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Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama (T.L.C., J.L., H.S.), Birmingham, Alabama 35294-0019; ProPath Laboratory, Inc. (R.T.M.), Dallas, Texas 75207-4009; and Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center (R.A.W.), Dallas, Texas 75390-9032
Address all correspondence and requests for reprints to: Dr. Trudy L. Cornwell, VH Room G019, Department of Pathology, University of Alabama, 1670 University Boulevard, Birmingham, Alabama 35294-0019. E-mail: cornwell{at}path.uab.edu
Abstract
Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.
PREGNANCY IS ASSOCIATED with adaptations of uterine smooth muscle function such that the uterus remains quiescent during most of gestation. Although a number of structural and biochemical modifications have been described, the mechanisms responsible for maintaining uterine smooth muscle in a relaxed state for 37–40 wk are not completely known (1, 2, 3, 4, 5). Smooth muscle contraction results from thick (e.g. myosin) and thin (e.g. actin) filament interactions, initiated by calcium/calmodulin-dependent myosin phosphorylation. The thin filament proteins, calponin and caldesmon (2, 6, 7, 8, 9, 10, 11, 12, 13), and the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway (14, 15, 16) are believed to inhibit actomyosin interactions through distinct cellular mechanisms. The potential of these pathways to serve as regulators of uterine smooth muscle relaxation in pregnancy has not been fully explored.
Caldesmon and calponin inhibit actin-activated myosin adenosine triphosphatase (ATPase) activity in vitro (6, 7, 8), and both proteins are proposed to play an important role in the regulation of cross-bridge cycling in smooth muscle (6, 12, 13). Caldesmon is localized to contractile domains of smooth muscle cells (9, 10), whereas calponin is predominantly localized to the cytoskeletal domain (10, 11). Major changes in the expression of contractile proteins and their isoform distribution occur as smooth muscles undergo adaptations during physiological (17, 18) and pathophysiological (19, 20) conditions. For example, in uterine smooth muscle during pregnancy, the high mol wt, smooth muscle-specific isoform of caldesmon (h-CDM) is disproportionately increased relative to smooth muscle myosin or actin (2). Although in vitro studies established that h-CDM inhibits contractile filament interactions, it is not understood how these interactions function in vivo, nor is the significance of increased h-CDM levels during gestation understood.
Myosin and actin filament interactions are also opposed by decreases in the amount of Ca2+-dependent myosin phosphorylation. Activation of soluble guanylate cyclase by NO leads to an increase in tissue cGMP levels. A major mechanism of cGMP-induced vasorelaxation is activation of PKG, which results in decreased free intracellular Ca2+ (14, 15, 16) and increased myosin phosphatase activity (21) in vascular smooth muscle cells. Although evidence supporting a role for this signaling pathway in the maintenance of uterine quiescence during pregnancy has been reported (22, 23, 24, 25), experimental evidence from other studies conducted both in vivo and in vitro (3, 26, 27, 28) is conflicting. Previously, we reported that high concentrations of NO donors or cGMP analogs are required to elicit relaxation in myometrial tissues from pregnant rats, in part due to progesterone-mediated decreases in PKG (3). This was of major concern in view of the potential use of NO donors as tocolytic agents (23) and the nonspecific effects of high concentrations of NO (29, 30). Considering the well documented differences in the reproductive biology and endocrinology of parturition among species (1), a goal of the current study was to examine the regulation of the components of the NO/cGMP signaling pathway in the human uterus. Specifically, the levels and subcellular distribution of PKG in human uterine specimens were determined relative to those of h-CDM and calponin. The results suggest that mechanisms other than cGMP-induced activation of PKG regulate uterine smooth muscle contraction and relaxation during human pregnancy. We also report a unique subcellular redistribution of the thin filament-associated proteins in myometrial tissues from pregnant women.
Materials and Methods
Source of myometrial tissues
Normal human myometrial tissue was obtained from uteri of
nonpregnant women undergoing hysterectomy for benign gynecological
conditions of the myometrium or well differentiated malignancies not
involving the myometrium (Table 1
). In
uteri from nonpregnant women, normal myometrium was obtained from the
uterine fundus remote from the endometrium, cervix, or leiomyomas. In
pregnant women, myometrium was obtained either from the fundus at the
time of cesarean hysterectomy or from the superior margin of the
uterine incision at the time of cesarean section (Table 2). Informed consent in writing for the
use of tissue was obtained from the women undergoing surgery according
to protocols approved by the institutional review boards for human
experimentation at the University of Texas Southwestern Medical Center
and institutional review boards of the University of Alabama
(Birmingham, TX), St. Paul (Dallas, TX), and Brookwood (Birmingham, AL)
Hospitals. Tissues were dissected free of serosa, connective tissue,
and major blood vessels, rinsed three times in PBS, and snap-frozen in
liquid N2 before use.
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Extracts were prepared by homogenization of pulverized tissue using a Polytron (three 10-sec bursts, setting 50%) in 3 vol (0.4 ml) of PE buffer [20 mM potassium phosphate (pH 7.0), 150 mM NaCl, 0.32 M sucrose, 2 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin]. Crude soluble and particulate fractions were separated by centrifugation at 12,000 x g for 10 min. In certain experiments pellets were reextracted by sonication (setting 50%) in PE buffer containing detergents or additional salts as follows. Pellets from the initial extraction (supernatant = extract 1) were washed with 1 ml PE buffer, then reextracted by sonication in 0.4 ml PE containing 1% Triton X-100. After centrifugation at 12,000 x g for 10 min, supernatants (extract 2) were stored on ice, and pellets were washed with 1 ml PE buffer before further extraction in PE buffer containing 0.6 M KCl. After centrifugation as described above, supernatants (extract 3) were stored on ice, and pellets were washed in 1 ml PE buffer before a final reextraction in PE buffer containing 2% SDS (extract 4). Extracts 1–4 were subjected to Western blot analysis for caldesmon (10–15 µg/lane), PKG (15 µg/lane), and calponin (1–2 µg/lane in Fig. 5).
Western blot analysis
Protein in tissue extracts or purified PKG from bovine lung was
separated by SDS-PAGE on 10% gels and transferred to nitrocellulose at
70 V for 14–16 h in the presence of methanol (20%). Blots were then
briefly rinsed in Tris-buffered saline (TBS) and blocked with TBS
containing 0.1% Tween 20 (TBST) and 4% powdered milk for 1 h.
For analysis of h-CDM, PKG, and calponin under identical experimental
conditions, membranes were divided into three sections according to mol
wt determinations and incubated with appropriate dilutions of
antibodies. For PKG, blots were incubated overnight at 4 C with a
polyclonal PKG peptide antibody (StressGen Biotechnologies Corp.,
Victoria, Canada) diluted in TBS containing 2.5% BSA and 0.05% sodium
azide (1:5,000 or 1:10,000). Thereafter blots were washed three times
with TBST (5 min each) and incubated with horseradish
peroxidase-conjugated goat antirabbit IgG (1:20,000 to 1:40,000). After
extensive washing with TBST or TBS containing 0.05% SDS, 0.05%
Nonidet P-40, and 0.125% deoxycholate, the blots were developed with a
chemiluminescent detection system (Pierce Chemical Co.,
Rockford, IL). The polyclonal PKG antibodies used in this study were
made by immunizing rabbits against a C-terminal peptide of human PKG
I
. This region of the protein is identical in bovine and human
species and in both I and Iß isoforms of PKG. These antibodies
were used to quantitate PKG (I and Iß) in the human extracts by
comparison to standards of purified bovine PKG I. In the Western
blots presented, PKG I (76 kDa) and Iß (78 kDa) comigrate. A
monoclonal antibody to chicken gizzard caldesmon (CD21,
Sigma, St. Louis, MO) was used at a dilution of 1:30,000
to detect both high and low mol wt isoforms of caldesmon. A monoclonal
antibody to human uterine smooth muscle calponin (hcp,
Sigma) was diluted 1:80,000 for the detection of calponin.
Sheep antimouse horseradish peroxidase (1:50,000 to 1:100,000;
Jackson ImmunoResearch Laboratories, Inc., West Grove, PA)
was used as secondary antibody for caldesmon and calponin blots.
Relative amounts of immunoreactive proteins were quantified using a
Hewlett-Packard Co. ScanJet 3c (Palo Alto, CA) and Scion
Image analysis program (Scion Corp., Frederick, MD). Density units of
immunoreactive bands for samples appearing on different blots were
normalized by comparison to values of one extract that was included on
all blots.
Immunohistochemistry for PKG
Formalin-fixed, paraffin-embedded tissues were sectioned at 5 µm and mounted on slides in which both positive (human lung, kidney, adrenal, spleen, and placenta) and negative (human liver and skin) control sections for PKG were mounted (31). These controls were first used to optimize antigen retrieval conditions and antibody dilutions to obtain specific staining for PKG. After drying, the slides were deparaffinized in xylene and graded alcohols to water. Epitope retrieval was then performed in a pressure cooker using 0.25 M Tris-HCl (pH 9) as the epitope retrieval solution. Dilute egg white solution and skim milk were used to block endogenous avidin-biotin activity as previously described (32). Thereafter, slides were incubated in primary PKG antibody (StressGen Biotechnologies Corp.; 1:75) for 30 min at 25 C. After a similar incubation with biotinylated secondary antibody (Scytek, Logan, UT) for 15 min, slides were placed in 0.3% H2O2 in PBS for 10–13 min to quench endogenous peroxidase activity. Slides were then incubated with horseradish peroxidase-conjugated streptavidin (Scytek) for 15 min at 25 C. Reaction product was developed by immersing the slides in prepared diaminobenzidine solution (Research Genetics, Inc., Huntsville, AL) at 32 C for 4 min. The slides were then rinsed in tap water and placed in 0.5% copper sulfate in normal saline for 5 min at 25 C to enhance the appearance of the chromogen. Slides were rinsed in water, counterstained in hematoxylin, dehydrated in graded alcohols and xylene, and protected with a coverslip. Negative control sections of the same tissue were stained as described above, except the primary antibody was replaced with nonimmune rabbit IgG.
Assay for PKG enzyme activity
PKG activity was determined from crude soluble extracts.
Incorporation of 32P from
[
-32P]ATP (NEN Life Science Products, DuPont, Boston, MA) into histone F2b was monitored
using a filter paper assay (33). Extraction in 1% Triton
X-100 was used to obtain maximal cGMP-stimulated activity.
Statistics
Results were expressed as the mean ± SEM. Statistical comparisons between groups were conducted by t test. P < 0.05 was considered significant.
Results
Quantification of PKG in myometrial tissues from nonpregnant and pregnant women
Immunodetection of PKG in protein extracts from human
myometrial tissues was conducted by Western blot analysis (Fig. 1). First, standards of PKG purified from
bovine lung and protein extracts from selected uterine specimens (from
both nonpregnant and pregnant myometrium) were analyzed to determine
conditions for accurate quantitation (data not shown). Immunoreactivity
for purified PKG was linear between 5–25 ng. In extracts, linear
values were obtained between 6.25–25 µg protein (nonpregnant) and
12–50 µg (pregnant). Having validated the assay method for
determining quantitative amounts of PKG in myometrial tissue, the
levels of PKG in myometrium obtained from pregnant (n = 13) and
nonpregnant (n = 18) women were determined in three overlapping
sets of Western blots. The clinical characteristics of the women from
whom specimens were procured are summarized in Tables 1
and 2. A single
immunoreactive band of 78 kDa that comigrated with purified PKG was
apparent in all samples analyzed. Levels of PKG in myometrial tissues
from pregnant women were reduced 3-fold compared with those from
nonpregnant women (Fig. 1B; P < 0.05).
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; P = 0.86).
Next, we evaluated the possibility that the expression of PKG was
decreased in myometrial tissues at term relative to those obtained
earlier in gestation. As shown in Fig. 2B, PKG levels in myometrial
samples from women delivering preterm were not different than
specimens taken from full-term deliveries (P = 0.92).
It might be predicted that PKG levels would be reduced in laboring
myometrium to facilitate force generation during labor. Expression of
PKG in tissues obtained from women in labor, however, were similar to
those obtained from women before the onset of labor (Fig. 2C;
P = 0.38). These studies indicate that expression of
PKG is reduced significantly in myometrium from pregnant women, and
this reduction is not due to differences in tissue sampling, time in
gestation, or laboring status. In nonpregnant women, PKG expression was
similar in pre- and postmenopausal women (Fig. 2D; P =
0.26).
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To determine whether the decrease in immunoreactive PKG corresponds to a decrease in cGMP-stimulated kinase activity, cGMP-dependent enzyme activity was measured. In myometrial extracts from nonpregnant women, cGMP-stimulated PKG activity was 727 ± 136 pmol/min·mg (mean ± SEM; n = 3). cGMP-stimulated PKG activity was significantly decreased in myometrial extracts from pregnant women (246 ± 76 pmol/min·mg; n = 3; P < 0.05).
Cellular localization of PKG in human myometrium
Immunohistochemistry was used to determine the distribution of PKG
expression in myometrium from nonpregnant and pregnant women (Fig. 3). In nonpregnant women,
immunoreactive staining was prominent in the cytoplasm of uterine and
vascular smooth muscle cells. Immunoreactivity was more intense in the
arterial smooth muscle cells compared with uterine myocytes, and PKG
expression was absent in stromal cells surrounding myometrial muscle
bundles and vascular plexi (Fig. 3A). As in nonpregnant women,
vascular smooth muscle showed intense staining for PKG in myometrial
tissues from pregnant women (Fig. 3B). Staining was weak in uterine
smooth muscle cells, however, compared with vascular smooth muscle
cells in the same section (Fig. 3B) or myometrial cells from
nonpregnant women (Fig. 3A). The striking differences in staining
intensity between vascular smooth muscle and myometrial cells were
observed in uterine sections from three different pregnant women and in
all sections examined.
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The amounts of many proteins associated with the contractile
system are similar in myometrium from pregnant and nonpregnant women
(2, 5). The level of h-CDM, however, is increased in
myometrium during pregnancy (2). To validate the finding
that PKG is reduced in myometrium from pregnant women, comparisons with
proteins shown to increase (h-CDM) or remain the same (calponin) in the
myometrium during pregnancy were performed (Fig. 4). As expected, Western blot (Fig. 4A)
and densitometric (Fig. 4B) analyses indicated a 3-fold increase in
h-CDM in extracts from pregnant uterus. Unexpectedly, the expression of
calponin was also increased 3-fold in tissues from pregnant women.
On the other hand, PKG levels were reduced in tissues from pregnant
women despite increased expression of h-CDM and calponin (Fig. 4).
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The cellular distributions of these proteins were analyzed further
by extracting pregnant and nonpregnant myometrial specimens
sequentially with salts and detergents (Fig. 5A). Relative amounts of each protein in
crude soluble (extracts 1 and 2) and myofilament/cytoskeletal (extracts
3 and 4) fractions were calculated and are shown in Fig. 5B. PKG
distribution did not differ between pregnant (n = 6) and
nonpregnant (n = 7) tissues in the soluble fraction
(P = 0.43) or myofilament/cytoskeletal fractions
(P = 0.43). Total PKG, however, was decreased
significantly in tissues from pregnant women relative to that from
nonpregnant women (Fig. 5C), consistent with the results shown in
Figs. 1–3. Although h-CDM was readily extracted with PE and Triton buffers
from both pregnant (n = 4) and nonpregnant (n = 3) tissues,
extraction with high salt or SDS was required to fully extract h-CDM
from nonpregnant tissues (Fig. 5, A and B). Like h-CDM, the percentage
of calponin extracted in the soluble fraction was significantly greater
in pregnant (n = 3) specimens compared with nonpregnant (n =
3) specimens (P < 0.01). Conversely, greater amounts
of calponin were distributed in the myofilament/cytoskeletal fractions
in nonpregnant tissues (P < 0.01). Total levels of
h-CDM (total immunoreactivity in all fractions) were increased
approximately 4-fold in pregnant myometrium (Fig. 5C). The total level
of calponin, however, was similar in nonpregnant and pregnant
myometrial tissues (Fig. 5C). Thus, in myometrial tissues during
pregnancy, both h-CDM and calponin appear to be redistributed to a more
readily extracted pool, indicating a lower affinity for cytoskeletal
and myofilament proteins.
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Myometrial contractions occur through a calcium-stimulated actin-myosin-based contractile protein apparatus, similar to other smooth muscle tissues. Many agents known to inhibit force in vascular smooth muscle also function to inhibit uterine smooth muscle contractility. However, uterine smooth muscle has unique properties, such that contractility is suppressed in pregnancy. In this study we investigated the potential of two distinct inhibitory pathways to be involved in the suppression of uterine contractility during pregnancy. First, we evaluated the expression and distribution of PKG (the downstream target of NO-induced increases in cGMP). Investigations regarding modulation of the NO/cGMP signaling system in uterine smooth muscle are controversial with data in support of (22, 23, 24, 25) and against (3, 26, 27, 28) a role for NO and cGMP in mediating uterine quiescence of pregnancy. Second, we compared the expression and distribution of PKG with those of two thin filament-associated proteins, h-CDM and calponin. These two proteins are believed to modify contractility by inhibiting actin-activated myosin ATPase activity (6, 7, 8, 9, 10, 11, 12, 13) and, in the case of caldesmon, by tethering actin to myosin and inhibiting the velocity of actin/tropomyosin filaments in the presence of nonphosphorylated myosin (34).
We found that PKG levels are significantly down-regulated in myometrial tissues during pregnancy, with no differences in preterm and term myometrium, lower uterine segment and fundus, or tissues obtained before and after the onset of labor. It is predicted that a decrease in PKG should result in compromised relaxation in response to NO-induced increases in cGMP. Indeed, Jones and Poston reported that myometrial tissues from pregnant women were refractory to relaxation by L-arginine, the precursor of NO (27). Poor responsiveness to cGMP during gestation has also been documented in the rat (3, 28). However, decreased responsiveness to NO is not consistent with previous reports by Bansal et al. (4) and Yallampalli et al. (22) that support a role for NO in myometrial quiescence based on the detection of inducible NO synthase in human myometrium and responses to L-arginine in pregnant rats, respectively. These studies prompted us to consider the possibility that in human myometrium, PKG may be sequestered in a membrane fraction during pregnancy. However, PKG was extracted predominantly in detergent-free buffer in all myometrial tissues, suggesting that decreases in PKG during pregnancy are probably not due to redistribution of PKG to an insoluble fraction (i.e. cytoskeleton, sarcoplasmic reticulum, or caveolae). Moreover, lower levels of PKG protein were confirmed by immunohistochemical studies. It should be noted that unlike uterine smooth muscle PKG, kinase levels in myometrial blood vessels do not appear to decrease during pregnancy. Thus, our estimates of low uterine smooth muscle PKG levels in pregnant myometrial tissues are probably overestimated due to the unavoidable contamination of vascular smooth muscle in myometrial tissue homogenates.
In contrast to PKG, h-CDM was significantly increased in pregnant myometrium, and both h-CDM and calponin were redistributed to a more readily extracted subcellular pool. In pregnant myometrium, both thin filament-associated proteins were extracted in a crude soluble fraction. Disruption of the myofilaments or cytoskeleton was necessary to recover greater amounts of h-CDM and calponin from nonpregnant tissues. These findings indicate that the affinity of caldesmon and calponin for myofilament and cytoskeletal proteins may be decreased in uterine smooth muscle during pregnancy. This decreased affinity may be secondary to phosphorylation (35, 36, 37, 38) or increased expression of other proteins that compete for myosin or thin filament protein binding. In intact smooth muscle, caldesmon is phosphorylated by MAPK (36, 38). MAPK-induced phosphorylation results in reversal of ATPase inhibition and reduced binding to actin (35, 36, 38). The relationship between MAPK activation, caldesmon phosphorylation, and redistribution of h-CDM during pregnancy, however, is not known. The preferential extraction of h-CDM and calponin by Triton X-100 in pregnant tissues further suggests that greater amounts of thin filament proteins associate with membrane-bound proteins during pregnancy. Future studies directed at identifying thin filament binding proteins in pregnancy might help elucidate the physiological significance of membrane-associated h-CDM and calponin.
The cellular signals that mediate these changes in PKG, h-CDM, and calponin in human myometrium are not known. In the rat, PKG is down-regulated by progesterone in the uterus (3) and by NO, natriuretic peptides, and cGMP in vascular smooth muscle in a negative feedback loop (39). In human myometrium from nonpregnant women, PKG levels are decreased in progestin-exposed tissues (40), consistent with the down-regulation in pregnancy reported in this study. The increase in uterine h-CDM levels during pregnancy appears to be mediated through mechanical distention, rather than steroid hormones (41). Further studies regarding the cellular signal transduction pathways that mediate the subcellular redistribution of h-CDM and calponin during pregnancy are needed.
In summary, we established that PKG is down-regulated in human myometrial cells during pregnancy independently of uterine vascular smooth muscle. These data provide evidence that cGMP-induced activation of PKG does not mediate uterine quiescence during gestation. We also identified a redistribution of two smooth muscle-specific, thin filament-associated proteins. These findings together with the biochemical properties, smooth muscle-specific localization, abundance, and subcellular distribution of caldesmon and calponin suggest that in pregnancy altered compartmentalization of these proteins plays an important role in the modulation of uterine smooth muscle contractility. These proteins may also be important in sustaining the cytoskeletal framework of myocytes during the substantial hypertrophy that accompanies all intrauterine pregnancies.
Acknowledgments
We acknowledge the contributions of Dr. Edward Wilson and the members of the pathology departments of University of Alabama, St. Paul, and Brookwood Hospitals. We also thank the University of Alabama Comprehensive Cancer Center Tissue Procurement Facility and personnel for assistance in procuring tissues. Thanks to Dr. Sharron Francis for providing purified PKG, and to Cheryl Van Epps-Fung, M.S., for technical contributions. Richardson’s Meat Processing and Greene’s Processing Centers are acknowledged for donating bovine lungs for the purification of PKG.
Footnotes
This work was supported by NIH Grants HD-32622 (to T.L.C.) and P01-HD-11149 (to R.A.W.), the March of Dimes Foundation (Grant 6-FY99-482; to T.L.C.), and the American Heart Association (Grant 9950820Y; to R.A.W.).
Abbreviations: ATPase, Adenosine triphosphatase; h-CDM, high mol wt, smooth muscle-specific isoform of caldesmon; NO, nitric oxide; PE buffer, 20 mM potassium phosphate (pH 7.0), 150 mM NaCl, 0.32 M sucrose, 2 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin PKG, cGMP-dependent protein kinase; TBS, Tris-buffered saline; TBST, TBS containing 0.1% Tween 20.
Received August 25, 2000.
Accepted April 8, 2001.
References
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; n = 18) and pregnant (
; n
= 13) women. *, P < 0.05.
