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5 Integrin (Itga5) Is Elevated in the Rat Myometrium During Late Pregnancy and Labor: Implications for Development of a Mechanical Syncytium1
Division of Basic Medical Sciences, Health Sciences Centre, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that 5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.
female reproductive tract, gene regulation, parturition, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prior to myometrial activation, the uterus undergoes dramatic growth during late pregnancy, primarily due to myometrial hypertrophy under the influence of progesterone and estrogen [3]. Uterine distension by artificial means or as a result of growing fetuses has also been demonstrated to stimulate growth, particularly hypertrophy [4, 5]. To support hypertrophy, cell-extracellular matrix (ECM) contacts must be reorganized to properly anchor growing cells to their ECM; consequently, a regulator of cell-ECM contact reorganization, named focal adhesion kinase (PTK2), is highly expressed and activated in rat myometrium during late pregnancy [6]. In smooth muscle, these cell-ECM contacts are termed dense plaques, which are analogous to the focal adhesions described in cultured cells in vitro (for simplicity, hereafter, dense plaques will be referred to as focal adhesions) [7, 8].
Late pregnancy is also a period marked by substantial remodeling of the ECM [3]. Type IV collagen, laminin, and fibronectin are deposited around smooth muscle cells in rat myometrium during late pregnancy [9]. At the molecular level, both fibronectin mRNA (Fn) and fibronectin protein (FN) expression have been reported to increase in the myometrium before labor [9, 10]. Of significance, the interaction of FN with its major receptor, 5ß1 integrin (ITGA5B1), is important for FN matrix assembly and strong intercellular cohesion [11–13]. Integrin receptors are integral components of focal adhesions. These ECM-binding, heterodimeric, transmembrane receptors are composed of and ß subunits, and the composition of some of these heterodimeric receptors can be quite specific. For instance, 5 integrin (ITGA5) partners solely with ß1 integrin (ITGB1) in cell membranes to form a fibronectin receptor [14]. Integrins can mediate tension transmission between the contractile apparatus of the cell and the ECM, yet they lack enzymatic activity, and therefore must associate with numerous adaptor proteins, cytoplasmic kinases, or transmembrane growth factor receptors to connect them to the actin cytoskeleton, or to trigger biochemical signaling pathways, or both [8, 15–17].
As in nonmuscle cells, there is evidence that integrins play a pivotal role in mediating functional adjustments in smooth muscle cells in response to changes in their external environment [8]. Functional adjustments can include changes in contractility resulting from the interaction of ECM proteins with appropriate integrin receptors. For example, ligand binding to ITGA5B1 can increase L-type Ca2+ current in arteriolar smooth muscle and alter contractility [18]. In addition, in renal vascular smooth muscle cells, the binding of ligands containing Arg-Gly-Asp peptides to integrins stimulates the release of intracellular calcium, which can alter smooth muscle contractility [19].
While the detection of integrins in nonpregnant human myometrium and leiomyomas has been reported [20], there have been no reports of integrin expression in the myometrium during pregnancy and labor. Because integrins are components of focal adhesions, and late pregnancy involves significant uterine growth, and ECM and focal adhesion remodeling, we hypothesized that Itga5 mRNA and ITGA5 protein expression would be induced in the myometrium of the rat during late pregnancy and labor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprague-Dawley rats were obtained from the Mount Scio Vivarium (Memorial University of Newfoundland, St. John's, NF, Canada). Animals were individually housed and cared for under standard environmental conditions (12L:12D) in the Animal Care Unit at the Health Sciences Center, Memorial University of Newfoundland. Rats were fed LabDiet Prolab RMH 3000 (PMI Nutrition International, Brentwood, MO) and water ad libitum. The institutional animal care committee approved all experiments under animal care protocols 02-02-DM and 02-03-DM. Virgin female rats (
220 g each) were mated with stud males, and observation of vaginal plugs the following morning was designated Day 1 postcoitum. The time of delivery under these standard conditions was Day 23 of gestation.
Experimental Design
Normal pregnancy and term labor All animals were killed by carbon dioxide inhalation on the desired day of sampling and pregnancy (e.g., nonpregnant [NP]; Days 6, 12, 15, 17, 19, 21, 22, and labor [L]; and 1 day postpartum [PP]). Labor samples were taken during active labor and only after the rat had delivered two to three pups.
Progesterone-delayed labor The onset of labor is coupled with a withdrawal of the inhibitory effects of progesterone on the myometrium following a decline in plasma levels of this steroid. To determine whether progesterone withdrawal might modulate Itga5 expression, pregnant rats were given either a daily injection of progesterone (4 mg s.c. in 0.2 ml of corn oil) to maintain elevated plasma levels of this steroid, or vehicle alone (0.2 ml of corn oil, s.c.) beginning on Day 20 of gestation [21]. Animals were killed by carbon dioxide inhalation on the desired day of sampling. Vehicle control animals were killed on Days 21, 22, and L (during delivery). Progesterone-treated rats were killed on the same gestation days and on Day 24; however, on Days 23 and 24, the rats were not in labor.
Tissue Collection
Uterine horns were removed, opened longitudinally, and fetuses and placentas were discarded. Myometrial tissue was subsequently obtained and stored exactly as previously described [6].
Northern Blot Analysis
Northern blot analysis for the normal pregnancy regime was performed on four separate independent sets of RNA samples (n = 4; i.e., four rats used per gestational time point), whereas analysis for the delayed labor regime was performed on three separate independent sets of RNA samples (n = 3). RNA was isolated from tissues using TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA) exactly according to the manufacturer's instructions. RNA quality and quantity (A260/280) were determined using a Shimadzu Bio-Mini Spectrophotometer (Mandel Scientific, Guelph, ON, Canada), and samples were stored at –70°C.
Ten micrograms of each RNA sample in sample buffer (50% formamide, 0.066 M formaldehyde, and 1x MOPS buffer in diethyl pyrocarbonate [DEPC]-treated double distilled (dd) H2O) were loaded on a 1% agarose gel containing 0.66 M formaldehyde and 1x MOPS buffer (0.02 M MOPS pH 7.0, 2 mM sodium acetate, and 10 mM EDTA pH 8.0) and electrophoretically separated at 80 V in 1x MOPS/0.22 M formaldehyde running buffer. RNA was transferred overnight to a nylon membrane (Hybond-XL; Amersham Biosciences, Little Chalfont, Buckinghamshire, England) by upward capillary action using 2x SSC (0.3 M sodium chloride and 0.03 M sodium citrate) in DEPC-treated ddH2O. RNA was cross-linked to nylon membrane with a UVC-508 ultraviolet cross-linker (Ultra-Lum Inc., Paramount, CA). All blots were stored at –20°C until required.
The pOTB7 vector containing the human Itga5 cDNA was purchased from the American Type Culture Collection (MGC-3697; Manassas, VA; www.atcc.org). Digestion of pOTB7 with the restriction endonucleases EcoRI and XhoI resulted in the production of four DNA fragments including a 2.4-kilobase (kb) Itga5 cDNA fragment that was subsequently isolated with a Qiagen Gel Extraction Kit (Qiagen, Inc., Mississauga, ON, Canada), according to the manufacturer's instructions, and used for the production of random primed, radiolabeled cDNA probes. The 2.4-kb fragment of the human Itga5 cDNA (GenBank accession number BC008786) was found to have 89% identity to the rat Itga5 cDNA (GenBank accession number XM_235707).
Membranes were prehybridized in hybridization buffer consisting of 50% formamide, 5x sodium chloride-sodium phosphate EDTA (SSPE; 0.75 M sodium chloride, 0.05 M sodium phosphate, and 0.005 M EDTA), 1% SDS, 5x Denhardt solution, and 0.1 mg/ml herring sperm DNA for 1–2 h at 42°C in a hybridization oven (Hybaid Instruments, Franklin, MA). Radiolabeled cDNA probes were prepared with a Megaprime DNA Labeling kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England) exactly according to the manufacturer's specifications. Hybridization was performed overnight at 42°C. Blots were then washed once for 15 min, and three times for 5 min at 65°C in 0.2x SSC and 0.2% SDS, and then exposed to x-ray film (Hyperfilm MP; Amersham Biosciences). 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 analyzed for expression of 18S RNA (Rn18s) as described above using a rabbit Rn18s 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 used in the past as a loading control for analysis of myometrial gene expression [10, 22, 23].
Immunoblot Analysis
Immunoblot analysis for both normal pregnancy and delayed labor regimes was performed on four separate, independent sets of protein samples (n = 4; i.e., four rats used per gestational time point) according to the method described by MacPhee and Lye [6]. 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, and 0.1% [wt/vol] SDS) containing 100 µM Na2VO3 and Complete, Mini EDTA-free protease inhibitors (Roche Molecular Biochemicals, Laval, QC, Canada). Samples were centrifuged at 15 000 x g at 4°C for 15 min, and the supernatants were collected. Protein concentrations were determined using the Bradford assay [24] with Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Mississauga, ON, Canada). Protein samples (50 µg/lane) were separated under nonreducing conditions by polyacrylamide gel electrophoresis in 9% resolving gels according to the method described by Laemmli [25], and gels were electroblotted to Pierce 0.45-µm nitrocellulose membrane (MJS BioLynx, Inc., Brockville, ON, Canada).
Membranes were rinsed in Tris-buffered saline (20 mM Tris base, 137 mM NaCl pH 7.6) with 0.1% Tween-20 (TBST) for 5 min. Unless otherwise stated, all incubations were performed at room temperature and with constant agitation. Blots were blocked in 5% BSA/TBST for 30 min. Rabbit polyclonal antisera raised against ITGA5 (AB1928; Chemicon International, Temecula, CA) or mouse monoclonal antisera raised against smooth muscle calponin (CNN) (C2687; clone hCP; Sigma-Aldrich, Oakville, ON, Canada) used at dilutions of 1:125 000 and 1:100 000, respectively, were incubated with blots for 1 h. Blots were rinsed once for 15 min in TBST, followed by two rinses for 5 min in TBST. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit imunoglobulin G (IgG) (H + L) or HRP-conjugated goat anti-mouse IgG (H + L) (31460 and 31430, respectively; Pierce, Rockford, IL) were used at dilutions of 1:100 000 and 1:150 000, respectively, and incubated with blots for 1 h. Blots were washed once for 15 min in TBST, then four times for 5 min each in TBST. Proteins were detected using the Pierce SuperSignal West Pico Chemiluminescent Substrate (MJS BioLynx, Inc.) detection system and multiple exposures were generated to ensure the linearity of the film exposures.
Following immunoblot analysis of ITGA5 expression, analysis of CNN protein expression was subsequently performed. We have determined that CNN is constitutively expressed in nonpregnant and pregnant rat myometrial tissue under our protein extraction conditions.
Immunocytochemistry
Two separate, independently collected sets of rat tissues (n = 2; i.e., two rats used per gestational time point) were used for immunocytochemistry experiments, and experiments were repeated four times. Tissues were fixed overnight at room temperature with constant agitation in zinc-buffered fixative (ZBF; 100 mM Tris pH 7.4, 3 mM calcium acetate, 27 mM zinc acetate, and 37 mM zinc chloride) [26], and then rinsed in PBS overnight at room temperature with constant agitation. Tissues were paraffin-embedded, sectioned, and mounted on microscope slides by the Histology Department of Memorial University of Newfoundland School of Medicine.
Slides were dried overnight at 37°C. Sections were dewaxed in xylene (three times, 5 min each), rehydrated in descending grades of ethanol, and soaked in 1x PBS. Heat-induced epitope retrieval was accomplished using a solution of 0.01 M SSC pH 6.0. This solution was heated for approximately 3 min (until boiling) using a microwave. Slides were immersed in the hot solution for 10 min and then air-dried for 5 min. This was repeated an additional three times, and then slides were rinsed in PBS. Sections were blocked in 5% normal goat serum/1% horse serum in PBS for 30 min at room temperature with constant agitation. Sections were then incubated for 1 h at room temperature in rabbit anti-ITGA5 (AB1928; Chemicon International) at dilutions of 1:500 (4 µg/ml) in blocking solution or rabbit IgG (011-000-003; Jackson Immunoresearch Labs Inc., West Grove, PA) at the same concentration to serve as a negative control. Tissue sections were washed in PBS (three times, 5 min each) and then incubated in fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (F7512; Sigma, St. Louis, MO) at a dilution of 1:250 in blocking solution for 30 min with constant agitation at room temperature. Sections were washed with cold PBS containing 0.02% Tween-20 (PBT) (three times, 5 min each) 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, NY).
Data Analysis
Densitometric analysis of Northern blots and immunoblots was performed with the aid of Scion Image software (Scion Image Corporation, Frederick, MD). Densitometric measurements of Itga5 mRNA were normalized to those of Rn18s RNA, whereas measurements of ITGA5 protein on immunoblots were normalized to those of CNN. Statistical analysis was performed with GraphPad Instat version 3.0 (GraphPad Software, San Diego, CA; www.graphpad.com) and data were graphed using GraphPad Prism version 4.0 (GraphPad Software). Data from Northern blot and immunoblot analyses of Itga5 and ITGA5 expression, respectively, during normal gestation were subjected to a one-way analysis of variance (ANOVA) and a Tukey-Kramer multiple comparisons test. Data from Northern blot analysis of Itga5 mRNA expression during progesterone-delayed labor were subjected to a two-way ANOVA and a Bonferroni post-test. Values were considered significantly different if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of Itga5 mRNA and ITGA5 protein To characterize the levels of Itga5 mRNA within myometrial samples, Northern blots of myometrial total RNA from NP; Days 6, 12, 15, 17, 19, 21, 22, and L; and PP were analyzed with radiolabeled probes generated from a human Itga5-specific cDNA (Fig. 1A). Our experiments demonstrated that Itga5 mRNA expression significantly increased during late gestation (one-way ANOVA, P < 0.0001; n = 4). Specifically, Itga5 mRNA expression was barely detectable from NP to Day 12, and then began to increase by Day 15 of gestation. Itga5 mRNA expression then became significantly elevated (Tukey-Kramer post-test, P < 0.05) between Days 17 and L, inclusive, compared with that at NP, Day 6, Day 12, and PP time points (Fig. 1, A and B).
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Immunoblot analysis using ITGA5-specific antisera demonstrated that ITGA5 was readily detectable, and ITGA5 levels in myometrial tissue lysates increased gradually during gestation (one-way ANOVA, P < 0.05; n = 4). Specifically, ITGA5 protein levels during labor were significantly elevated over NP time points (Tukey-Kramer post-test, P < 0.05; n = 4; Fig. 2, A and B).
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Immunocytochemical detection of ITGA5 In the longitudinal muscle layer of the myometrium, ITGA5 was readily and exclusively localized to myometrial cell membranes from NP to Day 6, and from Day 15 to PP (Fig. 3). A slight decrease in detection of ITGA5 expression was consistently observed at NP, Day 6, and PP. In the circular muscle layer of the myometrium, ITGA5 was barely detectable at NP and Day 6, then was primarily localized to cell membranes from Day 15 to PP (Fig. 4). However, detection of ITGA5 on Day 15 and Day 17 was low, with a more punctate staining pattern compared with the expression of ITGA5 at later gestational time points. As gestation progressed, detection of ITGA5 increased, and a more continuous membrane-staining pattern was observed.
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Progesterone-Induced Delayed Labor
Expression of Itga5 mRNA and ITGA5 protein Throughout the majority of pregnancy, circulating levels of progesterone in the rat are high, and they subsequently decline between Day 22 and L [27]. Because Itga5 mRNA expression became significantly elevated from Day 17 to L, this suggested that the effects of progesterone on the myometrium might influence Itga5 mRNA expression during late pregnancy. Animals treated with progesterone beginning on Day 20 of gestation did not exhibit labor on Day 23, and our experiments demonstrated that Itga5 mRNA expression was sustained through to Day 24, a full day after normal labor would have occurred and when Itga5 expression would have declined significantly PP. Specifically, there was no significant decrease in Itga5 mRNA levels in progesterone-treated animals on Day 24 compared with vehicle-treated animals during labor (two-way ANOVA, P > 0.05; n = 3; Fig. 5). Immunoblot analysis also demonstrated that ITGA5 protein levels in myometrial tissue lysates were maintained and were similar in progesterone-treated animals on Day 24 compared with vehicle-treated animals during labor (two-way ANOVA, P > 0.05; n = 4; Fig. 6).
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Immunocytochemical detection of ITGA5 ITGA5 in the longitudinal muscle layer was continuously localized around myometrial cell membranes, and detection was maintained at Day 24 (Fig. 7). In addition, ITGA5 in progesterone-treated animals consistently appeared to be accumulating at high levels in cell membranes on Days 23 and 24 compared with that in vehicle controls, giving the appearance of thicker membranes. In the circular muscle layer, ITGA5 was also primarily localized to myometrial cell membranes, and expression was sustained on Day 24 (Fig. 8). Similar to the longitudinal muscle layer, ITGA5 in progesterone-treated animals consistently appeared to be accumulating at high levels in cell membranes on Day 23 and Day 24 compared with vehicle controls; however, the increased detection was primarily in the form of punctate staining.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of Itga5 and Fn gene expression Our Northern blot analysis demonstrated that a significant increase in Itga5 mRNA expression by Day 17 of gestation preceded the reported increase in Fn mRNA expression just before labor [10]. MacPhee and Lye [6] previously reported that the tyrosine kinase PTK2, and a PTK2-binding adapter protein named paxillin (PXN), were highly activated and tyrosine-phosphorylated, respectively, in rat myometrium, by Day 21 of gestation, indicative of a significant period of focal adhesion remodeling at this time to support stretch and hormonally induced myometrial hypertrophy. Because integrins can be components of focal adhesions, induction of Itga5 mRNA expression may be required for subsequent translation into ITGA5 protein to aid this focal adhesion turnover and reorganization before labor.
Our immunoblot analysis demonstrated that ITGA5 was readily detectable in myometrial tissue lysates during late pregnancy and labor, reaching statistically significant levels at labor. Although immunocytochemical analysis demonstrated that ITGA5 in the longitudinal muscle layer was highly detected around individual myometrial cells (membrane localized) at all gestational time points examined, ITGA5 at NP and Day 6 was almost undetectable in the circular muscle layer, whereas detection on Day 15 and Day 17 was low, with a more punctate staining pattern compared with that of later time points. Therefore, detection of significantly more ITGA5 at labor versus NP by immunoblot analysis may reflect that ITGA5 is primarily expressed in only the longitudinal muscle in the nonpregnant rat myometrium, unlike the rat myometrium at labor. Furthermore, increased detection of ITGA5 by Day 19 in the circular muscle layer would appear to be relatively coordinated, with increased levels of Itga5 mRNA by Day 17 of gestation. However, the lack of significant changes in protein expression in the longitudinal muscle layer and after Day 19 in the circular muscle layer that would closely correlate with the significant elevation of Itga5 mRNA expression before and during labor, suggests that ITGA5 expression may be translationally regulated. It is also possible that an increase in ITGA5 protein turnover during late pregnancy and labor, perhaps in concert with focal adhesion remodeling during late pregnancy, may be occurring. Nonetheless, the immunoblot and immunocytochemistry data do compare favorably with the reported increased immunofluorescent detection of FN around individual hypertrophic smooth muscle cells by Day 16 and Day 19 of rat pregnancy, in accordance with increased thickness of basement membranes [9].
One day PP, ITGA5 continued to be readily detectable and localized around individual myometrial cells, particularly in the longitudinal muscle layer, in contrast to analysis of FN localization 3 days postpartum in the rat, which demonstrated that FN became localized in discontinuous areas of myometrial cell membranes, similar to localization in nonpregnant and early pregnant (Day 7 and Day 10) rat myometrium [9]. We believe that these differences in immunolocalization patterns, compared with those of late pregnancy and labor, are simply due to the significant temporal differences in postpartum analysis of these proteins.
Because ITGA5 partners solely with ITGB1 in cell membranes to form a fibronectin receptor [14] and ITGB1 is detectable by immunoblot analysis in myometrial lysates from NP to PP time points (data not shown), our results as a whole suggest that ITGA5B1-FN interaction may be occurring during late pregnancy and labor in the rat. This interaction would likely be a significant event during late pregnancy and labor, because ITGA5B1 is unique among the FN-binding integrins in that it is the only integrin that naturally assembles FN into a fibrillar matrix [12].
Late pregnancy is a period of increasing uterine stretch due to growing fetuses and, as a result, stretch-induced myometrial hypertrophy [4]. Therefore, existing ITGA5 proteins in myometrial cell membranes may act as the initial mechanical stress gauges during late pregnancy and be involved in signal transduction pathways that ultimately induce additional Itga5 mRNA expression, cytoskeletal reorganization, and Fn gene expression. This process is likely aided by the known association of integrins with the actin cytoskeleton via adapter proteins [28–31], facilitating the sensing of mechanical forces both from the exterior and interior of the cell. Considerable evidence has accumulated that mechanical stress induces integrin gene expression and ECM synthesis/remodeling [32–36].
Progesterone-Induced Delayed Labor
In our study, we demonstrated that Itga5 mRNA expression was maintained at high levels on Day 24 during progesterone-induced delayed labor. Further work is clearly required to determine whether progesterone or other biochemical signals (or both) are regulators of Itga5 mRNA expression during late pregnancy, labor, or postpartum; however, it is possible that the prolongation of the gravid state of the uterus may be a mechanism underlying sustained Itga5 mRNA expression. Additional stretch and stretch-induced hypertrophy as a result of delayed labor may maintain Itga5 mRNA expression at Day 24, because recent work by Shynlova et al. [10] has demonstrated that gravidity positively regulates Fn mRNA expression.
While our immunoblot analysis demonstrated the maintenance of ITGA5 levels in myometrial tissue lysates at Day 24 during delayed labor, immunocytochemistry experiments showed ITGA5 was consistently detectable at higher levels in cell membranes of both myometrial layers at Day 23 and Day 24 in progesterone-treated animals compared with vehicle controls. These results suggest an increased recruitment of ITGA5 to cell membranes on Day 23 and Day 24 of delayed labor, likely to facilitate increased ITGA5B1-FN interaction during a period of extended gravidity, subsequent myometrial stretch and hypertrophy, and increased focal adhesion remodeling marked by sustained PTK2 activation during delayed labor [6]. The increased localization of ITGA5 in cell membranes was not reflected in increased total ITGA5 levels from our immunoblot analysis. We postulate that these results might be explained by increased ITGA5 turnover during delayed labor. Newly translated ITGA5 might be quickly recruited to membranes to replace ITGA5 proteins that were degraded during focal adhesion remodeling and maintain relatively comparable total myometrial ITGA5 levels in the process.
The Role of 5 Integrin in Promoting Contractility
While there is evidence that integrins and their ligands can regulate smooth muscle contractility, largely through changes in intracellular calcium concentration [18–19], our results demonstrate that considerable expression of Itga5 mRNA and ITGA5 protein occur many days before labor occurs, implying that ITGA5 may have a broader role during late pregnancy. We propose that ITGA5, partnered with ITGB1, may be important during late pregnancy in promoting cellular cohesion. Robinson et al. [12] have elegantly demonstrated that ITGA5B1 confers stronger cohesivity to three-dimensional tissue aggregates than that conferred by N-cadherin; a member of the cadherin family that is traditionally regarded as a primary regulator of tissue cohesivity. This cohesivity appears to be dependent on FN matrix assembly [13], a process that requires activation by binding to ITGA5B1 and integrin clustering [11].
Recently, Kuo and Seow [37] used electron microscopic and functional evidence to report that airway smooth muscle cells in a tissue bundle work as a mechanical syncytium during contraction, dependent on correct cytoskeletal filament organization, focal adhesion formation, and cell-cell and cell-ECM interaction. Therefore, in the rat myometrium during late pregnancy, ECM remodeling, focal adhesion turnover, increased expression of Itga5, and subsequent ITGA5B1-FN interaction may facilitate proper smooth muscle cellular cohesion in myometrial tissue before labor. These processes may be components of myometrial activation by contributing to the development of a mechanical syncytium that will facilitate efficient force transduction of the sustained, coordinated, and powerful contractions of labor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Daniel MacPhee, Division of Basic Medical Sciences, Health Sciences Centre, Room 5335, 300 Prince Philip Drive, St. John's, NL, Canada A1B 3V6. FAX: 709 777 7010; dmacphee{at}mun.ca
Received: 30 August 2004.
First decision: 22 September 2004.
Accepted: 23 December 2004.
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REFERENCES |
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5ß1 integrin mediates strong tissue cohesion. J Cell Sci 2003 116:377-3865ß1-mediated cell cohesion. Mol Biol Cell 2004 15:973-9815ß1 integrin requires signaling between focal adhesion proteins. J Biol Chem 2001 276:30285-30292-actinin and the ß1 integrin subunit in vitro. J Cell Biol 1990 111:721-729Vß3-integrin turnover regulates focal adhesion behavior. J Cell Biol 2001 155:1319-1332This article has been cited by other articles:
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  O. Shynlova, S. J. Williams, H. Draper, B. G White, D. J MacPhee, and S. J Lye Uterine Stretch Regulates Temporal and Spatial Expression of Fibronectin Protein and Its Alpha 5 Integrin Receptor in Myometrium of Unilaterally Pregnant Rats Biol Reprod, November 1, 2007; 77(5): 880 - 888. [Abstract] [Full Text] [PDF]
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 O. Shynlova, P. Tsui, A. Dorogin, B L. Langille, and S. J Lye The expression of transforming growth factor {beta} in pregnant rat myometrium is hormone and stretch dependent Reproduction, September 1, 2007; 134(3): 503 - 511. [Abstract] [Full Text] [PDF]
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B E Cross, H M O'Dea, and D J MacPhee Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour Reproduction, April 1, 2007; 133(4): 807 - 817. [Abstract] [Full Text] [PDF]
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O. Shynlova, P. Tsui, A. Dorogin, B. L. Langille, and S. J. Lye Insulin-like Growth Factors and Their Binding Proteins Define Specific Phases of Myometrial Differentiation During Pregnancy in the Rat Biol Reprod, April 1, 2007; 76(4): 571 - 578. [Abstract] [Full Text] [PDF]
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M. Breuiller-Fouche and G. Germain Gene and protein expression in the myometrium in pregnancy and labor. Reproduction, May 1, 2006; 131(5): 837 - 850. [Abstract] [Full Text] [PDF]
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P.A. Elustondo, G.E. Hannigan, I. Caniggia, and D.J. MacPhee Integrin-Linked Kinase (ILK) Is Highly Expressed in First Trimester Human Chorionic Villi and Regulates Migration of a Human Cytotrophoblast-Derived Cell Line Biol Reprod, May 1, 2006; 74(5): 959 - 968. [Abstract] [Full Text] [PDF]
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O. Shynlova, P. Tsui, A. Dorogin, M. Chow, and S. J. Lye Expression and Localization of Alpha-Smooth Muscle and Gamma-Actins in the Pregnant Rat Myometrium Biol Reprod, October 1, 2005; 73(4): 773 - 780. [Abstract] [Full Text] [PDF]
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