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
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ABSTRACT |
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protein kinase C; uterine smooth muscle; parturition
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 ,
1,
2, and
; novel PKC isoforms (nPKC) including
,
,,
,
,
and
; and atypical PKC isoforms comprising
,
, and
.
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
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
(31).
We previously demonstrated that among the six isoforms of PKC (,
1,
2,
,
, and
) found in
the pregnant human myometrium, only PKC
is required for ET-1-induced
proliferation of human myometrial cells
(12). There is indeed some
evidence that PKC
and/or PKC
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
is involved in the contractile potentiation of ET-1 in the
porcine coronary artery (33),
whereas PKC
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 and
in human
myometrial contractility, we first studied the role of each PKC isoforms in
the contractile process by using a selective inhibitor of PKC
(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 (peptide Z) (11). An
additional approach has consisted in targeting PKC
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 was questioned in this process.
These different approaches provide the first evidence that only PKC
activation mediates ET-1-induced myometrial contraction and, furthermore, that
ET-1 is able to induce the translocation and the association of PKC
to a
structure coincident with actin cytoskeleton.
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MATERIALS AND METHODS |
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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 was from Sigma-Aldrich and raised against peptides 577592.
The antibody against smooth muscle
-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 (812
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) 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 9095% 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
-actin.
Oligonucleotide treatments. Anti-sense phosphothiorate PKC
oligonucleotide (AS-ODN) and scrambled PKC
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
mRNA, were complementary to positions 587601 and
12601278 of the PKC
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
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 510 separate experiments, where the percentage of contraction is the percentage of decrease in terms of the original surface area.
Immunofluorescence analysis. PKC 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
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
and
actin. Cells were incubated overnight at 4°C with the first monoclonal
antibody against smooth muscle
-actin and the first antibody against
PKC
. 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 500555 nm for FITC and 568 and 600640
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 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.
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RESULTS |
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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
(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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Effects of a PKC antisense oligonucleotide on
ET-1-mediated collagen lattice contraction. Two AS-ODN directed against
human PKC
, AS-1 ODN and AS-2 ODN directed against human PKC
mRNA,
and their corresponding scrambled PKC
-ODN control were used to determine
the role of PKC
in ET-1-induced myometrial cell contraction. Collagen
lattices were incubated alone or with increasing concentrations of AS-ODN
(0.54 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 (14 µM) for 48 h, and the
protein levels were assessed by Western blotting to determine whether AS-ODN
actually reduced the amount of PKC
. Immunoblot analysis confirmed that a
concentration-dependent reduction in PKC
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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Immunofluorescent localization of PKC. Immunocytochemistry
using antibody against PKC
confirmed the presence of PKC
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
antibody (data not shown). Untreated myometrial cells showed a
diffuse distribution of PKC
throughout the whole cytoplasm including the
extremities. A short incubation (210 min) with ET-1 (100 nM) resulted
in a shift of PKC
toward the perinuclear area, whereas the staining of
the extremities of the cells was less visible. The immunofluorescent
localization of PKC
did not change after ET-1 incubation for 20 or 30
min.
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As shown in Fig.
3B, the effect of ET-1 on PKC 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 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
. By
contrast, we confirmed that ET-1 had no detectable effect on the increase in
immunoreactive PKC
in the particulate membrane fraction as we previously
observed in myometrial cells from nonpregnant women
(39).
Association of activated PKC 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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We assessed the effect of the AS-1-ODN directed against human PKC
mRNA to determine whether PKC
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
-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
and an anti-
-smooth muscle actin revealed that the immunostaining of
PKC
is somewhat similar to that of actin. Note that in untreated cells,
PKC
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
immunofluorescence
colocalized with that of actin in the perinuclear area
(Fig. 5B).
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DISCUSSION |
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We first used rottlerin, an inhibitor of PKC. This compound was
reported to show preference for PKC
(IC50 = 36
µM), whereas higher concentrations of rottlerin were required to inhibit
the activity of other PKC isoforms with IC50 values for
PKC
,
of 3042 µM and PKC
,
of 80100
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
is not
implicated in the ET-1-induced myometrial contraction. Little is known about
the role of PKC
in uterine function at the end of pregnancy, but
several studies demonstrated that PKC
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
(12), it is uncertain whether
PKC
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 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
(31) and that PI3
kinase substrate, PDK-1, phosphorylates PKC
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
-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 (
,
) and PKC
were without
effect. The direct involvement of PKC
in ET-1-induced contraction was
confirmed both by using the PKC
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
markedly blocked the myometrial contractile response of ET-1.
Additionally, the localization of PKC was investigated in human
myometrial cells. The translocation of PKC
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
to a structure coincident with the actin cytoskeleton. However, we
did not observe membrane-associated PKC
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
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
to show that inhibition of PKC
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. PKC2 seems to be closely associated with the actin component of
the cytoskeleton in NIH-3T3 fibroblasts, whereas PKC
accumulates in
Golgi organelles and PKC
associates with nuclear membranes
(16). The microtubules play an
important role in the translocation of PKC
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
and
(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
and actin is also observed. The same group
proposed a role for PKC
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
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
mediates
nuclear responses such as gene transcription and proliferation
(7), but ET-1 increased
membrane-associated PKC
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 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
(43). Whether PKC may
phosphorylate in vitro cytoskeletal proteins in human myometrial cells
requires clarification.
In conclusion, we have shown that PKC 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)-
/IL-1
and premature
childbirth has been proposed
(1). Additionally, aPKCs are
important components of the TNF-
/IL-1
signaling pathway that
control NF-
B activation
(8), and recent evidence also
indicates that targeted disruption of the gene of PKC
results in the
impairment of the NF-
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
to uterine contraction at term pregnancy
in humans.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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