A critical role for PKC{zeta} in endothelin-1-induced uterine contractions at the end of pregnancy

G. Di Liberto,1 E. Dallot,1 I. Eude-Le Parco,1 D. Cabrol,2 F. Ferré,1 and M. Breuiller-Fouché1

1Institut National de la Santé et de la Recherche Médicale (INSERM) U361, Université René Descartes, Pavillon Baudelocque, and 2Maternité Port-Royal, Hôpital Cochin, 75014 Paris, France

Submitted 28 January 2003 ; accepted in final form 30 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that protein kinase C (PKC) {zeta} and/or PKC{delta} are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567–1573, 2000). Here, we report that the selective inhibitor of PKC{delta} isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKC{zeta} or with an antisense against PKC{zeta} resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKC{zeta} to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKC{zeta} has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKC{zeta} plays a role in myometrial contraction in pregnant women.

protein kinase C; uterine smooth muscle; parturition


ALTHOUGH BEST RECOGNIZED as a potent vasoconstrictor, endothelin-1 (ET-1) can exert a wide spectrum of biological actions in many different tissues, including uterine smooth muscle. Like other uterotonic agents, ET-1 is effective for stimulating the force and frequency of human myometrial contractions in vitro. The uterine contractile responsiveness to ET-1 is markedly increased at the end of pregnancy (44) and is accompanied by an elevation of the density of myometrial ETA receptors functionally coupled to the phospholipase C (PLC)/calcium system (20, 34).

Activation of the polyphosphoinositide-signaling cascade plays a key role during generation of ET-1-stimulated myometrial contractions. Among the multigene family of serine/threonine kinases, protein kinase C (PKC) has been shown to participate in the regulation of uterine smooth muscle contraction. Three classes of PKC isoforms have been defined: conventional PKC isoforms (cPKC) consisting of {alpha}, {beta}1, {beta}2, and {gamma}; novel PKC isoforms (nPKC) including {delta}, {epsilon},, {eta},{theta}, and {gamma}; and atypical PKC isoforms comprising {zeta}, {lambda}, and {tau}. Atypical PKC (aPKC) are distinct from other members of the PKC family in that they are neither activated by calcium nor by diacylglycerol or phorbol esters but are regulated by other phospholipidic mediators (32). So far, little is known about the physiological activators of aPKC. PKC{zeta} was thought to act downstream of phosphatidylinositol 3-kinase (PI3 kinase). Furthermore, the products of PI3 kinase (phosphatidylinositol 3,4-biphosphate and phosphatidylinositol 3,4,5-triphosphate) stimulate autophosphorylation of PKC{zeta} (31).

We previously demonstrated that among the six isoforms of PKC ({alpha}, {beta}1, {beta}2, {delta}, {epsilon}, and {zeta}) found in the pregnant human myometrium, only PKC{alpha} is required for ET-1-induced proliferation of human myometrial cells (12). There is indeed some evidence that PKC{delta} and/or PKC{zeta} is necessary for ET-1-induced myometrial contractions at the end of pregnancy (13). However, few biological functions have been defined in smooth muscle for these particular PKC isoforms. Additionally, previous studies in vascular smooth muscle documented that PKC{delta} is involved in the contractile potentiation of ET-1 in the porcine coronary artery (33), whereas PKC{zeta} has been described as a possible partner in the sustained phase of the contraction of the bovine carotid arterial smooth muscle (40).

In the present study, we have attempted to individualize the PKC isoform able to selectively elicit uterine contraction at the end of pregnancy. To elucidate the contribution of isoforms of PKC{delta} and {zeta} in human myometrial contractility, we first studied the role of each PKC isoforms in the contractile process by using a selective inhibitor of PKC{delta} (rottlerin) (18) and a pharmacological inhibitor of PI3 kinase (LY-294002) (42).

We extended the analysis to a complementary model of cultured myometrial cells that could be utilized for identifying potential regulatory pathways that modulate normal uterine activity. However, whether human myometrial cells preserve their contractile properties in culture remains uncertain. We previously demonstrated by two in vitro systems that subcultured myometrial cells preserve a "contractile" phenotype (6). First, myometrial cells distorted a silicone rubber sheet in response to ET-1. Additionally, we confirmed that ET-1 via selective activation of the ETA receptor was able to modify myometrial cell tension in a conventional collagen gel retraction assay. When myometrial cells were incorporated into collagen lattices, the cumulative contraction of the cells may have pulled the collagen fibers together, causing a decrease in lattice area. Moreover, the regular arrangement of stress fibers seemed to be indispensable for coordinated contraction. This technique was therefore utilized to assess the contractility of cultured human myometrial cells. To elucidate the precise contribution of the protein kinase pathways in myometrial cell contraction, we examined the effects of PKC inhibitors with different profiles of selectivity and a myristoylated peptide corresponding to part of the pseudosubstrate region of PKC{zeta} (peptide Z) (11). An additional approach has consisted in targeting PKC{zeta} by an antisense oligonucleotide (AS-ODN).

Recent interest has focused on the implication of PKC as an important regulator of the cytoskeletal function. In an abundant literature, activation of PKC has been shown to result in cytoskeletal rearrangement in association with the phosphorylation of different cytoskeleton components (for a review, see Ref. 22). Last, we used a confocal microscopy approach to investigate whether the potent uterotonic agent ET-1 regulates cytoskeletal actin organization in myometrial cells and, if so, the potential role of PKC{zeta} was questioned in this process.

These different approaches provide the first evidence that only PKC{zeta} activation mediates ET-1-induced myometrial contraction and, furthermore, that ET-1 is able to induce the translocation and the association of PKC{zeta} to a structure coincident with actin cytoskeleton.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and biological samples. Biopsies of myometrium were obtained from 15 pregnant women who presented normal, uncomplicated pregnancies but were delivered by elective caesarean section performed before the onset of labor (range 38.5–40 wk gestation) because of diagnosed cephalopelvic disproportion. Myometrial strips were taken from the upper edge of the hysterotomy in the transverse lower uterine segment during caesarean section. Myometrial tissue from the outer uterine wall at an extraplacental site was removed by sharp dissection, leaving behind the decidua, and then carefully minced with fine scissors and immediately placed on ice. Written informed consent was obtained from all donors. This study was approved by the Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale (Paris-Cochin, France).

Chemicals. Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, penicillin-streptomycin mixture, phosphate-buffered saline (PBS) with and without calcium, and fetal calf serum (FCS) were supplied by In Vitrogen Life Technologies (Cergy-Pontoise, France). Collagen was purchased from Becton Dickinson Biosciences (Bedford, MA). ET-1 was from Neosystem Laboratories (Strasbourg, France). Gö-6976, rottlerin, and peptide Z were from Calbiochem (La Jolla, CA). LY-294002 and phorbol dibutyrate (PDBu) were from Sigma-Aldrich (St Louis, MO). Electrophoresis reagents were obtained from Bio-Rad (Richmond, CA). Hybond-C membranes, enhanced chemiluminescence detection system (ECL), and X-ray film were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK). Rabbit antibody against PKC{zeta} was from Sigma-Aldrich and raised against peptides 577–592. The antibody against smooth muscle {alpha}-actin was from Novocastra (Newcastle, UK). A donkey anti-rabbit IgG conjugated to horseradish peroxidase was from Amersham International. Tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin conjugate was from Sigma-Aldrich. The rhodamine isothiocyanate-labeled conjugated (RITC) antibody against mouse IgG1 and the fluorescein isothiocyanate-labeled conjugated (FITC) antibody against mouse IgG were from Southern Biotechnology Associates (Birmingham, UK), and the RITC antibody goat anti-rabbit IgG1 was from Tebu (France). Alexa Fluor 488 donkey anti-rabbit IgG was from Interchim (France). The fluorescent mounting medium was obtained from DAKO (Carpinteria, CA). Leupeptin, Nonidet-P40, phenylmethylsulfonyl fluoride (PMSF), and other drugs and chemicals used were of the highest quality available from Sigma-Aldrich.

In vitro contractile studies. Myometrial segments (8–12 x 3 x 2 mm) were suspended in parallel for isometric tension recordings using Bioscience UF1 tension transducers (Marty Technologie, Marcilly sur Eure, France), in 5-ml organ baths containing aerated (95% O2-5% CO2) Krebs buffer (in mM: 11.1 glucose, 6.2 KCl, 144 NaCl, 2.5 CaCl2, 0.5 MgCl2, 1 NaH2PO4, 30 NaHCO3) maintained at 37°C. A resting tension of 600 mg was applied to each segment, and a spontaneous tone was allowed to develop. As previously described (13), the myometrial strips were allowed to equilibration for 2 h until spontaneous contractions became regular in frequency and intensity. Muscle strips, which did not develop spontaneous contractions at this stage, were discarded. After equilibration, ET-1 was applied as a single dose. Rottlerin (inhibitor of PKC{delta}) and LY-294002 (inhibitor of PI3 kinase) were added to the bathing medium 30 min before the addition of ET-1. We verified that pretreatment with rottlerin and LY-294002 did not alter the spontaneous contractions. PDBu was added to the bath 4 h before the addition of ET-1. Measurements were processed by MacLab/8e software package (AD Instruments). The area under the tension curve was measured for a given time (contraction area). Results are expressed as grams per 5 or 20 min.

Myometrial cell culture. After collection, the myometrial biopsies were placed in DMEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Human myometrial cells were obtained by the explants method as previously described (4). Cells were cultured in DMEM supplemented with antibiotic solution and 10% FCS and routinely passaged when 90–95% of the cells were confluent. The experiments presented in this report were performed with cells between their fourth to fifth passages, with no noticeable difference between results obtained with cells from individual passages and with cells obtained from different uteri. Each population of myometrial cells studied had been taken from a different patient. Confluent myometrial cells were identified by their typical "hill and valley" microscopic appearance and their positive reaction to a monoclonal antibody against smooth muscle {alpha}-actin.

Oligonucleotide treatments. Anti-sense phosphothiorate PKC{zeta} oligonucleotide (AS-ODN) and scrambled PKC{zeta} oligonucleotide (S-ODN) resembling each other in size and G/C base content were designed and manufactured by Biognostik (Göttingen, Germany) and used to treat cells essentially as described by Schlingensiepen et al. (36). Two sequences of the AS-ODN used, AS-1 ODN (5'-CGTCCTCGTTCTTG-3') and AS-2 ODN (5'-TCTCCCCGCAGGATTTCG-3'), designed to specifically hybridize human PKC{zeta} mRNA, were complementary to positions 587–601 and 1260–1278 of the PKC{zeta} mRNA, respectively (2). The S-ODN with the same base composition but a randomized sequence was used as a control sense. Stock solutions (100 µM) were prepared in Tris-EDTA buffer (pH 7.2) and stored at –20°C. They were diluted with culture medium immediately before use to give a final concentration of 4 µM. Uptake into the cells was monitored using fluorescein-labeled oligonucleotide; uptake into the cells was uniform after 8 h. Comparable results were obtained with one or two applications of S-ODN and AS-ODN. Consequently, all experiments were performed with one application of oligonucleotide. Myometrial cells cultured in DMEM containing 10% FCS for 24 h were then exposed to either 4 µM S-ODN or increasing concentrations of AS-ODN against PKC{zeta} for 48 h. The cells were then washed and transferred to serum-free medium for 72 h.

Preparation of three-dimensional hydrated collagen lattices. When human pregnant myometrial cells were included into collagen lattices as recently described by Dallot et al.,(6), their cumulative contraction pulled the collagen fibers into a dense arrangement and decreased lattices areas. Briefly, confluent cells harvested with 0.05% trypsin-0.02% EDTA were centrifuged at 1,000 g for 5 min and resuspended in 10% FCS-DMEM at the required cell density. A type I collagen solution (4.1 mg/ml in 0.1 N HCl) was adjusted to pH 7.2 with 0.1 N NaOH. The final concentration of collagen was 1.5 mg/ml. The appropriate concentration of myometrial cells (150,000 cells/well) was then added to the neutralized collagen solution. Collagen gel-cell suspensions were incubated in 35-mm-diameter untreated culture dishes for 2 h at 37°C to allow gelling, and then 2 ml of fresh DMEM supplemented with 10% FCS were added over the cell-collagen lattice. When tested, the AS-ODN or S-ODN were introduced at this step. Two days later, the culture medium was replaced. The lattices were then gently detached from the sides and lifted off the bottom of the well containing 2 ml of serum-free medium and the agents to be tested. When PKC inhibitors were used, the myometrial cell preparations were exposed for 30 min to one of these drugs at the appropriate concentration and then to ET-1 (50 nM). We have verified that the growth rate of myometrial cells cultured into collagen gels, as well as cell viability, was similar to that of cells cultured on plastic dishes (data not shown). Images of the floating gels were captured and digitized using a scanner (Studio Scan IISI, Agfa) before the test agents were added and after incubation for 24 h. The lattice was assimilated to an ellipse, and the area was calculated after measuring the major and minor diameter of the gel. Collagen contraction was expressed as the percentage of contraction ± SE of triplicate determinations from 5–10 separate experiments, where the percentage of contraction is the percentage of decrease in terms of the original surface area.

Immunofluorescence analysis. PKC{zeta} was localized in quiescent subconfluent cells cultured in 24-well dishes with coverslips as previously described (12). Briefly, cells were incubated with 100 nM ET-1 in serum-free medium at 37°C and fixed for 15 min with 4% paraformaldehyde in PBS. Cells were then rendered permeable by incubation in 0.1% Triton X-100 in 10% FCS-PBS for 15 min. They were incubated with the first anti-PKC{zeta} antibody (dilution 1:50) for 1 h and with the second FITC-labeled anti-rabbit IgG solution (dilution 1:100) for 45 min. Cells were then washed to remove excess label. Nuclei were labeled with Hoescht 33342 and diluted 1:500 in water for 15 min. Polymerized F-actin was visualized with FITC-conjugated phalloidin (dilution 1:8,000), on the basis of the ability of this phallotoxin to stain filamentous actin. For colocalization studies, myometrial cells were simultaneously double stained for PKC{zeta} and actin. Cells were incubated overnight at 4°C with the first monoclonal antibody against smooth muscle {alpha}-actin and the first antibody against PKC{zeta}. Secondary antibodies used were FITC antibody against mouse IgG and RITC-labeled anti-rabbit IgG solution (dilution 1:100). Coverslips were mounted on slides by using fluorescent mounting medium. A Nikon E-600 inverted microscope was used for conventional fluorescence microscopy, and photographs were taken using Coolsnap Software (RS Photometrics, Evry, France). Confocal microscopy was performed with a Leica TCSSP model. The excitation and emission wavelengths were 488 and 500–555 nm for FITC and 568 and 600–640 nm for RITC, respectively. Negative controls were carried out by omitting the primary antibody. At least 15 cells were examined from a minimum of 6 experiments under each experimental condition.

Cell stimulation and extraction of PKC. Confluent myometrial cells were incubated for 3 days in serum-free medium in the absence and presence of 100 nM ET-1 for the indicated times. Subcellular fractions were obtained as previously described (39). Briefly, cells were lysed and scraped into a protein lysis buffer (20 mM Tris · HCl, pH 7.5, 250 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM mercaptoethanol, 2 mM PMSF, 5% glycerol, and 40 µg/ml leupeptin), sonicated, and centrifuged at 100,000 g for 60 min. The resulting supernatant was designated as the cytosolic fraction. The pellet was resuspended in protein lysis buffer containing 1% Nonidet P-40 and ultracentrifuged at 100,000 g for 30 min. The resulting supernatant was the solubilized particulate membrane fraction, and the remaining Nonidet P-40 insoluble pellet resuspended in the protein lysis buffer was designated as cytoskeletal fraction. Protein concentrations were determined using the Bio-Rad protein assay 96-well microtiter plate method.

Western blot analysis. Equal amounts of protein (20 µg protein/lane) from crude cell homogenates, particulate, and cytoskeletal fractions were separated by SDS-PAGE on 8% gels by the method of Laemmli (24), and the separated proteins were transferred to a nitrocellulose membrane overnight (13, 39). Nonspecific binding sites were blocked by incubating the membrane with 5% fat-free dried milk in TBST (10 mM Tris · HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20). Polyclonal antibody against PKC{zeta} was added at the appropriate concentration (1:5,000) and incubated for suitable times at room temperature. Membranes were washed with TBST and incubated with the secondary antibody, a donkey anti-rabbit IgG (dilution 1:5,000) conjugated to horseradish peroxidase (Amersham International). The blots were developed with ECL reagents and visualized on Kodak X-ray films. Molecular weight markers were run in parallel, and rat brain extract was used as positive control. Specific blocking in the presence of the respective antigen peptide against which the antibody had been raised showed the specificity of the immunoreactive band of PKC isoform.

Statistical analysis. Results were expressed as means ± SE. Groups of data were evaluated by ANOVA. A Bonferroni correction was performed to adjust for multiple comparisons of gel areas. Values of P < 0.05 are considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of inhibition of PKC{delta} and -{zeta} isoforms on ET-1-induced myometrial contraction. As we previously observed (13), administration of 30 nM ET-1 produced a marked tetanic contraction (Fig. 1A). To assess the role of PKC{delta} in ET-1-induced contraction, myometrial strips were incubated with the selective inhibitor rottlerin for 30 min before a single dose of 30 nM ET-1 was added. We found that a concentration of rottlerin as high as 3 µM did not affect the response of ET-1 (Fig. 1B). Because it was previously reported that PIP3, a PI3 kinase product, activates PKC{zeta} in vitro, we also evaluated the role of this atypical isoform of PKC in ET-1-mediated contraction by incubating myometrial strips with LY-294002, a PI3 kinase inhibitor. Thirty minutes after application of 5 µM LY-294002 (Fig. 1C), ET-1 only increased the rhythmic activity with negligible apparent modification of the tonus. We also evaluated whether phorbol ester-responsive PKC isoforms were required for ET-1-induced contraction by exposing myometrial strips to a prolonged treatment with PDBu (1 µM for 4 h). These conditions are required to substantially deplete cPKC/nPKC isoforms in myometrium (13). Myometrial strips responded to 1 µM PDBu by developing a gradual increase in contractile force that reached a plateau (Fig. 1D) and were still able to respond to ET-1.



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Fig. 1. A: typical records of the effects of endothelin-1 (ET-1) (30 nM) on human isolated myometrial strips from pregnant women. Each drug was added at the point indicated by arrowheads. Similar records were obtained in 10 other experiments. B: representative trace of the response to ET-1 (30 nM) of myometrium pretreated with 3 µM rottlerin for 30 min. Myometrial strips were then washed (W) and returned to spontaneous contractions. Similar records were obtained in 5 other experiments. C: representative trace of the response to ET-1 (30 nM) of myometrial strips pretreated with 5 µM LY-294002 for 30 min. Similar records were obtained in 5 other experiments. D: representative traces of the response to ET-1 (30 nM) of myometrial strips pretreated with 1 µM phorbol 12,13-dibutyrate (PDBu) for 4 h. Similar records were obtained in 5 other experiments.

 

Effect of PKC isoform inhibition on ET-1-mediated collagen lattice contraction. When myometrial cells were cultured in a three-dimensional (3D) collagen gel and detached from the underlying surface, cells contracted the gel over 24 h in serum-free DMEM (SF-DMEM), demonstrating their basal contractile tone (control: 21% contraction). We previously demonstrated that ET-1 is able to enhance collagen gel contraction by human myometrial cells. The reduction in the area of the lattices on day 1 was significant at ET-1 concentration as low as 1 nM, and maximal stimulation of myometrial cell contraction occurred at 50 nM (6). To determine whether the ET-1 effect is mediated through the PKC pathway, collagen gel-incorporated myometrial cells were treated with various inhibitors of PKC isoforms. As shown in Table 1, when ET-1 (50 nM) was added to the medium, a subsequent contraction of the lattice was observed (40.3 ± 7%). Neither the cPKC inhibitor Gö-6976 (29) (0.5 µM) nor rottlerin (3 µM) had an effect on myometrial cell-mediated gel contraction induced by ET-1. In contrast, the PKC inhibitor Ro-318220 (0.5 µM) and LY-294002 (5 µM) blocked the response to ET-1. It is noticeable that a cell-permeable myristoylated peptide corresponding to the autoinhibitory domain of PKC{zeta} (peptide Z) clearly blocked the contractile effects induced by ET-1 in myometrial cells on collagen lattices. The concentration used (33 µM) is well within the range of that reported to inhibit PKC in other cell systems (11).


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Table 1. Effects of PKC inhibition on ET-1-promoted gel contraction

 

Effects of a PKC{zeta} antisense oligonucleotide on ET-1-mediated collagen lattice contraction. Two AS-ODN directed against human PKC{zeta}, AS-1 ODN and AS-2 ODN directed against human PKC{zeta} mRNA, and their corresponding scrambled PKC{zeta}-ODN control were used to determine the role of PKC{zeta} in ET-1-induced myometrial cell contraction. Collagen lattices were incubated alone or with increasing concentrations of AS-ODN (0.5–4 µM) for 2 days and then with ET-1 (50 nM). AS-1 ODN treatment resulted in a dose-dependent decrease of ET-1-induced contraction (Fig. 2A). Cells were incubated in parallel with AS-1 ODN (1–4 µM) for 48 h, and the protein levels were assessed by Western blotting to determine whether AS-ODN actually reduced the amount of PKC{zeta}. Immunoblot analysis confirmed that a concentration-dependent reduction in PKC{zeta} immunoreactive band (78 kDa) occurred in AS-1 ODN-treated cells (Fig. 2B). Similar results were obtained when myometrial cells were treated with AS-2-ODN (data not shown), whereas treatment of cells with 4 µM S-ODN had no apparent effect on ET-1-mediated collagen lattice contraction (Fig. 2C).



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Fig. 2. A: reduction in ET-1-induced contraction of collagen lattices by myometrial cells after concentration-dependent PKC{zeta} antisense oligonucleotide (AS-ODN) treatment. Human pregnant myometrial cell collagen lattices were incubated without or with increasing concentrations of AS-1-ODN (0.5–4 µM) for 48 h before the addition of 50 nM ET-1. Results are expressed as the mean percentage contraction of collagen lattices of duplicate determinations. The data are representative of 2 separate experiments. B: PKC{zeta} depletion by AS-ODNs. Human myometrial cells were exposed without (control) or with increasing concentrations of AS-1-ODN (1–4 µM) for 2 days, and protein extracts were analyzed by gel electrophoresis and Western blotting (n = 3). C: effects of AS-ODN or scrambled PKC{zeta} oligonucleotide (S-ODN) on contraction of collagen lattices. Myometrial cell collagen lattices were incubated without or with 4 µM S-ODN or AS-1-ODN for 48 h before the addition of 50 nM ET-1. Results are expressed as mean percentage contraction ± SE of collagen lattices of duplicate determinations for 5 independent experiments, each performed on a different population of myometrial cells (subculture 4, 5) from 4 different uteri. *P < 0.05 compared with control.

 

Immunofluorescent localization of PKC{zeta}. Immunocytochemistry using antibody against PKC{zeta} confirmed the presence of PKC{zeta} in human myometrial cells (Fig. 3A). To ensure the specificity of the staining, negative controls were included. Unlabeled myometrial cells displayed no autofluorescence, and no staining was found in cells not incubated with PKC{zeta} antibody (data not shown). Untreated myometrial cells showed a diffuse distribution of PKC{zeta} throughout the whole cytoplasm including the extremities. A short incubation (2–10 min) with ET-1 (100 nM) resulted in a shift of PKC{zeta} toward the perinuclear area, whereas the staining of the extremities of the cells was less visible. The immunofluorescent localization of PKC{zeta} did not change after ET-1 incubation for 20 or 30 min.



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Fig. 3. A: analysis by immunofluorescence of the redistribution of PKC{zeta} after ET-1 treatment of human myometrial cells. Cells were stimulated with ET-1 (100 nM) for the indicated times. Magnification, x40 (n = 6). B: cells were exposed first to peptide Z (33 µM) for 30 min with or without 100 nM ET-1 for 10 min. Immunofluorescence staining of PKC was employed using rabbit anti-PKC{zeta} and FITC-conjugated anti-rabbit IgG (green fluorescence). C: representative Western blot of particulate (P) and cytoskeletal (CSK) immunoreactive PKC{zeta} in myometrial cells from pregnant women after stimulation with ET-1. Cells were incubated without or with 100 nM ET-1 for 2–20 min. This is a typical result of 3 independent experiments.

 

As shown in Fig. 3B, the effect of ET-1 on PKC{zeta} localization was clearly blocked when myometrial cells were pretreated with peptide Z. The intensity and the staining pattern were not different from that observed in untreated cells.

Figure 3C shows a representative immunoblot of myometrial cell fractions from a series of three experiments. The specific antibody to PKC{zeta} recognized an immunoreactive band at 78 kDa. Incubation of myometrial cells in the presence of 100 nM ET-1 elicited a transient enhancement of cytoskeletal immunoreactive PKC{zeta}. By contrast, we confirmed that ET-1 had no detectable effect on the increase in immunoreactive PKC{zeta} in the particulate membrane fraction as we previously observed in myometrial cells from nonpregnant women (39).

Association of activated PKC{zeta} with actin microfilaments. In the absence of ET-1, myometrial cells labeled with RITC-phalloidin showed actin stress fibers that were generally distributed in parallel arrays along the length of the cells (Fig. 4A). In some cells, there appeared to be a loss of actin fibers in the perinuclear space. ET-1 treatment (100 nM) provoked a rapid effect on actin organization. Contractile cells display well-defined actin stress fibers that extended the entire length of the cell, crossing over the nuclear region and aligned in a nonparallel manner (Fig. 4B).



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Fig. 4. Fluorescent immunochemistry of human myometrial cells using rhodamine (RITC)-conjugated phalloidin. Cells were treated and analyzed without (A) or after 10 min ET-1 (100 nM) in the absence (B) or presence of AS-1-ODN directed against human PKC{zeta} mRNA (4 µM) (C) or S-ODN (4 µM) (D). Original magnification, x40 (n = 6).

 

We assessed the effect of the AS-1-ODN directed against human PKC{zeta} mRNA to determine whether PKC{zeta} undergoes association with actin in myometrial cells after ET-1 treatment. After application of 100 nM ET-1 for 10 min, all the actin stress fibers were oriented along the longest axis of PKC{zeta}-deprived myometrial cells (Fig. 4C). By contrast, when the cells were treated with S-ODN before the addition of ET-1 (Fig. 4D), the stress fiber arrangement followed a pattern similar to that observed in ET-1-treated cells.

Double immunofluorescence studies on myometrial cells with anti-PKC{zeta} and an anti-{alpha}-smooth muscle actin revealed that the immunostaining of PKC{zeta} is somewhat similar to that of actin. Note that in untreated cells, PKC{zeta} gives a diffuse cytoplasmic signal with no overlap with actin (Fig. 5A). When cells were treated with 100 nM ET-1 for 10 min, PKC{zeta} immunofluorescence colocalized with that of actin in the perinuclear area (Fig. 5B).



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Fig. 5. Colocalization of PKC{zeta} and {alpha}-smooth muscle actin in ET-1-stimulated myometrial cells. Myometrial cells were treated without (A) or after 10 min ET-1 (100 nM) (B) and analyzed by confocal laser scanning microscopy with polyclonal anti-rabbit PKC{zeta} and RITC-conjugated anti-rabbit IgG (red fluorescence) and monoclonal anti-{alpha}-smooth muscle actin and FITC-conjugated anti-mouse IgG (green fluorescence). Cells are shown at the same magnification (x40). Diverse combinations in the order of staining steps were tried, and all resulted in identical staining (data not shown). We chose the combination that gave the brightest staining for both fluorochromes with minimal background. In addition, both combination of red-green with actin-PKC{zeta} were tested, giving similar results (n = 6).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study suggests strong evidences for the coupling of ET-1 to PKC{zeta} activation in human myometrium as a possible pathway leading to contraction at the end of pregnancy. Furthermore, our results confirm that activation of cPKC ({alpha}, {beta}1, and {beta}2) and nPKC ({delta} and {epsilon}) are not implicated in this process.

We first used rottlerin, an inhibitor of PKC{delta}. This compound was reported to show preference for PKC{delta} (IC50 = 3–6 µM), whereas higher concentrations of rottlerin were required to inhibit the activity of other PKC isoforms with IC50 values for PKC{alpha},{beta} of 30–42 µM and PKC{epsilon},{zeta} of 80–100 µM (18). Our results show that a concentration of rottlerin of 3 µM failed to modify ET-1-induced myometrial contraction, strengthening the proposal that PKC{delta} is not implicated in the ET-1-induced myometrial contraction. Little is known about the role of PKC{delta} in uterine function at the end of pregnancy, but several studies demonstrated that PKC{delta} might be involved in the regulation of cell growth and differentiation, as well as play a role in apoptosis and tumor development (17). Unlike PKC{alpha} (12), it is uncertain whether PKC{delta} is a regulator of human uterine growth during pregnancy. Interestingly, Robin et al. (35) recently demonstrated that cPKC/nPKC and aPKC, which are activated downstream of phospholipase C and PI3 kinase, respectively, contribute equally to ET-1-induced extracellular signal-regulated kinase (ERK) activation and DNA synthesis in rat myometrial cells. Some PKC isoforms should be able to form a complex regulating network for controlling the physiological uterine growth associated with gestation.

We therefore hypothesized that PKC{zeta} is one of the signaling pathways leading to contraction. To demonstrate this hypothesis, the downstream effect of ET-1-induced myometrial contraction was determined by using PI3 kinase inhibitor. It has been shown that PI3 kinase product, PIP3, activates PKC{zeta} (31) and that PI3 kinase substrate, PDK-1, phosphorylates PKC{zeta} in the activation loop site (25). Our results showed that PI3 kinase inhibitor LY-294002 strongly perturbed ET-1-induced myometrial contraction, which is consistent with another report in rabbit basilar artery (30). After PKC downregulation induced by long-term treatment with PDBu, the ET-1 contractile response was preserved. This finding suggests a role for a phorbol ester-insensitive PKC isoform in myometrial contractions. A convenient model of cultured human pregnant myometrial cells was needed for a complete understanding of the physiology of the uterine smooth muscle contraction. Cavaillé et al. (4) previously defined culture conditions of myometrial cells that allow preservation of expression of smooth markers of differentiation. Indeed, it was shown that smooth muscle {alpha}-actin and desmin content was enhanced by suppression of serum in culture medium after myometrial cells reached confluency. Additionally, by using a conventional collagen gel retraction assay, we recently demonstrated that myometrial cells do not undergo phenotypic modulation during the culture and are able to respond appropriately to ET-1 in terms of contractility via selective activation of the ETA receptor (6). Importantly, the concentration of ET-1 required to induce the maximum reduction in collagen gel size was similar to that able to produce maximum contraction in myometrial bath experiments. Taken together, these observations show that our culture model was appropriate for studying the mechanisms controlling uterine activity. The effects of ET-1 on myometrial cell contraction of 3D-collagen gels are mediated through PKC activation. Consistent with this, both Ro-318220, a general PKC inhibitor, and LY-294002 blocked the ET-1 effect. In contrast, inhibitors of cPKC ({alpha},{beta}) and PKC{delta} were without effect. The direct involvement of PKC{zeta} in ET-1-induced contraction was confirmed both by using the PKC{zeta} pseudoinhibitor peptide Z, which specifically blocked the contraction of collagen gels achieved by ET-1, and by using antisense technology. Incubation of collagen-gel lattices with an AS-ODN to PKC{zeta} markedly blocked the myometrial contractile response of ET-1.

Additionally, the localization of PKC{zeta} was investigated in human myometrial cells. The translocation of PKC{zeta} to the cytoskeletal fraction after ET-1 treatment suggests an interaction between this isoform and the actin-rich microfilaments of the cytoskeleton. It is thus not surprising that we detected using confocal microscopy, translocation, and association of PKC{zeta} to a structure coincident with the actin cytoskeleton. However, we did not observe membrane-associated PKC{zeta} in cultured myometrial cells (39). These results differ from those we previously obtained in myometrial tissue from pregnant women (13). This discrepancy must be one of many examples showing that the translocation of a specific PKC isoform may vary depending on the cell environment or on the differentiation state of the cells. It also appears that the presence of serum in culture media may alter the localization of PKC isoforms (9). PKC{zeta} started to move very quickly after ET-1 addition. This time of drug application is sufficient to induce an architectural reorganization of actin filament. Moreover, we used the AS-ODN toward PKC{zeta} to show that inhibition of PKC{zeta} results in a loss of ET-1-induced reorganization of actin filament. There were parallel actin fibers in the direction of the longest axis of the cells, although we observed a tangle of actin fibers when cells were treated with the S-ODN.

PKC has been implicated in rearrangement of microtubules (tubulin), intermediate filament proteins (vimentin, cytokeratin), membrane cytoskeletal cross-linking proteins (myrostylated alanin-rich C kinase substrates, ankyrin), and components of the actin filaments (F-actin) in a variety of cell types (for a review see Ref. 22). However, little information is available concerning which PKC isoform(s) are involved in this process. PKC{beta}2 seems to be closely associated with the actin component of the cytoskeleton in NIH-3T3 fibroblasts, whereas PKC{gamma} accumulates in Golgi organelles and PKC{epsilon} associates with nuclear membranes (16). The microtubules play an important role in the translocation of PKC{alpha} from the cytosol in cultured rat aortic smooth muscle cells (3). Evidence for an implication of aPKC isoforms in actin dynamics is also supported by several publications. For example, the reorganization of actin cytoskeleton of NIH-3T3 cells by oncogene Ras is reversed by expression of dominant-negative mutant of both PKC{zeta} and {lambda} (19). Uberall et al. (41) found that aPKC are involved in Ras-mediated disassembly of stress fibers, and Coghlan et al. (5) more precisely reported that Ras-dependant loss of stress fibers requires Cdc42. In lymphocytes, a colocalization of PKC{zeta} and actin is also observed. The same group proposed a role for PKC{zeta} in maintaining the integrity of the actin cytoskeleton in an IL-2-stimulated murine T cell line (14, 15). It is important to note that the association of PKC{zeta} with the cytoskeleton is not a consistent feature of translocation among smooth cell types or for the same agonist. As already reported in pulmonary artery smooth muscle cells, PKC{zeta} mediates nuclear responses such as gene transcription and proliferation (7), but ET-1 increased membrane-associated PKC{zeta} in mesangial cells (10).

Of particular interest is the conclusion that the actin cytoskeleton of uterine smooth muscle cells is dynamically regulated by uterine contractile agents, such as oxytocin, and is able to play a major role in the formation of stress fibers and focal adhesions, both of which have been shown to contribute to the maintenance of uterine contractions (45). The involvement of PKC (21, 38), tyrosine kinases (26), and RhoA/Rho kinase (23, 37) in Ca2+ sensitization has been reported in uterine smooth muscle. It is also commonly accepted that most of the uterotonic agents are able to modulate contraction by altering myofilament Ca2+ sensitivity or through Ca2+-independent pathway (27). Cross talk between different kinase pathways may be the key signaling event of Ca2+ sensitization of myofilaments. The present study allows us to propose that PKC{zeta} is particularly closely associated with the actin component of the cytoskeleton in human near-term myometrium. This isoform may induce either a phosphorylation of actin itself directly or of its binding proteins. In vascular smooth muscle cells, a thin filament associated protein such as calponin has been described as a poor substrate for PKC{zeta} (43). Whether PKC may phosphorylate in vitro cytoskeletal proteins in human myometrial cells requires clarification.

In conclusion, we have shown that PKC{zeta} may play a role in the regulation of ET-1-induced myometrial contraction at the end of pregnancy. The implication of this isoform in this process is not so surprising. The control of the onset of labor in women is as yet unknown, but modulation of the immune-inflammatory mechanisms, specifically a balance between Th1 (pro-inflammatory)/Th2 (anti-inflammatory) cytokine production, has been described at the time of parturition. It is now becoming evident that infection is concerned in the pathogenesis of preterm labor and delivery, and a link between tumor necrosis factor (TNF)-{alpha}/IL-1{beta} and premature childbirth has been proposed (1). Additionally, aPKCs are important components of the TNF-{alpha}/IL-1{beta} signaling pathway that control NF-{kappa}B activation (8), and recent evidence also indicates that targeted disruption of the gene of PKC{zeta} results in the impairment of the NF-{kappa}B pathway (28). Undoubtedly, additional work is necessary to elucidate the probable interactions between this aPKC isoform and the cascade of cytokines, but the results reported here unveil a novel mechanism that links PKC{zeta} to uterine contraction at term pregnancy in humans.


    DISCLOSURES
 
We are very grateful to Biognostik for financial support.


    ACKNOWLEDGMENTS
 
We are very grateful to Drs. Marie-Josèphe Leroy (INSERM U361) and Marc Bardou (Pharmacology Unit, UFR biomédicale des St. Pères, Paris) for constructive discussions. We thank R. Schwartzmann (Service Imagerie de l'Institut Fédératif de Recherche de Biologie Intégrative, Université Paris VI) for help with confocal analysis.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Breuiller-Fouché, INSERM U361, Pavillon Baudelocque, 123 Bld. de Port-Royal, 75014 Paris, France (E-mail: breuiller-fouche{at}cochin.inserm.fr).

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.


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