of TRIzol and then centrifuged at 12,000 g for 5 min at 4°C, and the supernatant was removed. The pellet was dried and dissolved in 30 µl of Tris-EDTA. RNA purity was determined by optical density at 260 and 280 nm. RNA integrity was assessed using 1% agarose gel electrophoresis.

Reverse transcription-polymerase chain reaction. After quantification by spectrophotometer, 100 ng of RNA were reverse transcribed using a Taqman Reverse Transcription Reagent Kit (Applied Biosystems, Foster City, CA) with final solution containing 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor, and either 1.25 U/µl reverse transcriptase or sterile water (for negative controls) to a final volume of 20 µl. The mixture was incubated for 10 min at room temperature, for 45 min at 48°C, then for 5 min at 95°C.

The cDNA samples were mixed with PCR reagents (Applied Biosystems), and PCR amplification was performed using an iCycler (Bio-Rad, Montreal, QC, Canada). Each reaction contained 2 µl of cDNA, SYBR Green buffer, 3 mM MgCl2, 0.2 µM primers, 0.01 U/µl AmpErase, 0.025 U/µl AmpliTaq, 1 mM dNTP mix with dUTP, and sterilized water to a final volume of 50 µl. Each PCR cycle consisted of a denaturing phase at 95°C for 30 s and an annealing and extension phase at 65°C for 60 s. The primers used for OTR were sense 5'-GTACCCATCCAGCGACCAG-3' and antisense 5'-GCGAACCTAAAGTTGACTCCC-3'. Primers for the control gene hypoxanthine guanine phosphoribosyltransferase (HPRT) were sense 5'-GACTTTGCTTTCCTTGGTCAGG-3' and antisense 5'-AGTCTGGCTTATATCCAACACTTCG-3'. Efficiencies were calculated for each of the PCR reactions. The relative quantity of OTR mRNA in each sample, normalized for expression of HPRT, was calculated according to the "{Delta}-{Delta}" method described by Pfaffl (19).

Radioligand-binding assay. ULTR cells were grown to confluence in 12-well plates and washed twice with buffer (PBS containing 10 mM MgCl2 and 0.2% BSA, pH 7.4). To assess total OT binding, we used the high-affinity and selective OTR ligand ornithine vasotocin analog (OVTA; 125I-labeled OVTA; PerkinElmer/New England Nuclear, Cambridge, MA) 0.03 pmol added to 500 µl of OT solution to attain a final concentration of 10 nM OT to saturate OTR (Kd ~1 nM). Nonspecific binding was determined by preaddition of OT (10 µM). Cells were incubated for 1 h at room temperature, and then excess OT was removed by washing three times with ice-cold PBS. To maximize counting efficiency, cells were lysed with 0.25 ml of 1 N NaOH per well, and radioactivity was measured in 2 ml of scintillation cocktail (Scintisafe Plus 50%; Fisher Scientific, Ottawa, ON, Canada) using the Beckman Scintillation System, LS 5000 TD (Beckman-Coulter, Fullerton, CA). Specific binding was calculated by subtracting the nonspecific binding from the total binding.

Adenoviral infection. The adenoviral constructs (Ad-c-Jun, Ad-c-Fos, Ad-tTA) have been described previously (32). The vectors were propagated to the following titers: the infection control vector expressing green fluorescent protein (Ad-GFP, 8.1 x 109 pfu/ml); the control containing only the tetracycline-regulated promoter (Ad-tTA, 1.06 x 1010 pfu/ml); the vector expressing c-Jun (Ad-c-Jun, 3.2 x 1010 pfu/ml); and the vector expressing c-Fos (Ad-c-Fos, 5.15 x 108 pfu/ml). In preliminary experiments, we determined that ULTR cells were optimally infected when exposed to a multiplicity of infection (or pfu/cell) of 30–40 for 6 h. There was no evidence of cell toxicity from the viral infections. The infected cells were washed and examined using immunofluorescence and Western immunoblotting over a time course of 24–72 h to allow for gene expression.

Immunofluorescence. The primary antibody for phosphorylated c-Jun was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at dilution of 1:500. Cells were grown overnight on eight-chamber slides. After treatment, cells were fixed with 3.7% formaldehyde and 0.5% Triton X-100 in PBS for 15 min. Cells were then washed with PBS, exposed to blocking buffer (5% BSA in PBS) for 30 min, and incubated with primary antibody overnight. After being washed three times, cells were exposed to the secondary antibody for 30 min in the dark (1:200, anti-mouse FITC conjugated; Sigma Aldrich, St. Louis, MO). Chambers were removed, and ~60 µl of 4,6-diamidino-2-phenylindole fixation solution were spotted onto the slide. Coverslips were secured with nail polish, and the slide was left to dry in the dark for ~15 min and then examined using a fluorescence microscope.

Western immunoblots. Total cellular protein was extracted with 100 µl/well of sodium dodecyl sulfate (SDS) lysis buffer containing 62.5 mM TrisHCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol (DTT), and 0.01% wt/vol bromophenol blue. Samples were sonicated for 10 s and stored at –80°C until used. At the time of analysis, 20 µg of protein were mixed with 5x sample buffer, boiled for 5 min, and run on a 10% denaturing polyacrylamide gel. The gel was transferred to a nitrocellulose membrane, which was then washed in 5% skim milk powder in TBS-0.1% Tween for 1 h. The primary antibodies for phospho-c-Jun (1:200; Cell Signaling Technology, Beverly, MA) and c-Fos (1:800; Oncogene Science, Cambridge, MA) were incubated overnight at 4°C. After washing three times, membranes were exposed for 1 h to the HRP-conjugated secondary antibody (Santa Cruz Biotechnology; for phospho-c-Jun, 1:3,500 of goat anti-rabbit; for c-Fos, 1:5,000 of goat anti-mouse). The membrane was then rinsed with TBS-0.15% Tween several times and exposed using ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were exposed on Super RX Fuji Medical X-Ray film for ~5–30 min.

Nuclear extraction. Nuclear proteins were extracted as described previously (28). Cells were washed two to three times in ice-cold PBS, scraped into 1.5-ml microfuge tubes, and then centrifuged at 1,500 g for 3 min at 4°C. The pellet was gently resuspended in 400 µl of ice-cold buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF for 15 min at 4°C. Twenty-five microliters of Nonidet 10% NP-40 was added. The homogenate was centrifuged for 30 s at 14,000 g, and the pellet was resuspended in ~50 µl of ice-cold buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, and vigorously rocked for 15 min at 4°C. After centrifugation for 5 min at 12,000 g, the supernatant was stored at –80°C.

Enzyme-linked immunoassay. The TransAM AP-1 p-cJun ELISA-based kit (ActiveMotif, Carlsbad, CA) was used for measuring binding of nuclear phosphorylated c-Jun protein to its oligonucleotide consensus sequence. This sequence was preadhered to the 96-well plate, and nuclear extracts were exposed to the wells. Binding was then measured according to the manufacturer's instructions, using a simple colorimetric assay.

Statistics. ELISA data were analyzed using a paired t-test. PCR and OT binding data were normalized to their appropriate time controls and analyzed using ANOVA followed by a Tukey-Kramer multiple comparisons test if P ≤ 0.05. Numbers of experiments are noted in the figure legends. Significance was defined as P ≤ 0.05. All data are expressed in the text and figures as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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In preliminary studies, we determined the effect of withdrawal of serum from the media for 24 h prior to treatment with TPA. Serum-deprived cells had lower concentrations of mRNA for OTR and lower OT binding. They also appeared thinner and generally less healthy. The response to TPA was similar qualitatively and quantitatively regardless of the presence or absence of serum; we therefore chose to continue serum supplementation throughout the experiments. Treatment of ULTR cells with TPA caused a rapid increase in expression of phosphorylated c-Jun, demonstrated by immunofluorescence, and increases in c-Fos and c-Jun assessed using Western analyses (Fig. 1, A and B). There also was increased AP-1 binding to its consensus sequence as demonstrated using the ELISA technique (Fig. 1C). This stimulation of AP-1 activity was followed by a decrease in mRNA for OTR, which was apparent by 6 h, and a decrease in OT binding, which was significant after 24 h (Fig. 2).


Figure 1
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  		Fig. 1. Treatment of ULTR cells with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 ng/ml) for 0.5 h caused an increase in expression of c-Jun and c-Fos and an increase in nuclear binding to the activating protein-1 (AP-1) consensus response element. A: immunofluorescence demonstrating phosphorylated (p-)c-Jun in control (i) and treated (ii) cells with respective controls that received 4,6-diamidino-2-phenylindole (DAPI) nuclear stain but no primary antibody (iii and iv). Increased p-c-Jun appears to be associated with the nucleus. Micrographs are representative of 3 such experiments. B: Western analyses (representative of 3 such experiments) show an increase in phosphorylated and nonphosphorylated c-Jun with peak levels by 30 min after treatment. The increase in c-Fos peaked at 15 min. C: ELISA showing severalfold increase in binding to consensus AP-1 response elements after TPA treatment for 0.5 h; n = 4 experiments. *P ≤ 0.05 by unpaired t-test.

 

Figure 2
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  		Fig. 2. Treatment of ULTR cells caused a significant decline in mRNA for oxytocin receptors (OTR; filled bars) as well as OT binding (gray bars); n = 6–10 experiments at each time point. Each time point has been normalized to control incubations, which are 1.0. Decline in mRNA was maximal by 6 h of treatment and decrease in OT binding at 24 h. *P ≤ 0.05, **P ≤ 0.01 vs. control values by 1-way ANOVA.

 
To confirm that the changes in OTR expression were secondary to stimulation of PKC, these experiments were repeated in the presence of two PKC inhibitors. Surprisingly, neither calphostin C nor BIM had any inhibitory effects on the TPA-induced suppression of OTR expression (Fig. 3).


Figure 3
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  		Fig. 3. Pretreatment of ULTR cells with PKC inhibitors bisindolylmaleimide hydrochloride (BIM, 50 nM) or calphostin C (CAL, 10 nM) had no effect on TPA-induced (100 ng/ml or 162 nM) suppression of mRNA for OTR (filled bars) or OT binding (gray bars); n = 6 in each group. The 12-h time point is illustrated for mRNA and the 24-h time point for OT binding, as these were the times of maximal suppression. *P ≤ 0.001 vs. control groups by 1-way ANOVA.

 
To determine whether the increase in AP-1 activity mediated the suppression of OTR, we infected ULTR cells with adenovirus expression vectors for c-Jun and c-Fos as well as GFP as an infection control. These infections resulted in increased phosphorylated c-Jun in the nucleus and increased cellular c-Jun and c-Fos protein that was evident by 24 h and continued to increase for up to 72 h (Fig. 4). Despite this, there was no change in concentrations of OTR mRNA throughout this time.


Figure 4
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  		Fig. 4. Infection of ULTR cells with adenovirus expression plasmids resulted in an increase in c-Jun and c-Fos expression without changes in mRNA for OTR. A: infection with Ad-c-Jun plasmid resulted in increased immunofluorescence for p-c-Jun (ii) vs. infection with plasmid containing empty plasmid (i). iii and iv: DAPI nuclear stained controls for i and ii, respectively. B: Western analyses after infection of ULTR cells for 24, 48, or 72 h using expression plasmids for c-Jun (Ad-c-Jun), c-Fos (Ad-c-Fos), or empty vector (Ad-tTA) with multiplicities of infection as indicated. Infections resulted in increases in c-Jun and c-Fos, as expected. Data from A and B are representative of 4 such validation experiments. C: There were no changes in relative amounts of mRNA for OTR at 48 h after infection with Ad-c-Jun (left) or both Ad-c-Jun and Ad-c-Fos (right) vs. controls in the absence of infection or with plasmid containing GFP only. Four experiments were performed and data analyzed using 1-way ANOVA.

 
Relevant uterine myocytes from the fundal portion of the uterus are difficult to obtain from human pregnancies. However, the choriodecidual layer of the fetal membranes lies immediately adjacent to the myometrium, and this readily available tissue is subject to the same endocrine/paracrine influences as the neighboring myocytes. We reasoned that, if AP-1 subunits were important in regulation of human uterine myocyte activation, there might be detectable changes associated with labor onset in their concentrations in choriodecidual tissues. By use of Western analyses of separated cytosolic and nuclear subfractions of the choriodecidual membrane, the concentration of c-Fos in cytosol was significantly reduced in uterine tissues obtained after labor onset (108.8 ± 16.4 optical density units) compared with tissues collected prior to labor (153.4 ± 8.9, P = 0.028; Fig. 5). There were no detectable changes in c-Fos or c-Jun concentrations in the nuclear fractions of these tissues around the time of labor onset.


Figure 5
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  		Fig. 5. Data from Western analyses showing relative concentration of c-Jun (left) and c-Fos (right) in cytosolic or nuclear compartments of cells from human choriodecidual tissues collected at term pregnancy either before (filled bars; n = 10) or after (gray bars; n = 10) spontaneous onset of labor. There were no significant changes except for a slight decrease in cytosolic c-Fos around the time of labor onset. *P ≤ 0.05 between before- and after-labor onset groups by unpaired t-test.

 
To confirm that the PKC antagonists did inhibit PKC activity, we assessed the concentrations of AP-1 in the nuclear extracts by use of the ELISA assay after treatment of ULTR cells with TPA and the inhibitors calphostin C and BIM. TPA treatment increased AP-1 concentrations, but, to our surprise, there was no significant effect of calphostin C or BIS on either basal or TPA-stimulated concentrations of AP-1 (Fig. 6).


Figure 6
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  		Fig. 6. Concentrations of phospho-c-Jun in nuclear extracts from ULTR cells treated with PKC antagonists BIM (50 nM) CAL (10 nM), or TPA; n = 5 in each group. *P ≤ 0.05 vs. control incubations by 1-way ANOVA. There were no significant effects of BIM or CAL on either control or TPA-treated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Treatment of human myometrium-derived ULTR cells with phorbol ester caused a significant increase in nuclear concentrations of Jun and Fos. This was followed by a clear and significant decrease in content of mRNA for OTR and in OT binding ability of the cells. The time course suggests an initial decrease in transcription of mRNA for OTR followed by a decrease in translation of OTR protein. Although we considered that the decreased expression of OTR was mediated through the increase in concentrations of AP-1, it appears that this was not the case, since there was no effect on OTR expression by increased concentrations of AP-1 following transfection with Jun and Fos expression plasmids.

The AP-1 binding sites in the OTR promoter are located at –870 and –880 nucleotides from the transcription start site. Their function, whether repressive or stimulatory, is unknown. However, we have noted that deletion of this segment of the promoter region nullifies the inhibitory effects of proinflammatory cytokines on OTR expression in transfected HeLa cells (27). This would be in keeping with an inhibitory effect of AP-1. In contrast, in a mammary tumor cell line (Hs578T), transfection with c-Fos/c-Jun expression plasmids, along with GABP{alpha}/beta expression plasmids, resulted in a 10-fold increase in expression of an OTR promoter-reporter construct (10). The differences in the responses between the ULTR and Hs578T cells may reflect simple differences in OTR regulation in different cells.

There are limited choices for model systems to study molecular mechanisms that regulate human uterine myocyte physiology. Uterine myocytes from the fundal region of the uterus of pregnant women are not readily available. Myocytes from the lower segment, obtained at the time of cesarean section, may not reflect the phenotype of the more relevant cells in the contractile fundus. For convenience and relevance, we chose the ULTR cell line, which was derived from human myometrial cells that were transformed using a recombinant retroviral construct containing the E6 and E7 open reading frames of the human papillomavirus type 16. We have found these cells to have similar morphological characteristics to primary human uterine myocytes in culture, and they express all genes that we have studied relevant to human myometrium, including receptors for OT, prostaglandin F2{alpha}, estrogen ({alpha}-isoform), and progesterone. Thus we believe that our findings are relevant to the human uterine myocyte.

There is considerable controversy regarding the role of PKC in contractile function of the uterine myocyte. Several studies using myographic techniques with strips of myometrium have explored the role of PKC on contractile activity. Savineau and Mironneau (26) demonstrated that phorbol ester increased the contractile response of rat myometrium to an electrical stimulus. However, the effects on K+- and OT-induced contractions were more complex. At lower concentrations of phorbol ester (100 nM), the effect was stimulatory for the first 20 min. At higher concentrations or for longer durations, the effects were inhibitory. The inhibitory effects were attenuated with the PKC antagonist H7. However, this antagonist is equipotent at inhibiting cAMP- and cGMP-dependent protein kinases, and its effects cannot be assumed to be due to inhibition of PKC alone (9). Phillippe (22) showed that phorbol ester at concentrations greater than 200 nM suppressed the response of rat myometrial strips to OT. However, at concentrations less than 100 nM, the effects were less clear. Pharmacological approaches to increase endogenous DAG concentrations also caused inhibition of OT-induced contractions in rat myometrium, and the effect was markedly increased in tissues obtained from late-pregnant compared with nonpregnant animals (4). High doses of phorbol ester suppressed rat myometrial tension induced by a high K+ solution but only one-half of this was blocked using a PKC inhibitor (13). Conversely, using human myometrial tissues phorbol ester treatment (1 µM) caused a significant stimulation of spontaneous and K+-induced contractility, and again this effect was much greater in tissues from pregnant compared with nonpregnant women (17). This augmented activity in tissues from pregnant women was completely attenuated by treatment with PKC inhibitors. In summary, the effect of phorbol ester treatment in vitro on myometrial contractile activity may be species specific and appears to be dependent on dose and time of exposure.

The molecular mechanism mediating the observed decrease in OTR mRNA and protein remains unclear. Prolonged exposure to TPA can deplete cells of PKC (31), but it is unlikely that this would have such a significant effect on OTR by 6 h. It has been demonstrated that downregulation of OTR caused by prolonged exposure to OT in uterine myocytes is accompanied by a decrease in mRNA for OTR (20). Phosphorylation of OTR by PKC or by PKC-activated G protein-related kinases could target OTR for internalization and breakdown (24). This mechanism is unlikely to be responsible for the decrease in OTR in the present studies, since the decrease in OTR binding was preceded by a decrease in mRNA for OTR. Recently, it has become clear that phorbol ester treatment can stimulate a variety of other kinases that contain the C1 DAG binding site. These kinases include the RasGRPs that activate the MAPK pathway, protein kinase D, the chimaerin pathways, and others (1, 5). Our data suggest that one of these non-PKC pathways may be at least partially responsible for the inhibitory effects of phorbol ester treatment on OTR expression in human uterine myocytes.

Our experiments using the PKC antagonists calphostin C and BIM were designed to confirm the role of PKC in the suppression of OTR. The doses that we used (5–50 nM) were based on earlier work in cultured rat uterine myocytes that demonstrated significant inhibition of serotonin-induced increases in matrix metalloproteinase-13 synthesis (30). We were surprised that neither of these PKC antagonists decreased basal or TPA-stimulated nuclear concentrations of AP-1. Subsequent studies in the literature have used manyfold higher concentrations (0.5–1 µM) of these antagonists to inhibit PKC activity in uterine myocytes (12, 17, 29). Unfortunately, our experiments using the PKC antagonists do not help to decipher the role of PKC in the suppression of OTR.

The time course of our Fos/Jun overexpression experiments was designed to allow for maximal cell infection (48–72 h) and sufficient time for the AP-1 transcription factor to influence genomic mechanisms. Because the expression of Fos and Jun appeared near maximal by 48 h and the maximal effect on inhibition of mRNA for OTR occurred within 6 h of the initial increase in these subunits (Figs. 1 and 2), we chose the 48-h time point at which to assess the effects of Fos/Jun overexpression on mRNA for OTR. The lack of effect of overexpression of c-Fos and c-Jun suggests that AP-1 activity is not rate limiting, although it may still be requisite in regulation of uterine myocyte expression of OTR. However, it is unknown whether pharmacological stimulation of PKC by treating uterine myocytes with phorbol ester mimics the physiological effects induced by stimulation of GPCRs. Previous studies have shown a close temporal relation between AP-1 expression and appearance of the contraction-associated proteins that are part of the process of uterine activation prior to parturition in rat (16, 23) and human (6) myometrium.

In myometrial tissue from the lower uterine segment obtained at the time of cesarean section, expression of myometrial c-jun appears to increase in human pregnancy and increase further at the time of parturition (8, 25). However, the critical changes in the uterine myocytes that precede parturition may be very different in the lower uterine segment, which relaxes during labor, and the contractile uterine fundus. Because the human decidua is also part of the uterine activation process prior to parturition, and because we cannot obtain human fundal uterine myocytes during pregnancy, we attempted to detect significant changes in expression of the AP-1 subunits in the human choriodecidual tissues around the time of spontaneous labor onset. We question whether the observed 30% reduction in cytosolic Fos, as measured by Western analysis, represents a biologically relevant change. The lack of significant changes in choriodecidual nuclear Fos and Jun fails to support a role for AP-1 in human uterine activation. However, these data may only reflect the choriodecidual tissue and may not apply to the myometrium, which may have different regulatory mechanisms. Clearly, more direct observations are required before such conclusions can be made.

In summary, these studies have demonstrated a significant decline in mRNA for OTR and in OT binding capacity in human uterine myocytes after treatment with phorbol ester. However, these effects were not duplicated by overexpression of AP-1 subunits. We conclude that phorbol ester-induced suppression of OTR is not caused by AP-1 activation but may be mediated by other PKC-induced pathways or by alternate targets for phorbol esters. Future studies examining other potential pathways or downstream targets could reveal novel mechanisms of OTR regulation. Further studies are required to clarify the controversial role of the PKC isoform pathways in uterine function.


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 ABSTRACT
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This research was supported by the Institute of Human Development, Child and Youth Health of the Canadian Institutes of Health Research and by the University of Alberta Faculty of Medicine and Dentistry.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the gift of the ULTR cell line from Dr. J. MacDougall of the Fred Hutchinson Cancer Research Center in Seattle, WA. We also are grateful for the assistance of the Perinatal Research Centre and the medical and nursing staff of the Royal Alexandra Hospital Obstetrical Unit in Edmonton, AB, Canada, for collection of the human tissue samples.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. F. Mitchell, Perinatal Research Centre, 220 HMRC, Univ. of Alberta, Edmonton, Canada T6G 2S2 (e-mail: brymitch{at}ualberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alfaidy N, Li W, MacIntosh T, Yang K, and Challis J. Late gestation increase in 11beta-hydroxysteroid dehydrogenase 1 expression in human fetal membranes: a novel intrauterine source of cortisol. J Clin Endocrinol Metab 88: 5033–5038, 2003.[Abstract/Free Full Text]
  2. Ali H, Fisher I, Haribabu B, Richardson RM, and Snyderman R. Role of phospholipase Cbeta3 phosphorylation in the desensitization of cellular responses to platelet-activating factor. J Biol Chem 272: 11706–11709, 1997.[Abstract/Free Full Text]
  3. Angel P and Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072: 129–157, 1991.[Medline]
  4. Ascher-Landsberg J, Saunders T, and Phillippe M. The role of diacylglycerol as a modulator of oxytocin-stimulated phasic contractions in myometrium from pregnant and nonpregnant rats. Am J Obstet Gynecol 182: 943–949, 2000.[CrossRef][Web of Science][Medline]
  5. Brose N and Rosenmund C. Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters. J Cell Sci 115: 4399–4411, 2002.[Abstract/Free Full Text]
  6. Geimonen E, Jiang W, Ali M, Fishman GI, Garfield RE, and Andersen J. Activation of protein kinase C in human uterine smooth muscle induces connexin-43 gene transcription through an AP-1 site in the promoter sequence. J Biol Chem 271: 23667–23674, 1996.[Abstract/Free Full Text]
  7. Gimpl G and Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81: 629–683, 2001.[Abstract/Free Full Text]
  8. Gustavsson I, Englund K, Faxen M, Sjoblom P, Lindblom B, and Blanck A. Tissue differences but limited sex steroid responsiveness of c-fos and c-jun in human fibroids and myometrium. Mol Hum Reprod 6: 55–59, 2000.[Abstract/Free Full Text]
  9. Hidaka H, Inagaki M, Kawamoto S, and Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036–5041, 1984.[CrossRef][Medline]
  10. Hoare S, Copland JA, Wood TG, Jeng YJ, Izban MG, and Soloff MS. Identification of a GABP alpha/beta binding site involved in the induction of oxytocin receptor gene expression in human breast cells, potentiation by c-Fos/c-Jun. Endocrinology 140: 2268–2279, 1999.[Abstract/Free Full Text]
  11. Hurd WW, Fomin VP, Natarajan V, Brown HL, Bigsby RM, and Singh DM. Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium. Am J Obstet Gynecol 183: 1525–1531, 2000.[CrossRef][Web of Science][Medline]
  12. Karteris E, Hillhouse EW, and Grammatopoulos D. Urocortin II is expressed in human pregnant myometrial cells and regulates myosin light chain phosphorylation: potential role of the type-2 corticotropin-releasing hormone receptor in the control of myometrial contractility. Endocrinology 145: 890–900, 2004.[CrossRef][Web of Science][Medline]
  13. Kim B, Kim YS, Ahn J, Kim J, Cho S, Won KJ, Ozaki H, Karaki H, and Lee SM. Conventional-type protein kinase C contributes to phorbol ester-induced inhibition of rat myometrial tension. Br J Pharmacol 139: 408–414, 2003.[CrossRef][Web of Science][Medline]
  14. Kim HP, Lee JY, Jeong JK, Bae SW, Lee HK, and Jo I. Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae. Biochem Biophys Res Commun 263: 257–262, 1999.[CrossRef][Web of Science][Medline]
  15. Mitchell BF and Schmid B. Oxytocin and its receptor in the process of parturition. J Soc Gynecol Investig 8: 122–133, 2001.[CrossRef][Web of Science][Medline]
  16. Mitchell JA and Lye SJ. Differential expression of activator protein-1 transcription factors in pregnant rat myometrium. Biol Reprod 67: 240–246, 2002.[Abstract/Free Full Text]
  17. Ozaki H, Yasuda K, Kim YS, Egawa M, Kanzaki H, Nakazawa H, Hori M, Seto M, and Karaki H. Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestation. Br J Pharmacol 140: 1303–1312, 2003.[CrossRef][Web of Science][Medline]
  18. Perez-Reyes N, Halbert CL, Smith PP, Benditt EP, and McDougall JK. Immortalization of primary human smooth muscle cells. Proc Natl Acad Sci USA 89: 1224–1228, 1992.[Abstract/Free Full Text]
  19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45, 2001.[Abstract/Free Full Text]
  20. Phaneuf S, Asboth G, Carrasco MP, Europe-Finner GN, Saji F, Kimura T, Harris A, and Lopez Bernal A. The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA. J Endocrinol 154: 7–18, 1997.[Abstract/Free Full Text]
  21. Phaneuf S, Carrasco MP, Europe-Finner GN, Hamilton CH, and Lopez Bernal A. Multiple G proteins and phospholipase C isoforms in human myometrial cells: implication for oxytocin action. J Clin Endocrinol Metab 81: 2098–2103, 1996.[Abstract]
  22. Phillippe M. Protein kinase C, an inhibitor of oxytocin-stimulated phasic myometrial contractions. Biol Reprod 50: 855–859, 1994.[Abstract]
  23. Piersanti M and Lye SJ. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activiator protein-1, c-fos. Endocrinology 136: 3571–3578, 1995.[Abstract]
  24. Plested CP and Bernal AL. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium. Exp Physiol 86: 303–312, 2001.[Abstract]
  25. Roh CR, Lee BL, Oh WJ, Whang JD, Choi DS, Yoon BK, and Lee JH. Induction of c-Jun mRNA without changes of estrogen and progesterone receptor expression in myometrium during human labor. J Korean Med Sci 14: 552–558, 1999.[Web of Science][Medline]
  26. Savineau JP and Mironneau J. An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle. J Pharmacol Exp Ther 255: 133–139, 1990.[Abstract/Free Full Text]
  27. Schmid B, Wong S, and Mitchell BF. Transcriptional regulation of oxytocin receptor by interleukin-1beta and interleukin-6. Endocrinology 142: 1380–1385., 2001.[Abstract/Free Full Text]
  28. Schreiber E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with "mini-extracts", prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989.[Free Full Text]
  29. Shlykov SG and Sanborn BM. Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells. Cell Calcium 36: 157–164, 2004.[CrossRef][Web of Science][Medline]
  30. Shum JK, Melendez JA, and Jeffrey JJ. Serotonin-induced MMP-13 production is mediated via phospholipase C, protein kinase C, and ERK1/2 in rat uterine smooth muscle cells. J Biol Chem 277: 42830–42840, 2002.[Abstract/Free Full Text]
  31. Szallasi Z, Smith CB, and Blumberg PM. Dissociation of phorbol esters leads to immediate redistribution to the cytosol of protein kinases C alpha and C delta in mouse keratinocytes. J Biol Chem 269: 27159–27162, 1994.[Abstract/Free Full Text]
  32. Wang N, Verna L, Hardy S, Zhu Y, Ma KS, Birrer MJ, and Stemerman MB. c-Jun triggers apoptosis in human vascular endothelial cells. Circ Res 85: 387–393, 1999.[Abstract/Free Full Text]
  33. Yue C, Ku CY, Liu M, Simon MI, and Sanborn BM. Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C. J Biol Chem 275: 30220–30225, 2000.[Abstract/Free Full Text]




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