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Division of BioMedical Sciences,7 Health Science Centre, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6
Samuel Lunenfeld Research Institute,4 Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
Departments of Physiology,5 and Obstetrics & Gynecology,6 University of Toronto, Toronto, Ontario, Canada M5S 1A1
ABSTRACT
The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.
cellular cohesion, pregnancy, progesterone, uterus
The uterus undergoes dramatic physiological adaptations during the course of pregnancy, in order to accommodate the developing fetus, placenta, and amniotic fluid. The onset of labor requires triggering of the endocrine (the fetal hypothalamic-pituitary-adrenal-placental axis) and mechanical (stretch resulting from fetal growth during pregnancy) signaling pathways within the myometrium, to activate the synchronized and temporally co-ordinated labor contractions of the uterine smooth muscle. Both signals play a determinant role in the growth of the uterus during late pregnancy that precedes myometrial activation. Mechanical stretching has previously been shown to promote rapid and extensive uterine hypertrophy and remodeling in nonpregnant [1], unilaterally pregnant [2], and postpartum [3] animals. Our previous data have suggested that the hypertrophic changes in the pregnant myometrium are associated with significantly increased expression of extracellular matrix (ECM) proteins in the second part of gestation [4]. Specifically, we have reported a decrease in the expression of fibrillar collagens and a co-ordinated temporal increase in the expression of components of the basement membrane (type IV collagen, laminin) and fibronectin (FN) near to term [4].
These changes in ECM content reflect a newly reorganized uterine tissue architecture, which allows hypertrophied uterine myocytes to anchor properly to the surrounding ECM. MacPhee and Lye discovered earlier that the expression and activity of the major regulator of cell-ECM contacts, focal adhesion kinase (FAK or PTK2), was up-regulated in the rat myometrium at late gestation [5]. It is well known that the activation of PTK2 causes the formation of complex specialized structures, termed focal adhesions, which are associated with the plasma membranes of smooth muscle cells (SMCs). Integrin receptors are essential components of focal adhesions, as they form clusters at sites where SMCs make contact with the different ECM components [6]. These ubiquitous large transmembrane receptors can bind to a number of ligands, primarily ECM molecules, thereby establishing dynamic associations between the ECM and the actin cytoskeleton. Integrins are involved in many cellular processes, such as cellular adhesion and migration, regulation of the cellular phenotypes of many cell types, and stimulation of numerous signal transduction pathways [7]. The interaction of ECM ligands with their corresponding integrins also plays an important role in embryo development, morphogenesis, and tumorigenesis (reviewed in [8]). Each integrin is a heterodimer that contains 
and ß subunits. The /ß pairings specify the ligand-binding abilities of the integrin heterodimers. For example, the integrin 5 subunit (ITGA5) partners exclusively with the ß1 integrin (ITGB1). The repertoire of integrins expressed on particular cell types is unique and can vary temporally depending on many factors. Cells often display multiple integrins that are capable of interacting with a particular ECM. Some integrins, such as 5/ß1 (ITGA5B1), which is the classic fibronectin receptor, bind to a single ECM protein [9–11]. FN is a multifunctional, adhesive glycoprotein that is secreted by cells as a soluble dimer and can be assembled on the cell surface into mesh-like fibrils [12]. Interaction of FN with its receptor and the formation of fibrillar FN matrix are important events for strong intercellular cohesion [9, 13].
We have previously reported an increase in Fn gene expression in the gravid horn of unilaterally pregnant rats that were subjected to a passive biological stretch by growing fetuses [4]. Moreover, we have demonstrated earlier that Itga5 gene and ITGA5 protein expression are up-regulated in the rat myometrium, specifically during the second half of gestation when mechanical stretching of uterine walls is imposed by growing fetuses [14]. Therefore, we decided now to use the same animal model to understand the effect of a mechanical stimulus on the expression of the ITGA5 subunit in pregnant rat uterine tissue. In the present study, we apply real-time PCR, Northern blotting, and immunoblotting in combination with immunofluorescence analysis to investigate the effects of gravidity on Itga5 expression and ITGA5 subunit expression and localization, as well as on the spatial and temporal distribution of FN. We also investigate whether progesterone (P4) regulates ITGA5 expression in the myometrium. Previously, we have found that Fn gene expression is induced following early P4 withdrawal [4]. As P4 plays a major role in myometrial ECM remodeling, thereby influencing the expression of the Fn gene, we analyzed the effects of P4 receptor blockage on Itga5 mRNA expression and ITGA5 localization in the rat myometrium during RU486-induced preterm labor.
Wistar rats (Charles River Co., St. Constance, QC, Canada) or Sprague-Dawley rats (Memorial University of Newfoundland, St. Johns, NL, Canada) were housed individually under standard environmental conditions (12L:12D cycle) and fed Purina Rat Chow (Ralston Purina, St. Louis, MO) or LabDiet Prolab RMH 3000 (PMI Nutrition International, Brentwood, MO) and water ad libitum. Female virgin rats were mated with male rats. Day 1 of gestation was designated as the day on which a vaginal plug was observed. The Samuel Lunenfeld Research Institute Animal Care Committee and Memorial University of Newfoundland Animal Care Committee approved all the animal experiments.
Unilaterally pregnant rats. Under general anesthesia, virgin female rats underwent tubal ligation through a flank incision, to ensure that they subsequently became pregnant in only one horn [15]. Animals were allowed to recover from surgery for at least 7 days before mating. Pregnant rat myometrial samples from nongravid (empty) and gravid horns were collected on gestational Days 15, 19, and 23 (labor). Labor samples were taken during active labor and only after the rat had delivered 1–3 pups (n = 3–4).
RU486-induced preterm labor. On Day 19 of gestation, two groups of rats were treated with either RU486 (10 mg/kg, s.c., at 1000 h, in 0.5 ml corn oil that contained 10% EtOH, mifepristone, 17ß-hydroxy-11ß-[4-dimethylaminophenyl]-17-[1-propynyl]-estra-4,10-dien-3-one [Biomol International, Plymouth Meeting, PA]) or vehicle. Myometrial samples were collected from both groups of animals on Day 20. RU486-treated rats were killed during active labor when they had delivered 1–3 pups (n = 4).
Animals were killed by carbon dioxide inhalation. For RNA and protein extractions, the uterine horns were placed into ice-cold PBS, bisected longitudinally, and dissected away from both pups and placentas. The endometrium was carefully removed from the myometrial tissue by mechanical scraping on ice, which we have previously shown removes the entire luminal epithelium and the majority of the uterine stroma [16]. The myometrial tissue was flash-frozen in liquid nitrogen and stored at –70°C. For immunohistochemical studies, the intact uterine horns were placed in ice-cold PBS buffer, cut into 3–10-mm segments using a scalpel blade, and fixed immediately in 4% paraformaldehyde solution at 4°C for 48 h or overnight in zinc-buffered fixative (ZBF; 100 mM Tris buffer [pH 7.4], 3 mM calcium acetate, 27 mM zinc acetate, 37 mM zinc chloride [14, 17]) while shaking at room temperature. These segments were further cross-sectioned or sectioned longitudinally. For each day of gestation, tissues were collected from four different animals and processed for different experimental purposes.
Total RNA was extracted from the frozen tissues using Trizol (Gibco BRL, Burlington, ON, Canada) according to manufacturer's instructions. RNA samples were column purified using RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada), and treated with 2.5 µl DNase I (2.73 Kunitz U/µl; Qiagen) to remove genomic DNA contamination. Reverse transcription (RT) and real-time PCR (RT-PCR) were performed to detect the expression of Itga5 mRNA in the rat myometrium. Total RNA (2 µg) was primed with random hexamers to synthesize single-stranded cDNAs in a total reaction volume of 100 µl using the TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The thermal cycling parameters for RT were modified according to the Applied Biosystems manual. Hexamer incubation at 25°C for 10 min and RT at 42°C for 30 min were followed by reverse transcriptase inactivation at 95°C for 5 min. Then, 20 ng of cDNA from the previous step was subjected to real-time PCR using specific sets of primers (listed in the legend to Fig. 1) in a total reaction volume of 20 µl. Real-time PCR was performed in an optical 96-well plate with the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems), using the SYBR Green detection chemistry. The run protocol was as follows: initial denaturation at 98°C for 2 min, followed by 40 cycles of amplification at 95°C for 15 sec and 60°C for 1 min. After PCR, a dissociation curve was constructed by increasing the temperature from 65°C to 95°C, for the detection of PCR product specificity. In addition, a no-template control (H2O control) was analyzed for possible contamination of the master mix. The cycle threshold (Ct) value was recorded for each sample. PCR was set up in triplicate and the mean of the three Ct values was calculated. A comparative Ct method (
Ct method) was applied to the raw Ct values to evaluate relative gene expression. The expression of the Itga5 gene on a specific gestational day was normalized to the level of ribosomal 18S mRNA. For unilaterally pregnant animals, Itga5 gene expression was calculated as the fold-change relative to the Day 15 gravid horn mRNA level, and for RU486-treated animals, as the fold-change relative to the vehicle sample using an arithmetic formula (see ABI User Bulletin #2). Validation experiments were performed to ensure that the PCR efficiencies between the target genes and 18S were approximately equal.
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RNA isolation and Northern blot analysis of mRNA were performed according to methods previously described in detail [14]. The pOTB7 vector, which contains the human Itga5 cDNA, was purchased from the American Type Culture Collection (Manassas, VA). Isolation of a human Itga5 cDNA fragment for probe production has been described previously [14]. Probed membranes were exposed to x-ray film (Hyperfilm MP; Amersham Biosciences) with an intensifying screen at –70°C and analyzed by densitometry. Multiple exposures were produced for each Northern blot to ensure the results were within the linear range of the film. Following analysis of Itga5 mRNA expression, Northern blots were stripped and reprobed with a similarly labeled 18S (Rn18S) probe (rabbit Rn18S ribosomal cDNA template, generously provided by Dr. I. Skerjanc, University of Western Ontario, London, ON, Canada). Rn18S RNA is constitutively expressed in rat myometrial cells and has been utilized as a loading control for the analysis of myometrial gene expression [4].
Total protein samples were extracted from the frozen tissues using RIPA lysis buffer, as was described previously [14]. Protein samples (50 µg/lane) were separated under nonreducing conditions by polyacrylamide gel electrophoresis in 9% (for ITGA5) or 6% (for FN) resolving gels. Proteins were electroblotted to nitrocellulose (ITGA5) or polyvinylidene difluoride (FN) membranes (Millipore, Bedford, MA). The expression levels of ITGA5 were measured using a primary polyclonal rabbit antibody (1:125 000; Chemicon, Temecula, CA). FN protein expression was measured using a primary mouse monoclonal antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate primary antisera were incubated with blots for 1 h at room temperature under constant agitation, and the blots were then rinsed with Tris-buffered saline-Tween-20 (TBST; 20 mM Tris base [pH 7.6], 137 mM NaCl, 0.1% Tween-20). The immunoblots were incubated with horseradish peroxidase (HRP)-conjugated secondary antisera (1:100 000; Pierce, Rockford, IL; or 1:50; EnVision, DAKO) for 30–60 min at room temperature with constant agitation and then rinsed in TBST. Proteins were detected using the Pierce SuperSignal West Pico Chemiluminescent Substrate detection system (MJS BioLynx, Inc., Brockville, ON, Canada), and multiple exposures were generated to ensure the linearity of the film exposures. Membranes were stripped and reprobed with anti-calponin (1:3000, clone hCP; Sigma-Aldrich, Oakville, ON, Canada) or anti-ERK antibody (1:1000; Santa Cruz Biotechnology) using the probing conditions described above for primary antibodies, to control for loading variations. We have previously demonstrated that calponin protein (also known as CNN1) and ERK are constitutively expressed in nonpregnant and pregnant rat myometrial tissues, as detected with our protein extraction procedures [14, 18].
Immunofluorescence was performed according to previously described methods [14]. Two separate, independently collected sets of rat uterine tissues (n = 2, i.e., two rats used per time-point) were utilized for immunocytochemistry experiments, and the experiments were repeated twice. For experimental purposes, two uterine tissue sections per slide for each gestational time-point were utilized. All tissue sections used were 5-µm thickness, contained both longitudinal and circular smooth muscle layers, and were processed under identical conditions (e.g., the same volumes used for blocking and antiserum solutions). Heat-induced epitope retrieval was accomplished using a solution of 0.01 M SSC (pH 6.0). One tissue section on each slide was incubated for 1 h at room temperature in primary rabbit anti-ITGA5 antiserum (1:500; Chemicon) or mouse anti-FN antibody (clone FBN11, 1:100; NeoMarkers, Fremont, CA). Simultaneously, the remaining tissue section on each slide was incubated with affinity-purified IgG of the appropriate species, at the same concentration as the primary antiserum, to serve as a negative control. After three washes in PBS, all of the tissue sections were incubated with FITC-conjugated secondary antisera (1:250; Sigma). Sections were washed with cold PBS that contained 0.02% Tween-20 (PBT) with constant agitation. Tissues were mounted in Vectashield (Vector Laboratories, Inc., Burlington, ON, Canada) before viewing with an Olympus Fluoview laser scanning confocal microscope (Olympus Optical Company Ltd., Melville, New York) or Leica DMRXE microscope (Leica Microsystems, Richmond Hill, ON, Canada). With both instruments, the image acquisition settings for each experiment were precisely maintained to allow examination of specific detection differences across the gestational profiles analyzed.
Densitometric analyses of Northern blots and immunoblots were performed with the aid of the Scion Image software (Scion Image Corp., Frederick, MD). Densitometric measurements of Itga5 mRNA were normalized to those of Rn18S RNA, while measurements of ITGA5 protein on immunoblots were normalized to those of CNN1. Statistical analyses of Northern blots and immunoblots were performed with the GraphPad Prism version 4.0 software (GraphPad, San Diego, CA) and analysis of the real-time PCR results was carried out with SigmaStat version 2.01 software (Jandel Corp., San Rafael, CA). Data from the Northern blot, real-time PCR, and immunoblot analyses of Itga5 and ITGA5 expression in the unilaterally pregnant model were subjected to a two-way ANOVA, followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine differences between groups. The RU486 results were compared to vehicle using a t-test. Values were considered significantly different for P < 0.05.
Gravidity Regulates FN and ITGA5 Receptor Expression in Unilaterally Pregnant Rat Myometrium
Expression of Itga5 mRNA: Northern blot analysis.
To determine whether Itga5 gene expression is affected by mechanical stretching of the myometrium during late pregnancy, Northern blot analysis was performed. We used total RNA isolated from empty and gravid uterine horns of unilaterally pregnant Sprague-Dawley rats. As shown in Figure 1A, the expression of Itg5 gene was elevated in the gravid horn compared to the empty horn at Day 15, Day 19, and Day 23 (labor). Densitometric analysis revealed that this increase was statistically significant (P < 0.0001) (Fig. 1, A and B).
Expression of Itga5 mRNA: real-time PCR analysis. The levels of Itga5 mRNA were also analyzed in uterine tissues from gravid and empty horns of unilaterally pregnant Wistar rats by real-time PCR analysis. Similar to normal pregnant animals [14], the mRNA levels for Itga5 in the gravid horn of unilaterally pregnant rats remained high and relatively constant at late gestation (Day 15 to Day 19) and during labor (Day 23). In contrast, on all three gestational days, the Itga5 transcript levels were significantly lower in the empty horn myometria compared to the gravid horn (Day 23, 4.4-fold increase in gravid vs empty horn, P < 0.001) (Fig. 1C).
Detection of ITGA5 protein by immunoblot and immunofluorescence analyses. Immunoblot analysis was performed using specific anti-ITGA5 antibodies, to determine whether the expression of this integrin subunit in empty and gravid horns of unilaterally pregnant rat myometria at late gestation reflects its gene expression. The ITGA5 protein levels were significantly higher in gravid uterine horn on gestational Day 15, Day 19 (P < 0.01) and Day 23 (labor, P < 0.05) (Fig. 2, A and B) than in the empty horn. Therefore, the expression of ITGA5 in the rat myometrium is dependent upon gravidity.
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We also studied the spatial and temporal distributions of ITGA5 protein in the gravid and empty uterine horns at late gestation. ITGA5 was detected in situ in both the longitudinal and circular myometrial layers. Strong immunoreactivity for ITGA5 was observed in the longitudinal muscle layers of all myometrial samples examined, as compared to the circular muscle layers. We also consistently observed a small increase in ITGA5 detection on Day 15 and Day 19 in the gravid horns compared to the empty horns. In the circular muscle layers of the empty horns, ITGA5 immunostaining was virtually undetectable, while ITGA5 was readily immunodetected in the gravid horns (Fig. 3). However, we also observed a temporal alteration of ITGA5 detection in the circular muscle layers of gravid horns. ITGA5 detection in gravid horns was noticeably up-regulated in the circular muscle layers of late pregnant (Day 19) and laboring (Day 23) myometria (Fig. 3). These data indicate differential regulation of ITGA5 expression between two muscle layers, which could result in enhancement of the contractile activity of the circular muscle of the gravid horn during late pregnancy. Spatial distribution of ITGA5 was similar in both muscle layers and was not affected by gravidity. In particular, we found that ITGA5 was predominantly localized to the myometrial cell membranes in the form of punctate staining (Fig. 3).
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Detection of FN protein by immunoblot and immunofluorescence analyses. We have reported earlier that myometrial Fn mRNA levels are dramatically up-regulated starting from Day 15 and are significantly higher in the gravid horns than in the empty horns of unilaterally pregnant rats, suggesting that mechanical stretching may contribute to this change [4]. Using antibodies that specifically recognize fibrillar FN, we found that this ECM protein was also up-regulated at midpregnancy, exclusively in the gravid uterine horn (Fig. 4). Interestingly, this coincided with the beginning of hypertrophic transformation of myometrial SMCs during the "synthetic" phase of myometrial differentiation.
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To investigate the spatial and temporal distributions of FN protein in the myometria of gravid and empty uterine horns of unilaterally pregnant rats at gestational Days 15, 19, and 23, we applied immunofluorescence analysis (Fig. 5). In the empty horn, FN immunostaining in the longitudinal muscle layer was localized mostly around the SM bundles, particularly at Day 15 and Day 19, whereas in the circular muscle layer, staining was also found around individual SMCs. In the gravid horn, immunoreactive FN was found in both muscle layers surrounding the plasma membrane of each individual SMC, in connective tissue surrounding the SMC bundles, and in blood vessels. FN protein immunostaining was localized around individual SMCs, exhibiting a continuous, regular, bead-like pattern of staining associated directly with the cell surfaces of hypertrophic myocytes (Fig. 6).
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Effect of RU486-Induced Preterm Labor on ITGA5 Expression
Expression of Itga5 mRNA.
Circulating levels of P4 in rat maternal serum peak between Day 15 and Day 19, and thereafter decrease dramatically until Day 23, the day of delivery [19]. We have previously demonstrated a potential role for P4 in the regulation of Itg5 and ITGA5 expression using a P4-prolonged labor model [14]. In particular, treatment of pregnant rats from Day 20 with daily injections of P4 resulted in failure to initiate labor on Day 23 and maintained Itg5 and ITGA5 expression at Day 24. To examine further the relationship between P4 plasma levels and Itg5/ITGA5 expression, pregnant rats were treated with the P4 receptor antagonist RU486. Treatment of pregnant animals at Day 19 of gestation with RU486 induced preterm labor within 24 h. Real-time PCR analysis of myometrial tissues collected from vehicle-treated and RU486-treated rats indicated that the Itg5 mRNA levels were significantly up-regulated after P4 blockade (P < 0.05; Fig. 7). In contrast to the significantly elevated expression of Itg5 after administration of RU486, the ITGA5 protein levels were only slightly increased and the difference did not reach statistical significance (data not shown).
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Immunocytochemical detection of ITGA5. In the longitudinal muscle layer, staining of ITGA5 in RU486-induced labor samples appeared to be more intense than in the vehicle-treated controls (Fig. 8). Spatially, immunoreactive ITGA5 accumulated in regions closely associated with plasma membranes of hypertrophied uterine SMCs. Importantly, in the circular muscle layer, ITGA5 also appeared to accumulate at cell membranes following RU486-induced labor, as compared to the vehicle control, mimicking changes that are observed in normal term labor (Fig. 3; [14]). Moreover, the staining was more punctate compared to that of the longitudinal smooth muscle layer.
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In the present study, we have used the unilaterally pregnant rat model to investigate the role of biological mechanical stretching in the myometrial expression of FN and its unique FN-binding integrin partner ITGA5 during late pregnancy and labor. Our data primarily demonstrate that: 1) marked synchronous up-regulation of FN and ITGA5 occurs specifically in the gravid uterine horn of unilaterally pregnant rats; and 2) the increase in ITGA5 detection during late pregnancy occurs mainly in the circular muscle layer of the gravid horn, which is subject to mechanical stimulation.
Our previous studies have led us to conclude that the myometrium undergoes gradual changes in phenotype during pregnancy, characterized by an early proliferative phase, an intermediate synthetic phase of cellular hypertrophy and matrix elaboration, and a final contractile/labor phase [20, 21]. Myometrial hypertrophy occurs specifically in the second half of gestation (starting from gestational Day 15 in rats) when fetal growth is maximal. Several groups have suggested that in addition to endocrine factors, the enlarging fetus may induce uterine growth by placing the uterine wall under tension [2, 22]. Using a unilaterally pregnant rat model, Douglas and Goldspink have shown that cellular hypertrophy is dramatically increased in the gravid horn (subjected to stretch by the growing fetus), while remaining unchanged in the empty horn [2]. Using the same model, we report that the gestational changes in Fn and Itga5 gene and FN and ITGA5 protein expression described in the present study occur specifically in the gravid horn of unilaterally pregnant rats beginning around midpregnancy, at which time-point the myometrial SMCs acquire a synthetic phenotype. These changes in FN and ITGA5 protein expression coincided with the onset of hypertrophic changes in the gravid rat myometrium. Studies on the heart suggest that multiple factors are responsible for the hypertrophic response and that integrins are an important component of this process, with ITGB1 being directly linked to the hypertrophic cellular transformation of neonatal ventricular myocytes [23]. Moreover, ITGA1 and ITGB1 are up-regulated in the adult rat myocardium during aortic constriction-mediated cardiac hypertrophy [24–26]. During the initial phases of pressure overload, the expression levels of FN and ITGA5B1 in cardiomyocytes increase in parallel [11]. Therefore, we propose that the co-ordinated induction of FN and ITGA5 in the pregnant rat myometrium is an important factor in the development of myometrial hypertrophy in rats. Of note, both the ITGA1 and ITGB1 subunits are also increasingly detected at myometrial cell membranes in the second half of gestation (Williams, Shynlova, Lye, MacPhee, unpublished results).
FN is a large multifunctional glycoprotein with wide tissue distribution, and it is essential for normal development and tissue repair [12, 23, 26, 27]. FN plays an important role in stabilizing ECM material by directly attaching cells to various substrates, such as collagen fibers and proteoglycans [28]. Integrin–FN interactions allow unfolding of the soluble protein and its assembly into a detergent-insoluble fibrillar matrix that can modulate cell morphology, growth, and tissue architecture [29, 30]. In vascular tissues, FN also appears to be involved in connecting the SMCs to each other, as suggested by the presence of FN-positive layers that bridge the gaps between adjacent cells. In the present study, using antibodies that specifically recognize fibrillar FN, we have found that this ECM protein is up-regulated in a gravid horn at midpregnancy during the synthetic phase of myometrial differentiation. Likewise, it has been reported that the fibrillar form of FN is associated with the surfaces of dedifferentiated (synthetic) vascular SMCs. In vitro freshly isolated vascular SMCs adhere to a substrate of FN, and after a few days of serum-free culture are converted from a contractile to a synthetic phenotype [31, 32]. These experiments suggest that a substrate of FN may be sufficient to promote the shift in phenotype of vascular SMCs, and that other exogenous macromolecules are not required for these processes. FN exerts its effect by interacting with integrin receptors, and this not only produces a physical linkage to the cells but also generates signals that adjust their behaviors.
Ligand-induced clustering of integrin receptors results in the formation of focal adhesions [7]. These structures mediate tension transmission between the cytoskeleton and ECM and contain various adapter proteins and kinases that can promote cytoskeletal interaction. The resulting connection to cytoskeletal components ultimately allows focal adhesions to act as mechanosensors, while association with cytoplasmic kinases, such as FAK, facilitates triggering of biochemical signaling pathways or mechanotransduction [33, 34]. Mechanical force applied to integrins results in increased clustering, perhaps as a result of increased actin and myosin recruitment and cytoskeletal assembly [35, 36]. Similar to their functions in pressure-induced cardiac hypertrophy [23] and stretched bladder SMCs [35], myometrial integrins are also components of mechanosensors that can sense and respond to the increased stretching of the uterine walls imposed by the growing fetus(es). During early pregnancy, the uterus grows noticeably in the longitudinal direction, followed by mostly circumferential growth from midpregnancy onwards, to accommodate the increasing fetal growth. Thus, it is expected that tension on the uterine wall during late pregnancy is predominant in the circular SM layer. Indeed, in the present study, we observed a temporal change in ITGA5 protein detection in the circular muscle layers of the gravid horns compared to the longitudinal muscle layers. These data provide additional support for a role of mechanical signals in the expression of ITGA5. The two muscle layers exhibit quite different phenotypes during pregnancy. For example, our previous studies have shown that the circular layer of the myometrium is more responsive to mechanical stretching than the longitudinal layer [37–39]. Such data imply specific roles for these myometrial layers during gestation. The circular muscle would provide the primary contractile response. while the role of the longitudinal layer would be to shorten the uterus upon expulsion of each fetus (reviewed in [38]). We propose that stretch-induced expression of ITGA5 in the circular myometrial layer stimulates FN-ITGA5 interaction and facilitates adhesion of hypertrophied SMCs to the ECM at late gestation, thereby contributing to myometrial growth and remodeling. We hypothesize further that just prior to labor, binding of FN to its receptor activates integrin signaling and assembly of the FN fibrillar matrix around individual SMCs in circular muscle, which would contribute significantly to tissue cohesion [13, 40]; this supports the specific role of this muscle layer during labor contractions. This mechanism would give rise to increased integrin signaling through FAK [41]. Mechanical stretching induces tyrosine phosphorylation of FAK, which is a critical event for stretch-induced MAP kinase activation in cardiomycytes [42] and fibroblasts [43]. Importantly, we have discovered earlier that in vivo and in vitro static mechanical stretching of myometrial SMCs activates extracellular signal-regulated kinase (ERK) and p38 MAP kinase, allowing myometrial SMCs to sense mechanical stimuli and convert them to changes in gene expression [44].
We have previously documented that myometrial hypertrophic growth is dependent upon P4 and mechanical stretching. A fall in the P4 levels and an increase in 17-estradiol (E2) in rat maternal plasma at late gestation are believed to be essential to the activation of the myometrium at labor. Our present in vivo findings show that blockade of P4 signaling following administration of RU486 increases Itga5 expression in the myometrium, but does not significantly increase ITGA5 levels, although immunoreactive ITGA5 in the circular and longitudinal layers accumulated in regions associated with myocyte plasma membranes, as compared to the controls. If P4 withdrawal alone positively regulated Itga5 gene expression, then a significant increase in Itga5 mRNA and/or ITGA5 protein expression in gravid horns on Day 23, as compared to the expression levels in the gravid horns on Day 15 and Day 19, would have been expected, since the circulating P4 levels would have declined by Day 23. This pattern of expression was not observed and as a result, significant regulation of Itga5 gene expression by progesterone is unlikely. The accumulation of immunoreactive ITGA5 at myocyte plasma membranes following RU486-induced labor may reflect a requirement for ITGA5 at these sites during the labor process itself. These data suggest that the increased Itga5 expression levels in the myometrium during late pregnancy and labor [14] are more likely due to increased tension on the myometrium than changes in P4 levels. We have also reported earlier that blockade of P4 receptors by RU486 causes a significant up-regulation of Fn and the basement membrane components collagen IV and laminin ß2 [4]. Taking into account all our findings, we speculate that the decrease in P4 and the subsequent increase in E2, in addition to mechanical stimulation of the myometrium may be responsible for an increase in the expression of FN-ITGA5 complexes. We cannot rule out the possibility that at late gestation and during labor, stimulation of the FN-ITGA5 interaction could be triggered by other factor(s) derived from the maternal decidua, extraembryonic membranes or the fetus itself.
In summary, we suggest that increased expression of FN and ITGA5 may represent an important event in the preparation of the myometrium for the development of optimal contractions during labor. Initially, FN and ITGA5 promote rapid growth (hypertrophy) of the pregnant myometrium. This synthetic phase of myometrial differentiation is associated with the formation of fibrillar FN matrix around individual SMCs and is characterized by high FAK phospho-Tyr activity and increased focal adhesion turnover [5]. During the contractile phase of myometrial differentiation, a decrease in FAK activity stabilizes the SMC-ECM interactions, forming stable focal adhesions that connect the FN matrix and actin cytoskeleton through clusters of ITGA5 molecules. The reinforcement of the ligand-integrin interaction contributes significantly to anchorage of hypertrophied SMCs to the uterine ECM and facilitates proper smooth muscle intercellular cohesion before labor. During labor, the previously formed focal contacts serve as major points of force transduction, guaranteeing that the myometrium works as a mechanical syncytium through each contraction.
ACKNOWLEDGMENTS
We acknowledge the assistance of Anna Dorogin, Judy Foote, and Art Taylor for tissue collection and processing, and we thank Quang Xu and Yong Lu for processing the rat myometrial tissues for immunofluorescence analyses.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by CIHR (S.J.L.) and NSERC (D.J.M.).
Correspondence: 2Stephen J. Lye, Samuel Lunenfeld Research Institute at Mount Sinai, 600 University Avenue, Suite 870, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8587; e-mail: lye{at}mshri.on.ca
Received: 18 April 2007.
First decision: 9 May 2007.
Accepted: 9 August 2007.
REFERENCES
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  R. C Burghardt, J. R Burghardt, J. D Taylor II, A. T Reeder, B. T Nguen, T. E Spencer, K. J Bayless, and G. A Johnson Enhanced focal adhesion assembly reflects increased mechanosensation and mechanotransduction at maternal-conceptus interface and uterine wall during ovine pregnancy Reproduction, March 1, 2009; 137(3): 567 - 582. [Abstract] [Full Text] [PDF]
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