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Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Departments of Obstetrics and Gynaecology and Physiology, University of Toronto, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5
Address all correspondence and requests for reprints to: Stephen J. Lye, Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Departments of Obstetrics and Gynaecology and Physiology, University of Toronto, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5. E-mail: Stephen-Lye{at}compuserve.com
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Abstract |
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Introduction |
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Studies by numerous laboratories, including our own, have shown that activation requires triggering of two signaling pathways in the fetus. The first requires activation of an endocrine cascade involving the fetal hypothalamic-pituitary-adrenal-placental axis, the ultimate output of which in most species leads to a fall in circulating levels of progesterone and an increase in estrogen. While necessary, this endocrine pathway is not sufficient to bring about myometrial activation, and we have recently demonstrated that mechanical tension (or stretch) within the myometrium (as a result of fetal growth during pregnancy) is also required (2). Importantly, under normal circumstances, premature activation of the myometrium by stretch is prevented by growth of the uterus, which itself is stimulated by fetal growth under the influence of progesterone. This involvement of mechanical signals in the growth of the uterus and in the process of myometrial activation at the onset of labor requires a mechanism by which uterine myocytes can both sense the mechanical signal and also generate biochemical responses to these signals.
In many tissues, focal adhesions have been postulated as providing the
means for mechanotransduction. Focal adhesions are comprised of
clusters of integrins at sites where the cell makes contact with the
extracellular matrix substratum (3, 4, 5). These adhesions or contacts are
potential sites for protein tyrosine kinase mediated regulation of cell
behavior such as cell spreading, migration, and anchorage-dependent
survival and proliferation (5). Proteins such as focal adhesion kinase
(FAK), c-src (Src), paxillin, vinculin, as well as proteins such as
GRB2, talin, 
-actinin, and tensin are known to associate at focal
adhesion sites (6). Among these proteins, FAK is believed to play a
critical role in modulating formation and turnover of focal adhesions.
FAK is a 125-kDa cytoplasmic tyrosine kinase that was first described
as a putative substrate for pp60 v-src (7, 8), an oncogenic retrovirus
gene product. It is broadly expressed in cell lines, but there is a
paucity of information regarding its expression in tissues in
vivo. FAK can form a signaling complex with Src, a member of the
Src family of cytoplasmic tyrosine kinases, and signaling can lead to
activation of downstream enzymes such as MAPK (9). Src binds FAK via
its SH2 domain (6), and Src kinase activity appears to be necessary for
focal adhesion turnover but dispensable for translocation to focal
adhesions (10).
Integrin binding has been reported to induce/coordinate tyrosine phosphorylation (P-Tyr) of FAK and of its substrate, paxillin, in a number of cell types (7, 8, 11, 12). Paxillin is a 68-kDa adaptor protein that may have regulatory and structural roles. Tyrosine phosphorylation of paxillin by FAK is thought to recruit SH2 domain-containing proteins such as vinculin, a 117-kDa protein that is highly concentrated in focal adhesions and smooth muscle dense plaques (6). Vinculin also binds F-actin (13), therefore this may allow a physical as well as biochemical link between focal adhesions and the cytoskeleton. Changes in vinculin expression appear to have serious consequences on cellular attachment and motility (6).
During late pregnancy and especially at labor, adhesion of myometrial cells to the extracellular matrix is critical for myometrial growth and for the development of the intense, coordinated contractions of labor. With the exception of some integrins (14), there have been no published reports of focal adhesion-associated proteins detected in the myometrium of the uterus. Our intent was thus to 1) characterize the expression and/or tyrosine phosphorylation (or activity) of FAK, paxillin, vinculin and Src in the myometrium of the rat during late pregnancy; 2) determine whether changes in expression/phosphorylation of focal adhesion proteins were linked to the onset of labor; and 3) to investigate the mechanisms that might control these events. We report for the first time that not only are there dramatic changes in activity of FAK and its substrates in the myometrium during late pregnancy and with the onset of labor, but that the steroid hormone, progesterone, is an important modulator of FAK activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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250 g each) were mated and the day of
observation of a vaginal plug was designated day 1 post coitum. The
time of delivery under these standard conditions was day 23 of
gestation.
Tissue collection
All animals were killed by carbon dioxide inhalation on day 15,
21, 22, 23, and 1 day post partum (1dPP). The uterine horns were
removed, placed in ice-cold saline, and opened longitudinally. The
endometrium was carefully removed by scraping the luminal surface of
the uterus, and myometrial tissue was flash-frozen in liquid nitrogen
and stored at -70 C. Samples were not pooled. The number of animals
analyzed on each day of gestation (n) is documented in the legend of
each figure.
Dynamics of focal adhesion protein expression during pregnancy and
labor
SDS-PAGE and Western blotting. Frozen rat myometrial samples
were pulverized under liquid nitrogen with a mortar and pestle and
homogenized in RIPA lysis buffer [50 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (wt/vol)
sodium deoxycholate, 0.1% (wt/vol) SDS, 100 µM
Na2VO3 and COMPLETE, Mini
EDTA-free protease inhibitors (Roche Molecular Biochemicals , Laval, Québec, Canada)]. Samples
were centrifuged at 15,000 x g at 4 C for 15 min and
the supernatants collected. Protein concentrations were determined by
the Bradford Assay (15) using Bio-Rad Laboratories, Inc.
protein assay dye reagent (Bio-Rad Laboratories, Inc.
Mississauga, Ontario, Canada). Protein samples (100 µg/lane) were
separated by PAGE according to the method of Laemmli (16), using
4–12% gradient polyacrylamide gels (Helixx Technologies, Scarborough,
Ontario, Canada). All protein sample sets were checked for equal
loading with Coomasie Blue staining. Protein samples were
electroblotted to 0.45 µm polyvinylidene difluoride (PVDF) membrane
(Millipore Corp. Canada, Nepean, Ontario, Canada).
PVDF membranes were rinsed in methanol for 10 s then rehydrated in tap distilled water for 3 min. Unless otherwise stated, all incubations were done at room temperature and with constant agitation. Blots were blocked in 5% skim milk powder for 1 h (5% BSA for detection of phosphotyrosines), rinsed 1 x 15 min in Tris-buffered saline (20 mM Tris base, 137 mM NaCl, pH 7.6) with 0.1% Tween-20 (TBST) and 2 x 5 min in TBST. Primary antisera were diluted in blocking solution and incubated with blots for 1 h or overnight at 4 C. Membranes were rinsed 1 x 15 min in TBST, followed by two more washes for 5 min in TBST. Appropriate secondary antisera, conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Baie d’Urfé, Québec, Canada), were also diluted in blocking solution and incubated with membranes for 1 h. Membranes were washed 1 x 15 min in TBST then 4 X 5 min in TBST. Proteins were detected using the Amersham Pharmacia Biotech ECL detection system and multiple exposures were generated to ensure the linearity of the film exposures.
Antisera. Rabbit polyclonal antisera raised against FAK, Src and phosphotyrosines (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal antisera raised against activated MAPK (ERK1 and 2) was obtained from Promega Corp. (Promega Corp., Madison, WI). Monoclonal antisera to vinculin and paxillin were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and Transduction Laboratories (Bio/Can Scientific, Mississauga, Ontario, Canada) respectively. FITC-conjugated goat antirabbit IgG was purchased from Zymed Laboratories, Inc. (Markham, Ontario, Canada).
Immunoprecipitations
Prewashing Protein-A Sepharose 4B.
Protein-A Sepharose 4B
(Zymed Laboratories, Inc.) was washed with 5–10 x
volumes of cold RIPA buffer, vortexed, and centrifuged at 2500 rpm for
1 min. The supernatant was discarded and the pellet resuspended in the
original volume of RIPA buffer.
Immunoprecipitation.
Four hundred microgram protein samples
were diluted in RIPA buffer to a concentration of 1 µg/µl
(i.e. 400 µl for 400 µg), then precleared with 20 µl
of Protein-A Sepharose 4B for 30 min at 4 C on a nutator. Samples were
centrifuged at 1500 rpm at 4 C for 1 min, the supernatants were
retained on ice and the pellet discarded. 1 µg of antisera was added
to each sample and the mixtures incubated at 4 C on a nutator
overnight. 20 µl of Protein-A Sepharose 4B was added to each lysate
and incubated at 4 C for 1 h or overnight on a rotator.
Precipitates were collected by centrifugation at 2500 rpm for 5 min and
supernatants were collected and discarded. The Sepharose 4B pellets
were washed 4 times with 0.5 ml RIPA buffer with repeated
centrifugations at 2500 rpm. The final pellets were resuspended in
1 x SDS loading buffer, boiled 3 min, centrifuged, and analyzed
by PAGE and Western blotting.
FAK kinase assays. FAK kinase assays were based primarily on
a protocol by Guan and Shalloway (17). Immunoprecipitated FAK samples,
bound to protein-A beads, were washed twice with 1 x kinase
buffer (20 mM HEPES, pH 7.5, 10 mM
MnCl2, 1 mM DTT). Thirty microliters
of Poly Glu,Tyr (4:1) substrate beads (Sigma, St. Louis,
MO) were used per kinase reaction. The substrate beads were washed once
with 1 x kinase buffer then added to 2 µl (20 µCi) of
-32P-ATP and 8 µl of 2 x kinase buffer
(40 mM HEPES, pH 7.5, 20 mM
MnCl2, 2 mM DTT). The substrate
mixture was added to FAK/protein A beads and incubated at 30 C for 20
min. Reactions were terminated with the addition of 2 x SDS
loading buffer and samples run on 8–16% polyacrylamide gels. Gels
were dried and exposed to a phosphoimaging screen.
Src kinase assays. Ten micrograms of enolase
(Sigma) per reaction was acid denatured with 1 volume of
acetic acid for 5 min at 30 C then neutralized with 1/2 volume
of 1 M HEPES (pH 7.4) buffer. An equal volume of 2x kinase
buffer (40 mM HEPES, pH 7.5, 20 mM MnCl2, 2
mM DTT) was added, followed by 10 µCi of
-32P-ATP. This reaction buffer was added to
protein-A Sepharose beads, complexed to immunoprecipitated Src, that
had been washed twice in 1 x kinase buffer. Kinase reactions were
performed at 30 C for 15 min. Reactions were terminated with the
addition of 2 x SDS-loading dye and analyzed on polyacrylamide
gels. Gels were stained with Coomasie Blue and dried before exposure in
a phosphoimaging cassette. Densitometric analysis of images was
performed with Storm 860 phosphoimaging software (Molecular Dynamics, Inc.).
Paraffin embedding. Rat uteri were fixed in 4% paraformaldehyde in PBS overnight at 4 C with constant agitation. Tissues were rinsed with two washes of cold PBS and then dehydrated for one hour each through a graded series of ethanol (70%, 80%, 90%, 95%, 100%). Samples were cleared in xylene for 1 h and then infiltrated in paraffin overnight at 60 C. Uteri were then placed in moulds containing fresh paraffin before cooling.
Immunofluorescence detection of FAK. Five micrometer thick paraffin sections of rat uteri, from various stages of late pregnancy, were placed on SUPERFROST Plus (Fisher Scientific Ltd., Nepean, Ontario, Canada) slides and dried at 37 C overnight. Sections were dewaxed in xylene, rehydrated in descending grades of ethanol, and finally soaked in PBS. Tissues were permeabilized in 0.125% Trypsin in PBS for 10 min at room temperature and rinsed in PBS. Sections were blocked in 5% normal goat serum/1% BSA for 30 min at room temperature. Rabbit anti-FAK, at a dilution of 1:250, was used as a primary antibody and sections incubated overnight with antibody at 4 C. Tissue sections were rinsed in PBS and incubated for 1 h with FITC conjugated antirabbit IgG, at a dilution of 1:100, at room temperature on a shaker. Tissues were washed in cold PBS containing 0.02% Tween 20 and mounted in Vectashield (Vector Laboratories, Inc., Burlington, Ontario, Canada) before observation on an MRC-600 laser scanning confocal microscope (Bio-Rad Laboratories, Inc.).
Hormonal modulation of myometrial-cytoskeletal signaling
Progesterone-delayed labor experiments. The onset of labor
is associated with a withdrawal of the inhibitory effects of
progesterone on the myometrium consequent on a fall in plasma levels of
this steroid. To determine whether progesterone withdrawal might
modulate focal adhesion signaling, rats were randomized to receive
either a daily injection of progesterone (4 mg, sc, in 0.2 ml corn oil)
to maintain elevated plasma levels of this steroid or vehicle alone
(corn oil, 0.2 ml sc) beginning on day 20 of gestation (18). Control
animals were killed on day 21, day 22, and day 23 (during delivery).
Progesterone-treated rats were killed at the same gestation days;
however, on day 23 the rats were not in labor (since progesterone
blocks the normal onset of labor on day 23). Myometrial samples were
taken as described above. Focal adhesion signaling was monitored by
analysis of FAK P-Tyr/kinase activity.
Statistical analysis
Densitometric data were expressed as the mean densitometric
units ± SEM. Data were subjected to a one-way ANOVA,
followed by a Bonferroni or Student-Newman-Keul’s method multiple
comparison test to determine between-group differences. The level of
significance used was P < 0.05.
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Results |
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, A
and B), statistically significant (P < 0.05) changes
were detected in the expression of FAK and paxillin. FAK expression
significantly increased 3-fold (Fig. 1C) between days 15 and 22 of
gestation. In addition, FAK expression at 1dPP was 4-fold greater than
day 15 and 2-fold greater than day 23. Paxillin levels at 1dPP were
also significantly higher, increasing 1.9- and 1.7-fold over day 22 and
day 23 gestation stages respectively (Fig. 1D).
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and 3A). Greater expression was detected,
however, from day 21 onwards in both layers (Figs. 2, B–E, and 3,
B–E). FAK was detected in both cell/ECM contact sites and perinuclear
regions of myocytes (Fig. 4). In circular
muscle layers at day 23, however, FAK was primarily localized to
perinuclear regions (Fig. 2D). In the longitudinal muscle of the
myometrium, FAK was detectable in regions of close cell/ECM contact
(Fig. 3, B and C, E), but at day 23, longitudinal muscle also contained
significant amounts of FAK in perinuclear regions of myocytes (Fig. 3D).
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) during labor and delivery of the
fetuses (day 23). FAK kinase assays with an exogenous substrate
confirmed that the observed FAK P-Tyr levels represented an active
kinase (Fig. 6). Because paxillin is a
downstream substrate for FAK, we also analyzed the P-Tyr content of
paxillin as a potential marker of the activation of a focal
adhesion-cytoskeleton transduction pathway. Paxillin P-Tyr levels
followed an almost identical pattern to FAK, increasing 4-fold between
day 15 and day 21 and decreasing greater than 75% between day 21 and
day 23 (Fig. 5B). Src kinase activity did not significantly change
throughout late pregnancy (Fig. 5C).
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). Expression of both ERK1 and ERK2 (42 kDa)
was significantly elevated throughout late pregnancy compared with 1dPP
when levels fell by 75–80%.
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and 9). Moreover,
in these progesterone-treated rats in which the fall in FAK P-Tyr was
blocked, labor did not occur.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of focal adhesion proteins during late pregnancy
FAK, paxillin, Src, and vinculin were all detected by Western blot
analysis in the rat myometrium during late pregnancy, but only FAK and
paxillin displayed significant changes in expression with levels
continuing to increase through postpartum. The postpartum period is a
time of considerable change in the uterus, and in particular the
myometrium, as this tissue undergoes a process of dramatic remodeling
(termed involution) as it reverts to the nonpregnant state (20).
Involution involves considerable apoptosis, subsequent cell growth and
remodeling, processes that require reorganization of ECM-cytoskeletal
interactions at focal adhesions. In other systems, FAK and paxillin
have been implicated in the reorganization of focal adhesions
particularly during cell migration (21) and it is possible that they
play a similar role in the myometrium.
Modulation of FAK-P-Tyr and enzyme activity during late pregnancy
and the onset of labor
The tyrosine phosphorylation of focal adhesion-associated proteins
is of fundamental importance for regulating enzymatic activity and
protein-protein interactions. We found a dramatic increase in FAK P-Tyr
and enzyme activity between days 15 and 22, a time when we have shown
that the myometrium is undergoing maximal hypertrophy (Gleicher and
Lye, unpublished observations) likely as a consequence of exponential
increases in fetal growth. This period of late pregnancy is also a time
when there is substantial remodeling of the ECM, including increased
collagenolysis, the formation of elastic fibres within the circular
muscle of the myometrium, as well as marked increases in fibronectin
localization and thickness of continuous basement membranes (22).
Investigation of mice carrying a null mutation in the fak
gene suggest a positive association between FAK P-Tyr/activity and the
rate of focal adhesion turnover (23). While this mutation results in
embryonic lethality, fibroblasts from fak -/- null
embryos, when grown in tissue culture, exhibit very large focal
adhesions and significantly reduced cell migration, suggesting
decreased focal adhesion turnover. Thus the elevated FAK P-Tyr during
late pregnancy might mediate focal adhesion turnover and hence support
growth (hypertrophy) of the myometrium at this time.
The decrease in FAK phosphotyrosine content at day 23 suggest that focal adhesions are stabilized. This may be necessary for efficient and coordinated labor contractions to occur. In addition, because considerable FAK is detected in perinuclear regions of myocytes at day 23 and no cleaved forms of FAK, due to caspase activity (21), were detected on Western blots (data not shown), this perinuclear localization may represent newly synthesized FAK in the endoplasmic reticulum. It is possible that FAK is involved in additional function(s) during labor and in the postpartum period. Work by Frisch et al. (24) and Hungerford et al. (25) have shown that decreased FAK activity may be associated with increased apoptosis. Therefore, FAK deactivation near or at the termination of pregnancy may facilitate apoptosis within the myometrium and contribute to the involution process.
Our observation that exogenous administration of progesterone maintains elevated FAK P-Tyr levels and kinase activity on day 23 (when FAK P-Tyr normally falls) is the first indication that the level of tyrosine phosphorylation of FAK may be modulated by steroid hormones, in this case progesterone. These data also suggest that the decrease in FAK P-Tyr and enzyme activity with the onset of labor on day 23 is likely due to the endogenous fall in plasma progesterone levels that occurs at this time (1, 26). The mechanisms underlying this effect remain to be determined. One might speculate that, in the present study, progesterone may have modified the expression (or indirectly the activity) of a kinase for which FAK is a substrate. Because progesterone administration blocks the increase in estrogen normally seen during labor, it is also possible that the decrease in FAK activation is due to an action of estrogen (perhaps through induction of a phosphatase). Whatever the mechanism, these data provide an interesting area for future investigation.
The possible importance of focal adhesion assembly and signaling to physiologic events such as labor was underscored by the observation that in rats treated with progesterone not only was the elevated level of FAK P-Tyr and enzyme activity maintained but that labor was delayed in all of these animals.
FAK activation and downstream signaling
FAK activation has consequences on two major systems within the
cell; the recruitment and formation of the focal adhesion-cytoskeleton
complex, and the activation of downstream intracellular signaling
pathways. Paxillin is a substrate of FAK (11) and the close association
between its increased phosphorylation and the increase in FAK P-Tyr and
enzyme activity around day 20–22 is consistent with FAK
activation-induced paxillin phosphorylation within the myometrium at
this time. In its phosphorylated state, paxillin can interact with
other proteins, such as vinculin, through SH2 adaptor domains to
coordinate assembly of the focal adhesion-cytoskeleton complex (11, 27). Interactions between FAK and other kinases (e.g. Src at
the Tyr 397 site) leads to activation of signaling pathways
(e.g. MAPK pathway). Active ERK1 and ERK2 are present in
myometrial cells during late pregnancy to act as potential targets of
signaling initiated by FAK. In the present study, we found no major
change in Src kinase activity during the period of maximal FAK
activation. However, it would be premature to suggest that interactions
between FAK and Src are not occurring at this time because in this
study we analyzed total cellular Src rather than that component that is
associated with FAK.
FAK activation and the onset of labor
The onset of labor is associated with remarkable changes in
myometrial function, with this muscle switching from a state of
quiescence during pregnancy to intense activity during labor. This
activation process is characterized by the increased
expression of a cassette of genes encoding contraction-associated
proteins or CAPs, including gap junctions, agonist receptors, and ion
channels that confer increased contractility to this muscle. Recent
work from this laboratory suggests that the increased expression of
these CAPs requires both endocrine (estrogen and progesterone) and
mechanical (uterine stretch due to fetal growth) signals. Several
investigators have shown that increasing mechanical strain on vascular
endothelial cells or vascular smooth muscle cells activates
integrin-mediated signaling through focal adhesion complexes (9, 28, 29). In addition, stretching of mesangial cells has been shown to
increase FAK tyrosine phosphorylation, coincident with increased
production of ECM proteins such as fibronectin, collagen, and laminin
(30). It is possible that stretch of uterine myocytes during late
pregnancy might also modulate myometrial FAK expression/activity.
Our data suggest a role for FAK in the onset of labor. We speculate that during late pregnancy, fetal growth imposes mechanical tension on the myometrium, which in turn induces activation of FAK leading to focal adhesion turnover and supporting myometrial cell hypertrophy. The resulting growth of the myometrium acts to attenuate the tension induced by fetal growth. The fall in endogenous levels of progesterone at term leads to a fall in FAK P-Tyr/kinase activity, stabilization of focal adhesions (rather than turnover) and the cessation of myocyte hypertrophy. Because fetal growth continues, this places the uterine myocytes under significant tension leading to increased expression of CAPs, myometrial activation, and the onset of labor.
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Acknowledgments |
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Footnotes |
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Received July 28, 1999.
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