Institut National de la Santé et de la Recherche Médicale Unité 361, Université René Descartes, 75014 Paris, France
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ABSTRACT |
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The role of protein kinase C (PKC) in
endothelin-1 (ET-1)-induced proliferation of human myometrial cells was
investigated. ET-1 dose dependently stimulated DNA synthesis and the
number of cultured myometrial cells. Inhibition of PKC by calphostin C
or Ro-31-8220 or downregulation of PKC eliminated the proliferative effects of ET-1. The failure of two protein tyrosine kinase (PTK) inhibitors (tyrphostin 51 and tyrphostin 23) to affect ET-1-induced proliferation supports the hypothesis of noninvolvement of the tyrosine
kinase signaling pathway in this process. The expression and
distribution of PKC isoforms were examined by Western blot analysis.
The five PKC isoforms (PKC-,
-
1,
-
2, -
, -
) evidenced in
human myometrial tissue were found to be differentially expressed in
myometrial cells, with a predominant expression of PKC-
and PKC-
.
Treatment with phorbol 12,13-dibutyrate (PDBu) resulted in the
translocation of all five isoforms to the particulate fraction, whereas
ET-1 induced a selective increase in particulate
PKC-
1, PKC-
2, and PKC-
. Our
findings that multiple PKC isoforms are differentially responsive to
ET-1 or PDBu suggest that they play distinct roles in the myometrial
growth process.
endothelin; myometrium
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INTRODUCTION |
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ENDOTHELIN-1 (ET-1), originally isolated from endothelial cells, is a 21-amino acid peptide with potent vasoactive properties. Further studies have revealed the existence of additional isopeptides, ET-2 and ET-3, which differ from ET-1 by two and six amino acids, respectively. The three isopeptides are highly homologous and bind to distinct receptors, designated ETA and ETB (see Ref. 35 for review). Human uterine smooth muscle cells express the ETA receptor subtype, which is ET-1 selective (20, 25). This vasoactive peptide synthesized in intrauterine tissues (33) can affect myometrial cells in a paracrine fashion to induce contractility. In humans, ET-1-induced uterine contractions occur via the selective activation of ETA receptors (3, 21, 31), which are functionally linked to phospholipase C and protein kinase C (PKC) (7, 8). The discovery that ET-1 may also regulate proliferation of cultured human myometrial cells (6) provides new perspectives when the biological activity of ET-1 is considered. This hypothesis may be consistent with the presence of ETA receptors in leiomyomas, which involve abnormal myometrial cell growth and proliferation (9).
Despite accumulating evidence of the physiological-pathophysiological significance of ET-1 as a mitogen, the intracellular signaling pathways responsible for its mitogenic activity in myometrial cells remain incompletely understood. Many reports have proposed a key role for PKC in the activation of mitogen-activating protein kinases, which are components of the segmental cascade of events linking the hormone-receptor complex to the mitogenic effect in smooth muscle (30). In particular, PKC has been shown to be involved in the mitogenic action of ET-1 in glomerular mesangial cells (36) and in cardiomyocytes (11, 32). Moreover, studies on glomerular mesangial cells (36) and adrenal zona glomerulosa (26) have provided evidence for a role of PKC and the involvement of protein tyrosine kinases (PTK) in the control of ET-1-induced cell proliferation.
PKC is a large family of serine-threonine protein kinases involved in
the regulation of a variety of cellular processes such as
contractility, proliferation, differentiation, and tumorigenesis. To
date, 12 isoforms have been identified and classified into three groups
on the basis of their cofactor requirements. Conventional PKC isoforms
(,
1,
2, and
) are activated by
diacylglycerol (DAG) or phorbol esters in a
Ca2+-dependent manner. Novel PKC
isoforms (
,
,
,
, and µ) are also activated by DAG and
phorbol esters but in a
Ca2+-independent manner. Atypical
PKC isoforms (
,
, and
) are not activated by
Ca2+ or by DAG or phorbol esters
but are regulated by other phospholipidic mediators. Individual PKC
isoforms possess unique structural characteristics, tissue and
subcellular distribution, and substrate specificity. Accumulating data
suggest that each PKC isoform has a distinct role in cellular functions
(30). However, the role of a specific PKC isoform in a specialized
function has not been determined. We recently reported that human
uterine smooth muscle expressed several isoforms of PKC, including
PKC-
, PKC-
1,
PKC-
2, PKC-
, and trace
amounts of PKC-
, whereas PKC-
and PKC-
were undetectable. Their activation, measured by their translocation from the cytosol to
the particulate fraction, has been studied in response to different stimuli (8).
To elucidate the precise contribution of signaling pathways in human myometrial cells, we examined the effects of selective inhibitors of PKC (calphostin C, Ro-31-8220) and PTK (tyrphostin 51, tyrphostin 23) on ET-1-induced proliferation of human myometrial cells. A comparison with EGF-induced responses was made. Then we identified the various PKC isoforms present in cultured human myometrial cells and studied their regulation by a phorbol ester, phorbol 12,13-dibutyrate (PDBu), and ET-1. Another objective was to define, among the isoforms of PKC evidenced in cultured myometrial cells, those that are required for ET-1-induced cell proliferation.
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MATERIALS AND METHODS |
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Chemicals. Dulbecco's modified
Eagle's medium (DMEM) with or without phenol red and fetal calf serum
(FCS) was from GIBCO Life Technologies (Cergy-Pontoise, France).
[6-3H]thymidine (25 Ci/mmol), Hybond-C membranes, the enhanced chemiluminescence detection
system (ECL), and X-ray films were obtained from Amersham International
(Chalfont, Buckinghamshire, UK). ET-1 was from Neosystem Laboratoire
(Strasbourg, France). Antibodies against PKC-, PKC-
, PKC-
, and
PKC-
were from GIBCO Life Technologies. They were raised in rabbits
against peptides 313-326 from PKC-
, 662-673 from PKC-
,
726-737 from PKC-
, and 577-592 from PKC-
. Antibodies against PKC-
1 and
PKC-
2 were from Santa Cruz
Biotechnology (Le Perray-en-Yvelines, France). They were raised in
rabbits against peptides corresponding to amino acid sequences
656-671 and 657-673. The second antibody, donkey anti-rabbit
IgG conjugated to horseradish peroxidase, was purchased from Amersham
International, and prestained molecular-weight markers were from
Bio-Rad. Epidermal growth factor (EGF) was purchased from Chemicon
International (Temecula, CA). The PKC inhibitor Ro-31-8220 was a
generous gift of Dr. Bradshaw (Roche Discovery, Welwyn, Hertfordshire,
UK). Phorbol 12,13-dibutyrate (PDBu), leupeptin, Nonidet P-40,
phenylmethylsulfonyl fluoride (PMSF), and other drugs and
chemicals used were of the highest quality available from
Sigma (St. Louis, MO).
Preparation and culture of myometrial cells. Human myometrium was obtained from eight women (34-46 yr old) undergoing hysterectomy for benign gynecological indications. None of the patients was under hormonal treatment at the time of surgery. Tissue samples were excised in the uterine corpus from normal muscle (myometrial outer layer) in areas free of macroscopically visible anomalies. This study was approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale (Paris-Cochin, France).
Human myometrial cells were harvested as previously described (10) from
muscle explants and were cultured in 20% FCS in DMEM supplemented with
antibiotics. When cells could be observed growing out from the
explants, the culture medium was replaced by 10% FCS in DMEM. Cells
were confluent 21 days after tissue collection and then were
subcultured every 7 days by trypsinization. For selected cell growth
experiments, myometrial cells were obtained between
passages
3 and
6, with no noticeable difference in
results observed with cells from individual passages or with cells
obtained from different uteri at either the proliferative or the
secretory phase of the menstrual cycle. Confluent myometrial cells were identified by their positive reaction with monoclonal antibodies against smooth muscle -actin, smooth muscle-1 (SM1) and SM2 myosin heavy chains, and desmin and by the typical "hill and valley" microscopic findings. Each population of myometrial cells studied came
from a separate patient.
Assessment of proliferation. Myometrial cells (50,000 cells/well) were cultured to subconfluence in 24-well dishes in the presence of 10% FCS for 24 h. The cells in exponential growth were then transferred to serum-deprived media for 72 h to achieve quiescence. Quiescent cells were incubated for 48 h as previously described (6) in serum-free media in the presence of various concentrations of ET-1 or EGF dissolved in sterile water. In some experiments, PDBu and inhibitors of PKC and PTK were dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration was set in all cases at the maximal value of 0.1% and did not affect cellular growth. In combination experiments, protein kinase inhibitors were added 30 min before incubation with ET-1 or EGF. Serum-free DMEM with 0.1% DMSO was used as a negative control and DMEM with 10% FCS as a positive control. [3H]thymidine (0.4 µCi/well) was added during the final 24 h of incubation. After incubation, cells were washed twice with phosphate-buffered saline (PBS) without Mg2+ and Ca2+, 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.
Cell numbers were determined in separate experiments. Briefly, quiescent human myometrial cells were incubated with similar treatments. After 72 h, cells were trypsinized and counted with a hemocytometer. The viability of cells was assayed by trypan blue exclusion. Six replicate wells were used for each test condition.
Cell stimulation and extraction of PKC. Human myometrial cells (106 cells/75-cm2 flask) were cultured to subconfluence in the presence of 10% FCS for 48 h. Only cells at passage 4 were used here. Subconfluent cells maintained for 3 days in serum-free media were washed twice with PBS and incubated in culture medium containing 100 nM PDBu or 100 nM ET-1 for the indicated times at 37°C in a humidified atmosphere of 95% air-5% CO2. Incubation was stopped by aspiration of the medium, which was followed by two washes with cold PBS. The cells were then scraped into 20 mM Tris · HCl buffer, pH 7.5, containing 250 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM mercaptoethanol, 2 mM PMSF, 5% glycerol, and 40 µg/ml leupeptin and were sonicated twice for 10 s. The homogenates were centrifuged for 15 min at 1,000 g to remove cell debris and nuclei. After ultracentrifugation for 60 min at 100,000 g, the resulting supernatant was designated as the cytosolic fraction. The pellet was resuspended in the same buffer containing 1% Nonidet P-40 and was gently mixed for 45 min at 4°C. After centrifugation for 30 min at 100,000 g, the supernatant was saved and constituted the particulate fraction.
Protein contents were estimated with the Bradford protein assay (5).
In comparison studies, the same myometria were used for preparation of both crude homogenates and cell cultures. The PKC redistribution in human myometrial tissue was performed as previously reported (8).
Western blot analysis. Equal amounts of proteins (40 µg) from cytosolic and particulate fractions were separated by SDS-PAGE on 8% gels according to the method of Laemmli (23). The separated proteins were electrophoretically transferred to a nitrocellulose membrane overnight as previously described (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, 0.05% Tween 20). Anti-PKC antibodies were added at the appropriate concentration and incubated for different times at room temperature. The membrane was washed with TBST and incubated with the secondary antibody. The blots were developed with ECL reagents and visualized on Kodak X-ray films, and the immunoreactive bands were quantified by densitometric scanning (Studio Scann IISI, Agfa). Rat brain protein extracts were run in parallel as positive controls for the detection of PKC isoforms.
Statistical analysis. Data are presented as means ± SE. The statistical significance of differences in the results was evaluated by one-way ANOVA and Scheffé's multiple comparison test. Significance was set at P < 0.05.
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RESULTS |
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Effect of ET-1 on DNA synthesis and cell growth in
human myometrial cells. We had previously shown that
ET-1 stimulates the incorporation of
[3H]thymidine into DNA
in human myometrial cells cultured in the presence of 0.5% FCS and
increases the cell number (6). When added to quiescent cells cultured
in serum-free conditions, ET-1 exhibited a dose-dependent DNA synthesis
increase, with maximal stimulation (170% of the serum-free control)
occurring at 100 nM ET-1 (Fig.
1A).
The dose-response analysis gave an
EC50 value of 11 nM. The
concomitant increase in cell counts reached statistical significance
only at 100 nM ET-1, with maximal stimulation of 168% of the
serum-free control (Fig. 1B). The
increase in
[3H]thymidine
incorporation and cell number induced by 100 nM ET-1 was less than that
for 10% FCS.
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Effect of PKC and PTK inhibition on ET-1-induced
proliferation. To assess the contribution of PKC to
ET-1-stimulated DNA synthesis in myometrial cells, we examined the
effect of calphostin C and Ro-31-8220. Figure
2 shows that both compounds inhibited, in a dose-dependent manner, DNA synthesis induced by 100 nM ET-1 without affecting basal
[3H]thymidine
incorporation. The maximal inhibitory effect obtained with 1 nM of PKC
inhibitors was ~80% of ET-1 stimulation. Similarly, pretreatment of
myometrial cells with 1 nM calphostin C or 1 nM Ro-31-8220
significantly decreased the cell number induced by 100 nM ET-1
stimulation and evoked 80% inhibition (data not shown). In contrast,
the increase in DNA synthesis in response to 100 nM ET-1 was not
significantly affected by pretreatment with tyrosine kinase inhibitors
(tyrphostins 23 and 51) at any concentration tested (0.01-1 mM).
None of the inhibitors tested was toxic to human myometrial cells under
our conditions as assessed by trypan blue exclusion.
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Prolonged treatment with a phorbol ester is known to induce the
depletion of PKC. This was confirmed in myometrial cells by immunoblot
analysis of the conventional PKC isoforms (see Fig. 7). As shown in
Fig. 3, when myometrial cells were treated
for 36 h in the presence of 100 nM PDBu, ET-1 did not increase the cell
number. Under the same conditions, ET-1 had no significant effect on
[3H]thymidine
incorporation (data not shown).
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Effects of PKC or PTK inhibition on EGF-induced
[3H]thymidine
incorporation. Previous investigations demonstrated
that EGF (1-20 nM) stimulated
[3H]thymidine
incorporation in a concentration-dependent manner (6). The maximum
effect obtained at 15 nM EGF was ~280% of unstimulated serum-free
control. [3H]thymidine
incorporation in response to 15 nM EGF was weakly blocked by 1 nM
calphostin C, whereas 1 nM Ro-31-8220 had no effect (Fig.
4). Depletion of PKC by a 36-h treatment
with 100 nM PDBu did not prevent an increase in EGF-induced DNA
synthesis. By contrast, tyrphostin 51, which is a potent inhibitor of
the EGF receptor (16), reduced by ~58% the increase in
[3H]thymidine induced
by 15 nM of EGF.
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Expression and intracellular distribution of PKC
isoforms in human myometrial cells. Western blot
analysis with isoform-specific antibodies revealed the presence of at
least five PKC isoforms in cultured myometrial cells. Three
conventional PKC isoforms (PKC-,
PKC-
1, and
PKC-
2), one novel PKC isoform
(PKC-
) in small amounts, and one atypical PKC isoform (PKC-
) were
specifically detected. PKC-
and PKC-
were not identified
in our conditions, and the presence of other isoforms was not
investigated. The specificity of the immunobands was tested by
competition in the presence of the appropriate peptides used as
immunogens. Figure
5A shows a representative Western blot analysis of PKC isoenzyme expression in
human myometrial cells.
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Scanning densitometric analysis of the immunobands showed that PKC-,
PKC-
1, and PKC-
were nearly
equally distributed between the soluble (53.4 ± 2.9, 47 ± 3, and 59 ± 4%) and particulate fractions (46.6 ± 2.9, 53 ± 3, and 41 ± 4%, respectively; n = 3). In contrast, PKC-
2 and
PKC-
appeared to be entirely particulate. PKC-
,
PKC-
1, and PKC-
were
abundantly expressed, whereas
PKC-
2 and PKC-
were poorly represented.
A comparative study with results obtained with myometrial tissue was
achieved. No differences in PKC isoform expression were detected
between tissue and cultured cells. Scanning densitometric quantification of the Western blot PKC immunobands visualized in Fig.
5A showed that the amounts of total
immunoreactive PKC- and PKC-
were 2.5-fold and 2.2-fold greater,
respectively, in cells than in tissue. In contrast, the quantities of
the other PKC isoforms, PKC-
1,
PKC-
2, and PKC-
, did not
appear to be very different (136, 70, and 65%, respectively, compared
with tissue).
Comparative subcellular distribution of the various PKC isoforms in
myometrial cells and in myometrial tissue revealed some differences.
Whereas PKC-, PKC-
1, and
PKC-
were equally distributed in myometrial cells and myometrial
tissue, the other two PKC isoforms, PKC-
2 and PKC-
, were found
to be present solely in the particulate fraction (Fig.
5B). It should be noted that
PKC-
2 exhibited several lower-molecular-weight species despite the presence of various antiproteases and that PKC-
appeared to be poorly represented in
both myometrial tissue and myometrial cells.
Subcellular redistribution of PKC
isoforms. The effects of PDBu and ET-1 treatments on
the relative levels of various PKC isoforms in subconfluent
serum-deprived human myometrial cells are shown in Fig.
6.
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Acute treatment (2 and 20 min) with 100 nM PDBu induced an increase in
particulate-associated PKC immunoreactivity of all isoforms tested in a
time-dependent manner. Marked and sustained increases in particulate
PKC-, PKC-
1, and PKC-
were observed (330, 390, and 270% of the control level, respectively),
whereas a slight and more transient enhancement of particulate
immunoreactive PKC-
2 and
PKC-
was detected (150 and 250%, respectively). Interestingly, upon
PDBu stimulation, PKC-
was resolved as two bands. An additional band
of higher molecular weight appeared.
Incubation of myometrial cells in the presence of 100 nM ET-1 elicited
a different pattern in intracellular PKC redistribution. Whereas a
modest, transient change in immunoreactive particulates PKC-1 and
PKC-
2 was seen (150 and 170%
of the control level), a more pronounced and sustained increase in
particulate PKC-
(270%) was observed. However, ET-1 had no
detectable effect on the increase in immunoreactive PKC-
and PKC-
in the particulate fraction.
Long-term effect of PDBu on total PKC isoform
levels. To further characterize the role of PKC in the
mitogenic action of ET-1, we studied the effect of prolonged treatment
with PDBu on the PKC content of the cells. Downregulation of PKC
induced by prolonged exposure of myometrial cells to PDBu is shown in
Fig. 7. Long-term (36 h) incubation with 2 µM PDBu induced a decrease in the total immunoreactive amounts of the
main PKC isoforms detected. PKC- and
PKC-
1 were the most sensitive,
as 55 and 70% were lost, respectively, after this treatment. Under the
same conditions, PKC-
2 and
PKC-
immunoreactivities were decreased by only 10%.
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DISCUSSION |
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Our study demonstrates two principal findings. First, the mitogenic response of human myometrial cells to ET-1 appears to be mediated by activation of PKC. Second, the identification and the regulation of PKC isoforms were evidenced. We initially confirmed the mitogenic properties of ET-1 in terms of [3H]thymidine incorporation and cell proliferation in human myometrial cells. In addition, we demonstrated that ET-1 elicits an increase in DNA synthesis and cell growth in human myometrial cells through the PKC pathway. These observations were made with two chemically different PKC inhibitors that blocked [3H]thymidine incorporation and the increase in cell number stimulated by ET-1. Calphostin C was as potent as Ro-31-8220 at significantly reducing the mitogenic properties of ET-1. It is unclear whether PKC is sufficient for mitogenic signaling by ET-1, because ~20% of DNA synthesis was resistant to both PKC inhibitors. Because we had observed that 1 µM calphostin C as well as RO-31-8220 was toxic for human myometrial cells, higher concentrations were not tested. The failure of ET-1 to induce proliferative properties in PKC-depleted myometrial cells obtained after prolonged treatment with PDBu further supports a crucial role for PKC in this process. Although ET-1 has been shown to activate tyrosine kinases in mesangial cells (36) and in adrenal zona glomerulosa (26), the failure of two PTK inhibitors to affect the mitogenic properties of ET-1 in myometrial cells argues against a role for PTK in this process. Indeed, ET-1-induced [3H]thymidine incorporation was not significantly affected by tyrphostin 23 (a broad-range PTK inhibitor) or tyrphostin 51 (a potent inhibitor of the EGF receptor).
Next, we examined which PKC or PTK pathway was responsible for EGF-induced proliferation in myometrial cells. The expression of EGF receptors in human myometrial cells in culture has been described (34). In addition, EGF displays mitogenic properties (14, 38), and Harrison-Woolrych et al. (19) proposed the involvement of this growth factor in the uterine growth process during pregnancy and in the development of leiomyomas. We confirm that EGF showed greater mitogenic activity compared with the effect of ET-1. The observation that tyrphostin 51 significantly reduced EGF-induced DNA synthesis is consistent with EGF-mediated activation of receptor tyrosine kinase activity, as recently reported in an immortalized human myometrial cell line (2). In addition, our data indicate that the mitogenic response to EGF, in contrast to ET-1, is not dependent on PKC activation. Neither [3H]thymidine incorporation nor the cell number increase in response to EGF was significantly blocked by the two PKC inhibitors.
No information is as yet available concerning the identity of the PKC
isoform(s) responsible for mediating all or part of ET-1-stimulated
mitogenesis in human myometrial cells. In human myometrial tissue,
Western blot analysis has identified the presence of at least five PKC
isoforms: PKC-, PKC-
1,
PKC-
2, PKC-
, and trace
amounts of PKC-
(8). As expected, no differences in PKC isoform
expression were detected between intact myometrial tissue and cultured
myometrial cells. However, the levels of PKC expression differed, and
the two- to threefold higher levels of PKC-
and PKC-
observed in
myometrial cells compared with tissue were in accordance with the
proliferative phenotypic state of the cells. Indeed, specific PKC
isoforms were reported to be associated with smooth muscle cell
proliferation and differentiation. We cannot rule out that PKC-
and
PKC-
are critical for myometrial cell proliferation and may reflect
an activated state of the cells. The predominant expression of PKC
isoforms in the particulate fraction of myometrial cells is compatible
with previous work of Donnelly et al. (13), who reported a more
particulate PKC-associated form and a higher PKC activity in
proliferative cells than in quiescent cells. Similarly, the transformed
cells exhibit higher PKC activity compared with normal cells (22).
Cumulative data suggest that the individual PKC isoform may selectively
elicit specific cellular responses. PKC- has been shown to regulate
proliferative and antiproliferative effects in smooth muscle cells
(32). The expression of this ubiquitous isoform was modified during the
differentiation process and is correlated with the regulation of smooth
muscle cell proliferation and cell cycle progression (40). The novel
PKC-
isoform has been shown to induce cell growth and proliferation
in vascular smooth muscle (18), and
PKC-
2 seems to be associated
with actin microfilaments, suggesting a role in modulation of uterine
contractility (17). In various cell systems, the atypical PKC-
isoform appears to mediate nuclear responses such as maturation, gene
transcription, and proliferation.
In this study, we determined the activation of the various PKC isoforms
identified in human myometrial cells by measuring the increase in their
particulate fraction upon treatment by PDBu and ET-1. PDBu caused a
marked and sustained translocation of all the PKC isoforms detected in
these cells. However, as often reported, the magnitude and time course
of the particulate-associated PKC isoforms showed some differences
according to the isoform considered. Although PKC- contains a
single-system domain, its redistribution after exposure to PDBu may be
observed. No information is yet available as to whether its
translocation resulted from cross-reaction between conventional PKCs
and anti-PKC-
antibody (1) or was the consequence of the high dose
of phorbol ester used. Upon PDBu stimulation, we noted that PKC-
appeared to be present as a doublet, probably reflecting changes in the
phosphorylation state of the isoform as a consequence of its activation
(30). Under the same conditions, ET-1 can also induce rapid and
selective activation of PKC. As observed for phorbol ester,
differential sensitivity of the different PKC isoforms was noted.
Whereas ET-1 induced an increase in immunoreactive
PKC-
1,
PKC-
2, and PKC-
in the
particulate fraction, no variations in PKC-
and PKC-
were
observed. At all times tested, the ET-1 responses were of lower
magnitude than those induced by the direct PKC activator, as reported
in most cell systems.
The inability to detect an ET-1-induced increase in particulate PKC-
and PKC-
may be explained by a redistribution of the two PKC
isoforms into other intracellular sites, such as the perinuclear area
or within the nucleus as reported in several types of smooth muscle
cells, which suggests intranuclear functions and transcriptional control (12, 28, 32, 41, 42). Such a failure to detect translocation of
PKC-
and PKC-
in response to agonists has also been previously
observed for cardiomyocytes (11, 32). It may be considered that
individual isoforms of PKC should be associated with a particular
cellular structure that contains the specific protein substrates for
these isoforms.
Downregulation of PKC by prolonged treatment with PDBu abolished the
mitogenic action of ET-1 in myometrial cells. This observation is in
agreement with previous data that report inhibition of growth in
various cell types after depletion of PKC content (15, 37) and confirms
the role of PKC in the mitogenic properties of ET-1. Western blot
analysis revealed a marked decrease in the total amount of PKC- and
PKC-
1, suggesting that these
two PKC isoforms may be involved in the regulation of ET-1-induced
mitogenesis. However, we cannot exclude a role for the other isoforms
in this process. The failure to observe a consequent loss of
PKC-
2 and PKC-
during this
treatment may be explained by differential phorbol ester depletion of
PKC isoforms. More prolonged treatment in the presence of PDBu might be
necessary to induce complete downregulation of all PKC isoforms. It
should be pointed out that the mechanisms through which ET-1 modulates
cellular growth involve activation of multiple signal transduction
pathways. In addition to stimulating phosphoinositide hydrolysis
through phospholipase C activation (7), ET-1 is able to stimulate
phospholipase D (29) in the myometrium. We cannot rule out that both
pathways may also contribute to the proliferative action observed in
our cellular model.
These data suggest that ET-1-induced proliferation in human myometrial cells is mediated by a PKC-dependent pathway. The differential changes observed in the levels of PKC isoforms indicate that individual PKC isoforms may elicit specific cell responses. In relation to this problem, overexpression of PKC isoforms in various cell lines has been reported, and a correlation with the development of the malignant phenotype was observed (27). PKC activation appears to be involved in the smooth muscle cell-extracellular matrix interaction (18), suggesting a role for this enzymatic system in the metastatic process (24). Moreover, the PKC isoenzyme expression pattern has been associated with proliferative activity in endometrial tumor cells (4). Further studies are required to determine the role of each PKC isoform in human uterine smooth muscle in normal and pathological processes. In particular, the use of antisense oligonucleotides specific to an isoform may help to elucidate the role of each individual PKC isoform.
In summary, the results of this study indicate that PKC may regulate ET-1-induced proliferation in human myometrial cells. We also found that ET-1 caused selective intracellular redistribution of PKC isoforms, providing evidence that they regulate different functions. The main question that remains unanswered is the role of ET-1 in the development or regulation of hypertrophy and proliferation of the human myometrium during pregnancy and in uterine disorders such as leiomyomas.
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ACKNOWLEDGEMENTS |
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We thank E. Dallot for excellent help in cell culture and M. Verger for expert secretarial assistance. The authors are grateful to J. Bram for reviewing the English text and thank the Department of Obstetrics and Gynecology of Cochin-Port-Royal for assistance in obtaining uterine tissues.
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FOOTNOTES |
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We thank Dr. Bradshaw of Roche Products (Welwyn Garden City, UK) for the generous gift of Ro-31-8220.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. Breuiller-Fouché, Inserm U361, Pavillon Baudelocque, 123 Bld de Port-Royal, 75014 Paris, France.
Received 30 July 1998; accepted in final form 11 November 1998.
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