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Regular Article |
Is Required for Endothelin-1-Induced Proliferation of Human Myometrial Cells1
a INSERM U361, Université René Descartes, Pavillon Baudelocque, 75014 Paris, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with Gö 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKC efficiently reduced PKC protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKC revealed that there was no PKC immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKC to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKC is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKC by antisense nucleotides might be an important approach for the development of anticancer treatments.
female reproductive tract, growth factors, kinases, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein kinase C (PKC) has been implicated in the ET-1-induced proliferation of human myometrial cells [5]. PKC is a multigene family of serine/threonine kinases that is important in regulating normal and abnormal cell functions, such as growth, differentiation, and transformation [6]. The 13 members of the PKC family can be subdivided on the basis of structural and biochemical properties into 3 groups: the conventional (, ß1, ß2, 
), the novel (
, 
, µ, 
, 
, 
), and the atypical (
, 
, 
) isoforms. Of the 6 isoforms of PKC (, ß1, ß2, , , ) found in the human myometrium [7], PKC is more abundant in proliferating cultured human myometrial cells than in respective myometrial tissue [5]. PKC is a regulator of proliferation of many cell types; it controls cell cycle progression [8] and is essential for cell survival [9]. The overexpression of PKC causes dramatic alterations in the morphology and proliferation of human breast cells [10], and its inhibition reverses the transformed phenotype of human lung carcinoma cells [11]. The presence of PKC has been described for various cellular components, and growing evidence indicates the presence of active PKC in the nucleus. This isoform is associated with nuclear components deeper within the nucleus, such as proteins that bind chromatin and can directly regulate nuclear processes [12]. In the present study, we tested the hypothesis that PKC is required for the ET-1-induced proliferation of human myometrial cells.
Analysis of the role of PKC in the control of cellular growth is hampered by the fact that no isoform-selective inhibitor is available. The PKC inhibitor Gö 6976, which clearly inhibits conventional PKC (cPKC) isoforms and ß [13], is of considerable value for determining whether cPKC is involved in regulating the proliferation of human myometrial cells. However, an additional approach is needed to inhibit a particular protein, such as PKC. All these questions prompted us to selectively deplete PKC protein with an antisense oligonucleotide directed against PKC mRNA, thus preventing the production of PKC in human myometrial cells.
Some of the cPKC isoforms inhibited by Gö 6976 are involved in the ET-1-induced proliferation of human myometrial cells. The stimulation of myometrial growth by ET-1 is prevented by specifically depleting PKC with an antisense oligonucleotide. We also analyzed the distribution of PKC under basal and ET-1-activated conditions using immunofluorescence. PKC is translocated from the cytoplasm to the nucleus upon activation by ET-1. Thus, the mitogenic effect of ET-1 on human myometrial cells appears to be mediated by PKC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco modified Eagle medium (DMEM) with or without phenol red and fetal calf serum (FCS) were from Gibco Life Technologies (Cergy-Pontoise, France). The [methyl-3H]thymidine (25 Ci/mmol), Hybond-C membranes, enhanced chemiluminescence detection system (ECL), and x-ray film were obtained from Amersham International (Chalfont, Buckinghamshire, UK). ET-1 was obtained from Neosystem Laboratories (Strasbourg, France), Gö 6976 was from Calbiochem (La Jolla, CA), and phorbol myristate acetate (PMA) was from Sigma-Aldrich (St. Louis, MO).
Myometrial Cell Culture
Biopsies were obtained from the myometrium of cycling women 39–45 yr of age undergoing hysterectomy for benign gynecologic indications. All the women were in the follicular phase of their menstrual cycles. None had received hormone treatment for at least 3 mo before surgery. The uteri were examined by a pathologist to exclude adenomyosis or malignant changes. Tissue samples were excised in the uterine corpus from normal muscle in areas free of macroscopically visible anomalies. The collected samples of myometrium were placed in DMEM supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. This study was approved by the Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale (Paris-Cochin, France).
Human myometrial cells were cultured in 10% FCS-DMEM supplemented with antibiotics as described by Cavaillé et al. [14] and routinely passaged when 90%–95% of the cells were confluent. Cells (4000 cells/well) were plated in 96-well dishes and allowed to adhere for 48 h in DMEM plus 10% FCS. Cells were then washed with fresh medium and cultured for 72 h in serum-free DMEM to make them quiescent. Myometrial cells at passages 3–6 were used for assays, with no noticeable difference in results with cells from individual passages or with cells from different uteri. Myometrial cells were identified by their positive reaction with monoclonal antibodies against smooth muscle -actin, smooth muscle myosin heavy chains, and desmin and by the typical hill-and-valley microscopic findings. Each population of myometrial cells studied came from a different patient.
Oligonucleotide Treatments
Antisense phosphothiorate PKC oligonucleotide (AS ODN) and scrambled PKC oligonucleotide (S ODN) matched in size and guanine (G) and cytosine (C) content were designed and manufactured by Biognostik (Göttingen, Germany) and used to treat cells essentially as described by Schlingensiepen et al. [15]. The sequence of the AS ODN, designed to specifically hybridize with human PKC mRNA, was 5'CCTTTAGGTAAATCCG3' (which is complementary to positions 509–524 of the PKC mRNA) [16]. The S ODN with the same base composition but a randomized sequence was used as a control sens. Stock solutions (100 µM) were prepared in Tris-EDTA buffer (pH 7.2) and stored at -20°C. Solutions 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 and was uniform after 8 h. Comparable results were obtained with 1 or 2 applications of S ODN and AS ODN. Consequently, all experiments were performed with 1 application of oligonucleotide. Myometrial cells cultured for 24 h in DMEM containing 10% FCS were then exposed to 4 µM S ODN or AS ODN against PKC for 8 h. The cells were then washed and transferred to serum-free medium for 72 h.
Cell Proliferation Assay
Quiescent cells were incubated for 48 h in serum-free medium in the presence of various concentrations of ET-1 dissolved in DMEM, as previously described [2]. Gö 6976 dissolved in dimethylsulfoxide (DMSO) was used in some experiments. [Methyl-3H]thymidine (0.15 µCi/well) was added during the final 24 h of incubation. The incubated cells were washed twice with PBS without CaCl2 and MgCl2, fixed with 5% trichloroacetic acid, washed twice with 100% ethanol, and solubilized with 0.5 N sodium hydroxide. Cell-associated radioactivity was measured by scintillation counting. All experiments were performed in quadruplicate. Cells were counted with a hemocytometer in separate experiments. Quiescent cells were incubated for 72 h with similar treatments, trypsinized, and counted. The viability of cells was checked by trypan blue exclusion. Six replicate wells were used for each test condition.
Western Blot Analysis
Equal amounts of protein (20 µg/lane) from crude homogenates were separated by SDS-PAGE on gels by the method of Laemmli [17], and the separated proteins were transferred to a nitrocellulose membrane overnight [5]. Nonspecific binding sites were blocked by incubating the membrane with 5% fat-free dried milk in Tris-buffered saline with Tween 20 (TBST: 10 mM Tris HCl, pH 7.5, 0.15 M NaCl, 0.1% Tween 20). Polyclonal antibodies against PKC isoforms were added at the appropriate concentrations (PKC 1:20 000, PKCß 1:50, PKC 1:2000, PKC 1:15 000, PKC 1:5000) and incubated for suitable times at room temperature. Rabbit antibodies against PKC, PKC, and PKC (Sigma-Aldrich) were raised against peptides corresponding to amino acid positions 659–672, 726–737, and 577–592, respectively. Rabbit antibodies against PKCß and PKC (Santa Cruz Biotechnology, Le Perray en Yvelines, France) were raised against peptides corresponding to amino acid positions 656–671 and 656–673, respectively. Membranes were washed with TBST and incubated with the secondary antibody, a donkey anti-rabbit IgG (dilution 1:5000) conjugated to horseradish peroxidase (Amersham International). The blots were developed with ECL reagents and visualized on x-ray films (Kodak, Rochester, NY), and the immunoreactive bands were quantified by densitometric scanning (Studio Scan IISI; Agfa, Morstel, Belgium) processed by NIH Image 1.6 software (NIH, Bethesda, MD). Molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were run in parallel, and rat brain extract was used as a control. Specific blocking in the presence of the respective antigen peptides against which the antibodies had been raised showed the specificity of each immunoreactive band of PKC isoforms.
Immunofluorescence Analysis
PKC was localized in quiescent subconfluent cells cultured in 24-well dishes with coverslips. Some cells were incubated with 100 nM ET-1 or 100 nM PMA in serum-free medium at 37°C. Cells were then washed with PBS supplemented with CaCl2 (1 mM) and MgCl2 (1 mM) and fixed for 15 min with 4% paraformaldehyde in PBS. Excess fixative was quenched with 50 mM glycine 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 antibody (dilution 1:50) for 1 h and with the second fluorescein isothiocyanate-labeled anti-rabbit IgG solution (dilution 1:100) (Santa Cruz Biotechnology) for 45 min. Cells were then washed to remove excess label. Nuclei were labeled with Hoescht 33342 (Sigma-Aldrich), diluted 1:500 in water, for 2 min. Coverslips were mounted on slides with fluorescent mounting medium (DAKO, Carpinteria, CA). A Nikon E-600 inverted microscope was used for conventional fluorescence microscopy, and photographs were taken using Coolsnap software (RS Photometrics, Evry, France). No staining was found in cells not incubated with PKC antibody. At least 15 cells were examined from a minimum of 6 experiments under each experimental condition.
Statistical Analysis
Experimental results are presented as the means ± SEM of data obtained from 5 experiments or as the means ± SD of data obtained from 1 of 2 experiments yielding similar results. Groups were compared using a two-way ANOVA. Significance was defined at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As previously described [5], ET-1 increased thymidine incorporation and cell numbers. The increase in [methyl-3H] thymidine incorporation induced by 100 nM ET-1 was about 176% ± 24% that of serum-deprived cells (control) (P < 0.05) (Fig. 1). The concomitant increase in cell counts was significant at 100 nM ET-1, with a maximal stimulation of 146% ± 18% of the control (data not shown). The cPKC inhibitor Gö 6976 (1 µM) markedly inhibited the DNA synthesis induced by 100 nM ET-1 (Fig. 1). Similarly, pretreatment of myometrial cells with 1 µM Gö 6976 significantly decreased the number of cells induced by 100 nM ET-1 stimulation (data not shown). Other concentrations of Gö 6976 (1–100 nM) had no effect on ET-1-induced proliferation (data not shown). We verified that this inhibitor was not toxic to human myometrial cells under our conditions by using trypan blue exclusion.
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Effects of a PKC Antisense Oligonucleotide on ET-1-Induced Proliferation
We assessed the effect of an AS ODN directed against human PKC mRNA to determine whether PKC synthesis is required for ET-1-induced proliferation. Because AS ODN inhibits only de novo synthesis and not activation of existing PKC, effects were expected only after long-term treatment. We used AS ODN to investigate whether the specific depletion of PKC influenced the ET-1-induced increase in thymidine incorporation and number of human myometrial cells. Cells were incubated alone, with 4 µM S ODN, or with 4 µM AS ODN for 8 h and then with ET-1 (100 nM). AS ODN treatment resulted in the loss of ET-1-induced [methyl-3H]thymidine incorporation. Treatment of myometrial cells with S ODN had no effect on DNA synthesis induced by 100 nM ET-1 (Fig. 2A). Incubation of cells with 4 µM AS ODN abrogated the dose-dependent proliferative effect of ET-1, whereas incubation with S ODN did not alter its ability to increase DNA synthesis (Fig. 2B).
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Cells were incubated with AS ODN, and the protein levels were assessed by Western blotting to determine whether AS ODN actually reduced the amount of PKC. Immunoblot analysis confirmed that the level of PKC was specifically reduced in AS ODN-treated cells, whereas S ODN had no effect on PKC. AS ODN reduced PKC to 37% of the control concentration (Fig. 3). The AS ODN to PKC was also examined for nonspecific inhibition of other PKC isoforms. PKCß, , , and levels were unchanged after incubation with S ODN or AS ODN. Thus, the AS ODN to PKC had no effect on the synthesis of other PKC isoforms, indicating the specificity of the PKC inhibition in our cell model (Fig. 3).
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Immunofluorescent Localization of PKC
Immunocytochemistry using antibody against PKC confirmed the presence of PKC in human myometrial cells. PKC immunoreactivity was entirely absent from the nucleus of quiescent nonconfluent untreated cells, but it was evenly distributed throughout the cytoplasm (Fig. 4A). A short incubation with ET-1 resulted in a cytoplasmic staining pattern of PKC similar to that of untreated cells (Fig. 4B). Longer incubation with ET-1 caused a shift of PKC towards the perinuclear area at 15 min (Fig. 4C) and 30 min (Fig. 4D) and then towards the nucleus at 45 min (Fig. 4E) and 60 min (Fig. 4F). No staining was found in cells not incubated with PKC antibody (Fig. 4G). The absence of PKC immunoreactivity from the perimembrane region in stimulated cells is consistent with our previous finding that this isoform does not translocate from the cytosol to the particulate fraction [5]. Treatment with a phorbol ester such as 100 nM PMA mimicked ET-1-induced translocation of PKC to the nucleus (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is required for ET-1-induced growth of human myometrial cells. We used a PKC inhibitor specific for conventional PKC and an AS ODN directed towards PKC to show that inhibition of PKC results in a loss of ET-1-induced myometrial cell growth.
The mitogenic response of human myometrial cells to ET-1 appears to be mediated by activation of cPKC (PKC, PKCß1, and PKCß2) isoforms. Gö 6976, a selective inhibitor of cPKC isoforms, blocks [3H]thymidine incorporation in myometrial cells and the increase in cell number induced by ET-1. The inhibitor acts via the ATP binding site on the kinase, and even micromolar concentrations of Gö 6976 have no effect on the kinase activity of the Ca2+-independent subtypes of PKC [13]. The fact that PKC, PKCß1, and PKCß2 isoforms are found in cultured human myometrial cells [5, 18] and that the blockade of ET-1-induced growth is probably due to inhibition of cPKC indicates that PKC, PKCß1, or PKCß2 isoforms are involved in the growth of human myometrial cells. The ET-1-induced increase in particulate PKCß1 and PKCß2 in myometrial cells supports this explanation but does not exclude the participation of PKC, which is more concentrated in subconfluent myometrial cells than in myometrial tissue [5]. Although little is known of the mechanisms regulating human myometrial cell growth, a relationship of PKC expression, cell proliferation, and tumor growth has been clearly established in several cell lines [19–21].
We used antisense technology to obtain evidence that PKC is the isoenzyme involved in ET-1-induced proliferation of myometrial cells. Incubation of myometrial cells with an AS ODN to PKC for 8 h markedly reduced the level of PKC protein. The AS ODN to PKC also abolished the DNA synthesis and increase in cell number induced by ET-1. The S ODN had no effect on either parameter. Furthermore, there was no overproduction of the other PKC isoforms after incubation with AS ODN, suggesting that there was no compensation for the lack of PKC. Our results are in agreement with those of others who have reported that AS ODN does not alter the levels of other PKC isoforms [22, 23]. These data demonstrate that PKC mediates the proliferative effect of ET-1 on human myometrial cells in culture. Antisense nucleotides are increasingly used to specifically reduce the growth of many human cell lines and to inhibit the growth of human tumor xenografts in athymic mice (for a review see [24]). Targeting PKC in smooth muscle cells has provided evidence for its role in cell proliferation. For example, heparin blocks cell proliferation via the selective depletion of PKC [25], and cell proliferation was significantly decreased by exposing myoblasts to the AS ODN to PKC [26]. This finding agrees with numerous observations linking PKC to cancer-related processes such as invasion, metastasis, and multidrug resistance. A mutant form of PKC has been discovered in invasive human pituitary tumors and in human thyroid neoplasms. The mutation in the V3 hinge separating the regulatory from the catalytic domains of the enzyme (at residue 294) could be particularly involved in the progression of tumors [27, 28].
PKC itself may translocate to the nucleus and directly phosphorylate nuclear regulatory proteins [29]. PKC transport into the nucleus may occur via PKC-binding proteins, which can function as carriers. One probable candidate for the postulate nuclear carrier is the perinuclear binding protein protein-interacting C kinase 1, which contains a PDZ (PSD-95, disheveled, and ZO1) domain that is required for interaction with the COOH terminus of PKC (302–672) [30]. Thus, it is not surprising that we detected PKC translocation to the perinuclear region of human myometrial cells incubated with ET-1. PKC started to move after 15 min of incubation with ET-1, but 60 min were required to obtain almost complete migration from the cytoplasm to the nucleus. The exact function of PKC and which signals direct PKC isoforms to the nucleus are unknown. The correlation between nuclear localization and proliferation suggests that nuclear PKC influences growth regulation. PKC could act in the cytoplasm and cause nuclear effects indirectly by activating the linear Ras-Raf-mitogen-activated protein kinase cascade. Alternatively, PKC might itself be activated in the nucleus by either a cytoplasmic messenger or by an activator generated inside the nucleus, leading to the phosphorylation of nuclear proteins such as transcription factors (for review see [12]). Primary cultures of uterine smooth muscle cells show dramatic increases in their contents of c-Jun and c-Fos proteins, factors that bind to AP-1 sites, after PKC activation [31]. Although this interpretation is attractive, there is no indication that PKC acts like this in the human myometrium.
It has also been suggested that ET-1 acts as a promoter of myometrial cell growth, and this peptide may be involved in the development of a variety of neoplasms [32]. The exclusive presence of ETA receptors functionally coupled to phospholipase C in benign smooth muscle tumors of the uterus (leiomyoma) together with the growth in response to ET-1 of cultured human myometrial cells [2–4] led to the postulation that ET-1 is a new regulator of the growth of leiomyomas. Because PKC is present in leiomyoma tissue [33] and is one of the isoforms involved in the potentiating response of cultured leiomyoma cells to growth factors by ET-1 [34], PKC may also be involved in the growth of leiomyomas.
We have shown that PKC plays a critical role in the regulation of ET-1-induced human myometrial cell growth. Although PKC is linked to cancer, many questions about its action in human uterine tumors remain unanswered. Targeting of PKC by pharmacologic inhibitors or by antisense nucleotides might become part of anticancer therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants from the Fondation pour la Recherche Médicale (FRM, France). We acknowledge Biognostik GmbH for their financial support. 
2 Correspondence: Michelle Breuiller-Fouché, INSERM U.361, Pavillon Baudelocque, 123, Bld de Port Royal, 75014 Paris, France. FAX: 33 1 43 26 44 08; breuiller-fouche{at}cochin.inserm.fr
Accepted: August 2, 2001.
Received: June 7, 2001.
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in MCF-10A human breast cancer engenders dramatic alterations in morphology, proliferation and motility. Cell Growth Differ 1999; 10:343-352 reverses the transformed phenotype in human lung carcinoma cells. Exp Cell Res 1999; 250:253-263[CrossRef][Medline]. Nucleic Acids Res 1990; 18:2183 expression. J Biol Chem 1999; 274:1715-1722 expression by antisense RNA in transfected Jurkat cells. Eur J Immunol 1999; 29:466-476[CrossRef][Medline] and C-raf kinase: rationale and clinical experience in patients with solid tumors. In: Crooke ST (ed.), Antisense Research and Application. Berlin: Springer; 1998: 463–474 expression is required for heparin inhibition of rat smooth muscle cell proliferation in vitro and in vivo. J Biol Chem 1996; 271:25928-25935 protein kinase C. J Clin Endocrinol Metab 1993; 77:1125-1129[Abstract]This article has been cited by other articles:
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 P. Robin, S. Chouayekh, C. Bole-Feysot, D. Leiber, and Z. Tanfin Contribution of Phospholipase D in Endothelin-1-Mediated Extracellular Signal-Regulated Kinase Activation and Proliferation in Rat Uterine Leiomyoma Cells Biol Reprod, January 1, 2005; 72(1): 69 - 77. [Abstract] [Full Text] [PDF]
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 P. Robin, I. Boulven, C. Bole-Feysot, Z. Tanfin, and D. Leiber Contribution of PKC-dependent and -independent processes in temporal ERK regulation by ET-1, PDGF, and EGF in rat myometrial cells Am J Physiol Cell Physiol, April 1, 2004; 286(4): C798 - C806. [Abstract] [Full Text] [PDF]
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G. D. Liberto, E. Dallot, I. E.-L. Parco, D. Cabrol, F. Ferre, and M. Breuiller-Fouche A critical role for PKC{zeta} in endothelin-1-induced uterine contractions at the end of pregnancy Am J Physiol Cell Physiol, September 1, 2003; 285(3): C599 - C607. [Abstract] [Full Text] [PDF]
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G. Charpigny, M.-J. Leroy, M. Breuiller-Fouche, Z. Tanfin, S. Mhaouty-Kodja, Ph. Robin, D. Leiber, J. Cohen-Tannoudji, D. Cabrol, C. Barberis, et al. A Functional Genomic Study to Identify Differential Gene Expression in the Preterm and Term Human Myometrium Biol Reprod, June 1, 2003; 68(6): 2289 - 2296. [Abstract] [Full Text] [PDF]
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E. Dallot, M. Pouchelet, N. Gouhier, D. Cabrol, F. Ferre, and M. Breuiller-Fouche Contraction of Cultured Human Uterine Smooth Muscle Cells after Stimulation with Endothelin-1 Biol Reprod, March 1, 2003; 68(3): 937 - 942. [Abstract] [Full Text] [PDF]
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