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Biology of Reproduction 59, 862-870 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Thrombospondin-1 Expression in Human Myometrium before and during Pregnancy, before and during Labor, and in Human Myometrial Cells in Culture1

Taro Morimotoa, Judith R. Heada, Paul C. MacDonalda, , and M. Linette Casey2,a

a The Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics-Gynecology, Biochemistry, and Cell Biology-Neuroscience, The University of Texas Southwestern Medical School, Dallas, Texas 75235–9051


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of latent transforming growth factor ß (L-TGFß) is essential for the action of TGFß, which, in turn, is involved in the regulation of expression of some progesterone-responsive genes. One mechanism by which TGFß is activated involves thrombospondin (TSP), a protein that binds extracellular proteins. Immunoreactive TSP (irTSP) protein and TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women were localized by immunohistochemistry and in situ hybridization. IrTSP and TSP-1 mRNA were randomly distributed in myometrial smooth muscle cells of some, but not all, tissues of pregnant women at term before labor; but in some areas of most of these tissues, irTSP was intense and commonly localized extracellularly. Intense irTSP and TSP-1 mRNA in myocytes were more common in myometrium during labor. In myometrium from ovulatory women (n = 26), irTSP was localized primarily in vascular smooth muscle cells and was detected occasionally in scattered myocytes. Little TSP-1 mRNA was demonstrable by in situ hybridization in vessels or myocytes of myometrial tissue from ovulatory women (n = 7). By Northern analysis of total RNA, TSP-1 mRNA was detected in myometrial tissue of pregnant women and in human myometrial smooth muscle cells in culture. The levels of TSP-1 mRNA in myometrial tissues of pregnant women during labor (n = 18) were greater than those in myometrium at > 37 wk gestation before labor began (n = 25, p < 0.001). The ratios of TSP-1 to glyceraldehyde 3-phosphate dehydrogenase mRNAs in 3 myometrial tissues during oxytocin-induced labor were not statistically different from those in myometrium during spontaneous labor but were greater than those in myometrium before labor (p < 0.05). The level of TSP-1 mRNA in confluent human myometrial cells in culture was relatively high, was increased by treatment with fetal bovine serum, and was decreased by treatment with platelet-derived growth factor or activators of adenylyl cyclase or protein kinase C. Myometrial cells in culture constitute a useful model for studying the regulation of TSP-1 gene expression in human myometrium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matricellular is a term that has been used to describe the thrombospondins (TSP) and other extracellular proteins that function by binding simultaneously to extracellular matrix proteins and to cell surface receptors or other molecules that interact with the cell surface [1]. TSP is a large homotrimeric protein comprising subunits of ~180 kDa. TSP binds to at least 5 separate cell surface receptors [2] and is composed of multiple discrete domains that mediate binding to a variety of ligands, including heparin, calcium, fibrinogen, fibronectin, type V collagen, and sulfated glycolipids [3, 4].

Important biological functions of the TSPs in smooth muscle cells are of particular interest in investigations of the physiological functions of the human myometrium during pregnancy, at the time of parturition, and during puerperal involution of the uterus. First, TSP promotes replication of smooth muscle cells [5–9], and conversely, the expression of TSP is stimulated by growth factors, e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and serum [5–7, 10]. Second, TSP modifies smooth muscle cell responses to contractile agents [11, 12]. Third, TSP is involved in processes important to tissue remodeling similar to those in the uterus during labor, immediately postpartum, and during the puerperium, including the promotion of monocyte chemotaxis [13] and increased expression of monocyte chemoattractant protein (MCP)-1 [14]. Fourth, TSP promotes activation of the latent forms of transforming growth factors (L-TGFs)-ß [15–18]. TGFß, in turn, may facilitate the transition of the uterus from phase 0 to phase 1 of parturition, i.e., from a state of remarkable contractile refractoriness to one of preparedness for labor [19].

There are multiple potential roles of TGFß in the modifications of myometrial smooth muscle cell functions that are associated with the process of uterine awakening that precedes the onset of labor and during uterine involution postpartum [19]. TGFßs act 1) to inhibit selected actions of progesterone through progesterone receptor-independent mechanisms [20], 2) to down-regulate selected hepathelical receptors that activate the G{alpha}s-subunit of the G-proteins [21–24] and promote myometrial quiescence, 3) to oppose the actions of cAMP [25–31], and 4) to inhibit the formation of nitric oxide (NO) [32–36]. G{alpha}s-GTP and NO may promote myometrial quiescence by way of several diverse mechanisms, as is believed to be true in airway smooth muscle [37]. These actions of TGFßs, if operative in myometrium near the end of gestation, should favor the initiation of parturition. Hence, the process of activation of TGFß is of key importance.

TGFßs are secreted from most cells in one of two latent forms, which are biologically inactive. Little or no active (mature) TGFß is secreted directly from most cells. Consequently, the activation of L-TGFßs in vivo is a limiting step in TGFßs actions. TSP functions to activate L-TGFßs [15–18, 38], and TSP binds to plasminogen [39, 40], which may facilitate a plasmin-mediated activation of L-TGFßs. L-TGFßs and TSP co-localize on the cell surface [41]. In addition, TGFß stimulates the synthesis of TSP in multiple cell types [42, 43] and acts to cause a synergistic increase in PDGF-induced TSP expression in smooth muscle cells [44].

The action of TSP to activate L-TGFß prompted the conducting of this study to examine the levels of TSP-1 mRNA and irTSP in myometrial tissues before and during pregnancy, and before and during labor. In addition, the levels of TSP-1 mRNA in human myometrial cells in culture also were assessed, and studies were conducted to evaluate the regulation of the levels of TSP-1 mRNA in these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Myometrial Tissues

Myometrial tissues were obtained from the uteri of nonpregnant or pregnant women at hysterectomy or from the upper edge of the incision at the time of cesarean section conducted either before or during labor. Some of the tissues were immediately frozen in liquid nitrogen, others were frozen in liquid nitrogen using O.C.T.-embedding medium for histochemical evaluation, and some were placed in culture medium for use in isolation of myometrial cells. Gestational age was estimated from menstrual dates, sonography, well-defined milestones of pregnancy (uterine size increments, quickening, and auscultation of the fetal heart), and estimated gestational age from evaluations of the newborn. Informed consent for the use of tissues was obtained in writing from each woman. The consent forms and protocols were approved by the Institutional Review Board of this university.

Myometrial Cells in Culture

Myometrial smooth muscle cells were isolated from myometrial tissue of nonpregnant women and maintained in culture as previously described [45]. Briefly, the myometrial tissue was minced and incubated in a water bath with agitation for 4 h at 37°C with collagenase (1.5 mg/ml; ~150 U/mg), deoxyribonuclease I (0.1 ng/ml; ~2000 U/mg), and an antibiotic-antimycotic solution (2%, v:v) to disperse the smooth muscle cells. The dispersed cells were separated from nondigested tissue by filtration through gauze and collected by centrifugation of the filtrate at 400 x g for 10 min; suspended in Ham's F-12:Dulbecco's Modified Eagle's medium (F12:DMEM, 1:1, v:v) with fetal bovine serum (FBS, 10%, v:v), penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml), and amphotericin B (0.25 µg/ml); plated in plastic 75-cm2 culture flasks at a density of ~100 000 cells/cm2; and maintained at 37°C in a humidified atmosphere of air and CO2 (5%) until confluent (7–10 days after plating). The culture medium was changed every 72 h.

Confluent myometrial smooth muscle cells in primary culture were passed by standard methods of trypsinization, plated in plastic culture dishes (100-mm diameter or 24-well), and maintained in F12:DMEM with FBS (10%, v:v) and antibiotics-antimycotics until confluence was attained. Confluent, first-passage cells were used for all experiments. At the time confluence was attained, the culture medium was changed; and 24 h thereafter, the cells were pre-incubated for 24 h in serum-free F12:DMEM that contained BSA (1%, v:v) before the medium was changed to serum-free medium that contained the test agents.

Northern Analyses

Total RNA was extracted from tissues or myometrial cells in culture (100-mm dishes) by the guanidinium isothiocyanate method of Chirgwin et al. [46]. Total RNA was size-fractionated by electrophoresis on formaldehyde-agarose (1%) gels, transferred electrophoretically to Hybond-N+ membrane, and cross-linked to membranes by UV irradiation. Prehybridization was conducted for ~18 h at 42°C in prehybridization buffer comprising 5-strength SSC (single-strength SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), 10-strength Denhardt's solution, formamide (50%, v:v), dextran sulfate (5%, w:v), NaH2PO4 (50 mM), and salmon sperm DNA (0.5 mg/ml). Hybridizations were conducted for 16–24 h at 42°C in buffer comprising 5-strength SSC, double-strength Denhardt's solution, formamide (50%, v:v), dextran sulfate (10%, w:v), NaH2PO4 (20 mM), salmon sperm DNA (0.1 mg/ml), and human TSP-1 cDNA probe (~70–80 µCi) radiolabeled with [{alpha}-32P]deoxycytidine triphosphate (dCTP) by the random hexamer priming method. The human TSP-1 cDNA was synthesized by reverse transcription of total RNA from human myometrial cells in culture and polymerase chain reaction using oligonucleotides as primers: forward, 5'-TAC ACA CAG GAT CCC TGC TGG GCA-3'; reverse, 5'-CGC CTC AGC TCA TTG GCC AAC TCT-3'. The 979-base pair (bp) cDNA product was sequenced to verify identity with TSP-1 [47]. The blots were washed twice for 15 min at room temperature with 0.1-strength SSC and SDS (0.1%, w:v) and for 30 min at 42°C and 50°C with 0.1-strength SSC and SDS (0.1%, w:v). The membranes were blotted on filter paper, sealed in a plastic bag, and exposed to film for autoradiography at -70°C. In some studies, the membranes also were hybridized with an oligonucleotide probe for glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Radioactivity corresponding to TSP-1 mRNA or G3PDH mRNA was quantified by radioanalytical imaging, and the levels of TSP-1 mRNA are presented as the ratio of radioactivity in TSP-1 mRNA:G3PDH mRNA.

In Situ Hybridization

In situ hybridization was conducted by a modification of the method of Karr et al. [48] using digoxigenin (DIG)-uridine triphosphate (UTP)-labeled TSP-1 cRNA (sense and antisense) synthesized by T7 and SP6 RNA polymerase-driven transcription of a linearized plasmid (constructed in pGEM3; Promega, Madison, WI) that contained a 252-bp fragment of human TSP-1 cDNA in the Pst I site. Frozen sections (8 µm) were thaw-mounted onto charged slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA), dried in air briefly, and fixed for 60 min at room temperature in paraformaldehyde (3%) in PBS. The sections were washed with PBS, incubated for 10 min with triethanolamine (0.1 M) and acetic anhydride (0.25%), rinsed in double-strength SSC, dehydrated by rinsing in alcohols, and allowed to dry briefly in air. Hybridization was conducted overnight at 50°C in SET buffer (Tris [30 mM] buffer with NaCl [150 mM] and EDTA [2 mM]) with formamide [50% v:v], polyvinylpyrrolidone [5 mg/ml], ficoll [5 mg/ml], BSA [5 mg/ml], tRNA [1.25 mg/ml], and dextran sulfate [125 mg/ml]), with DIG-labeled TSP-1 cRNA (~100 ng/ml).

After hybridization, the sections were washed at 60°C for 30 min in double-strength SET buffer with formamide (50%) and twice at 37°C for 15 min in double-strength SET buffer. The sections were incubated at 37°C for 20 min with RNase A (10 µg/ml) in triple-strength SET buffer with BSA (100 µg/ml), washed sequentially for 15 min at 37°C with SSC (double-strength, single-strength, 0.2-strength), and then washed at room temperature for 10 min with sodium maleate buffer, pH 7.5 (maleic acid [100 mM] and NaCl [150 mM]). The sections were incubated at room temperature for 30 min and then for 2.5 h in a humidified chamber with sodium maleate buffer that contained Triton X-100 (0.05%, v:v), normal sheep serum (1%, v:v), and alkaline phosphatase-conjugated sheep anti-DIG Fab fragment antibody (Boehringer-Mannheim, Indianapolis, IN; 1:500). The sections were rinsed in sodium maleate buffer and with Tris (100 mM, pH 9.5) buffer that contained NaCl (100 mM) and MgCl2 (25 mM). The sections were incubated for 16 h in Tris (100 mM, pH 9.5) buffer that contained an alkaline phosphatase substrate, nitroblue tetrazolium/bromo-chloro-indolyl-phosphate (Promega, Madison, WI), and levamisole (100 mM; inhibitor of endogenous alkaline phosphatase). Thereafter, the sections were rinsed with Tris (10 mM, pH 8.0) buffer that contained EDTA (1 mM) and then with water, and allowed to dry in air. Coverslips were applied with an aqueous mounting medium (90% glycerol or Immu-mount [Shandon, Pittsburgh, PA]) for microscopic observation.

Immunohistochemical Analyses

Frozen sections (8 µm thick) of myometrial tissues were fixed in cold (-20°C) acetone for 10 min, then analyzed for TSP using an avidin-biotin immunoperoxidase technique (ABC kit; Vector Laboratories, Burlingame, CA) as described previously [49]. Diaminobenzidine was used as the chromagen, and the sections were counterstained lightly with hematoxylin. Identical results were obtained with two different mouse monoclonal antibodies to human TSP (clone 10 from Biodesign International, Kennebunk, ME, and clone 11.4 from Boehringer-Mannheim). To evaluate specificity, an isotype-similar, irrelevant antibody (IgG1; Sigma Chemical Co., St. Louis, MO) was used instead of a primary anti-TSP antibody. With the exception of a few leukocytes in some samples, positive staining was not detected with the irrelevant antibody. The tissue sections were examined with a Zeiss Photomicroscope II for TSP immunoreactivity by observers who had no knowledge of the origin of tissue samples. The relative staining of myometrial muscle bundles was scored according to the following criteria: 1—all or almost all negative; 2—mostly negative or weakly positive cells, with significant areas of strong perinuclear staining; 3—moderate staining throughout or mixed staining intensity with negative, weakly to moderately positive, and strongly positive bundles; 4—strongly positive staining of most or all bundles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TSP-1 mRNA in Myometrial Tissues from Pregnant Women

TSP-1 mRNAs (6.2 kilobases [kb] and 4.5 kb) are readily detected by Northern analysis of total RNA from myometrial tissue of pregnant (n = 48) and ovulatory (n = 18; n = 6 from each of follicular, early/mid-secretory [Day 15–Day 22], and late secretory [Day 23–28]) women. Among the myometrial tissues evaluated, the levels of TSP-1 mRNA varied widely. The levels of TSP-1 mRNA relative to G3PDH mRNA in myometrial biopsies from pregnancies before and during spontaneous labor (Fig. 1) and after administration of oxytocin were estimated by radioanalysis. The ratios of TSP-1 to G3PDH mRNA (mean ± SEM) from myometrial biopsies obtained after the spontaneous onset of labor (3.01 ± 0.36; n = 18) were significantly greater (p < 0.001, Mann-Whitney rank order sum test) than those in myometrial biopsies obtained before the onset of labor at > 37 wk gestation (1.40 ± 0.13; n = 25). The levels of TSP-1 mRNA at 40–42 wk gestation before labor (1.54 ± 0.16, n = 17) were greater (p < 0.05) than those at 38–39.5 wk gestation (1.10 ± 0.22, n = 8) and less than those during labor of spontaneous onset (p < 0.05; ANOVA, Dunn pair-wise comparison; Fig. 2). Oxytocin was administered after the spontaneous onset of labor in 4 pregnancies, and the values for the ratio of radiolabeled TSP-1 mRNA to G3PDH mRNA in these myometrial tissues were 1.52, 1.59, 4.47, and 2.54 (2.53 ± 0.69, mean ± SEM; not significant compared with that in myometrial tissues during spontaneous labor without oxytocin administration). In 3 cases, myometrial biopsies were obtained during oxytocin-induced labor (i.e., spontaneous labor had not begun when oxytocin was administered). The values for the ratios of TSP-1 mRNA to G3PDH mRNA in these three samples were 4.25, 4.47, and 13.2 (7.3 ± 2.9, mean ± SEM, Fig. 2).



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  		FIG. 1. TSP-1 mRNA in myometrial tissue from human pregnancies. Representative Northern blot of myometrial biopsies from pregnancies at term before (n = 10; NIL) and during (n = 10; SOOL) labor of spontaneous onset.



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  		FIG. 2. Relative levels of TSP-1 mRNA in myometrial biopsies from pregnancies (n = 38) at various stages of gestation, before or after labor onset or oxytocin administration. The level of TSP-1 mRNA was quantified by radioanalysis and normalized to the level of G3PDH mRNA in each sample. Each point represents data for myometrium from a single pregnancy; bars represent mean ± SEM. NIL, Not in labor; SOOL, spontaneous onset of labor; oxytocin-ind labor, oxytocin-induced labor.

In another study, the level of TSP-1 mRNA was evaluated in myometrial tissue pieces obtained from different sites of the same uterus obtained after cesarean hysterectomy at 38.5 wk gestation; the pregnancy was complicated by bleeding at the placental site and postpartum uterine atony, which was not resolved by administration of prostaglandin and oxytocin. Myometrium was obtained from 14 different sites in the uterus, 10 anterior (including sites in the lower uterine segment and near the incision site), 3 posterior, and 1 at the top of the uterine fundus. TSP-1 mRNA was detected in all samples of myometrial tissue from this pregnancy, and there was little difference in the levels among samples from different anatomic sites (data not shown).

Localization of Immunoreactive TSP (irTSP) and TSP-1 mRNA

The distribution of TSP protein in myometrial tissues from ovulatory women (n = 26) and from pregnancy at 37–42 wk gestation before (n = 29) and during (n = 19) labor was evaluated by immunohistochemical analysis. The relative staining of irTSP in these samples is summarized in Figure 3. In tissues from ovulatory women at all phases of the ovarian cycle, staining for TSP was often striking in the tunica media of arterial vessels (Fig. 4A). In contrast to the vascular muscle, irTSP was not detected in most myometrial myocytes in tissues from ovulatory women (16 of 26); in these tissues, irTSP was detected in the perinuclear region of a few myocytes widely dispersed throughout the myometrium. In other tissues from ovulatory (7 of 26) women, weak staining of irTSP was apparent in the myocytes, with more cells having strong perinuclear staining; in these tissues, the cell bundles near the endometrial-myometrial junction were usually more positive than those in other sites. A few tissues (3 of 26) had moderate levels of irTSP, some of which outlined the myocytes, suggestive of extracellular deposition. In many myometrial tissues, irTSP was detected in scattered cells of the connective tissue. The pattern of irTSP in myometrial tissue was not appreciably affected by the phase of the ovarian cycle.



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  		FIG. 3. Relative TSP immunoreactivity in myometrial samples from ovulatory women (open bars; n = 26) and pregnant women at term before (hatched bars; n = 29) and during (closed bars; n = 19) labor. Intensity ratings are explained in Materials and Methods.



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  		FIG. 4. Immunohistochemical localization of TSP in myometrium. Sections were counterstained lightly with hematoxylin. A) Strong TSP staining in the muscle of an artery in myometrium from an ovulatory woman but no significant staining in the myometrial myocytes (L, lumen); x200. B) Weak staining of myocytes in myometrium from a term pregnancy before labor, with stronger perinuclear staining (compare to C); x400. C) B incubated with an isotype-similar nonimmune antibody; only counterstain is visible; x400. D) Moderate intracellular staining in muscle bundles (transverse section) of myometrium from a pregnancy at term before labor, with some staining at the cell periphery (arrow); x200. E) Strong staining outlining the myocytes of muscle bundles in myometrium from a term pregnancy during labor; x400. F) Extensive irTSP in myometrial tissue (longitudinal section) from a term pregnancy before labor, x200. B, D, E, and F are from different pregnancies. Reproduced at 69%.

In myometrial tissues from term pregnancies, irTSP was detected in myometrial cells in widely varying levels, from barely detectable to extensive. IrTSP was most intense in myometrial tissues obtained during labor (Fig. 3). Of the tissues obtained before labor, 5 of 29 had no detectable irTSP in myocytes, except for occasional cells with distinct perinuclear staining, as in myometrial tissues from ovulatory women. In other myometrial tissues obtained before labor (6 of 29), irTSP was detected only in the perinuclear region of some myocytes (Fig. 4, B and C) or, in some bundles, intracellularly with some staining in the periphery of myocytes (Fig. 4D). In many of the myometrial tissues obtained before labor (15 of 29) and most of those obtained during labor (13 of 19), irTSP was either extensively moderate (Fig. 4D) or, more commonly, variable in the muscle bundles including some strongly staining areas surrounding the myocytes (Fig. 4, E and F). Within a single tissue section of such samples, irTSP was commonly undetectable in some muscle bundles, readily detectable in the perinuclear or intracellular regions of the myocytes of some muscle bundles, and intense in areas outlining the myocytes of other muscle bundles. In some myometrial tissues from term pregnancies before (3 of 29) and during (6 of 19) labor, irTSP was intense through most or all of the tissue (similar to the pattern in Fig. 4, E and F).

In situ hybridization was conducted to localize TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women. In 16 myometrial tissues, the distribution of TSP-1 mRNA was compared with that of TSP protein in the same tissues. Myometrial tissues from 7 ovulatory women and 9 pregnant women at term before (n = 5) and during (n = 4) labor were evaluated. In areas of myometrial tissues in which irTSP in myocytes was intense (such as in Fig. 4, E and F), there also was hybridization (of virtually all cells) with the antisense TSP-1 cRNA, localized in the perinuclear region (Fig. 5, A and B; compare to 5F). In areas of myometrial tissue with limited irTSP, there also was limited hybridization (Fig. 5C). In areas of myometrial tissue with moderate irTSP or widespread perinuclear irTSP, there was extensive hybridization with TSP-1 cRNA (Fig. 5D). There was little, if any, TSP-1 hybridization in muscle bundles in which little or no irTSP was detected, whether in myometrial tissues from ovulatory women or in myometrial tissues from term pregnancies (Fig. 5E). In all types of tissues evaluated, hybridization was detected often in cells of the connective tissue and was prominent in myometrial tissues in which irTSP in myocytes was relatively low (Fig. 5C). IrTSP was variable in these cells of the connective tissues. The singular and obvious dichotomy in the localization of TSP-1 mRNA and protein was in vascular smooth muscle cells. In myometrial tissues from term pregnancies in which irTSP was intense in myocytes, TSP-1 mRNA was detected in many vascular smooth muscle cells of some vessels (Fig. 5D). In myometrial tissues (from term pregnancies or ovulatory women) with little irTSP in myocytes and substantial irTSP in arterial vessels (such as in Fig. 4A), little or no TSP-1 mRNA was detected (Fig. 5E). Neither TSP-1 mRNA nor TSP protein was detected in infiltrating leukocytes that were present in some myometrial tissues from term pregnancies. TSP-1 mRNA and TSP protein were detected commonly in endothelial cells of vessels near accumulations of leukocytes (data not shown).



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  		FIG. 5. Identification of TSP-1 mRNA by in situ hybridization in myometrium using DIG-labeled TSP-1 cRNA detected with alkaline phosphatase-conjugated anti-DIG antibody. No counterstain. A) Extensive TSP-1 mRNA in myocytes (longitudinal and transverse orientation) of myometrium from a term pregnancy during labor; x100. IrTSP in myocytes of this myometrial tissue was strong. B) Extensive perinuclear TSP-1 mRNA in myocytes of myometrium from a term pregnancy before labor; x400. IrTSP in myocytes of this myometrial tissue was strong. C) Weak TSP-1 mRNA in scattered myocytes of muscle bundles (left) and stronger TSP-1 mRNA in cells dispersed throughout the connective tissue (arrow) in myometrium from a term pregnancy before labor; x200. IrTSP was detected in scattered myocytes. D) Strong TSP-1 mRNA in myometrial myocytes and in arterial smooth muscle cells (L, lumen) of myometrium from a term pregnancy during labor; x200. IrTSP was strong in perinuclear areas of myocytes and in vascular smooth muscle of this tissue. E) Weak TSP-1 mRNA in scattered myocytes of myometrium from a nonpregnant women. TSP-1 mRNA was not detected in an artery (L, lumen), despite strong irTSP in the same vessel; x200. F) No hybridization in a section of myometrial tissue (same as in A) incubated with DIG-labeled sense TSP-1 cRNA; x200. Reproduced at 69%.

TSP-1 mRNAs in Myometrial Cells in Culture

The levels of TSP-1 mRNA in confluent myometrial cells in culture were relatively high, increased in response to treatment with fetal bovine serum (Fig. 6), and decreased in response to treatment in serum-free medium with 12-O-tetradecanoylphorbol acetate (TPA; 100 nM) for 24 h (Fig. 6). Treatment of myometrial cells in serum-free medium for 24 h with PDGF, a mitogen in these cells (data not shown), also caused a decrease in the level of TSP-1 mRNA (Fig. 6). After shorter times of treatment with PDGF (e.g., 1–4 h) there was no change or a very slight increase in the level of TSP-1 mRNA (data not shown).



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  		FIG. 6. TSP-1 mRNA in myometrial cells in culture. Confluent cells in medium without serum (- serum) or with FBS (10%, v:v; + serum) were treated with TPA (T, 100 nM) or PDGF (P, 30 ng/ml) for 24 h. 0, Zero time; C, control, untreated cells. The ethidium bromide-stained 28S rRNA subunit is presented for reference. Total RNA (10 µg) was applied to each lane.

The level of TSP-1 mRNA decreased in cells treated for 8 h in serum-free medium with agents that are known to cause an increase in the levels of cAMP in these cells. The agents tested were isobutylmethylxanthine (IBMX; 50 µM), Nle8,18Tyr34-PTH1–34 amide (10-7 M; a potent analogue of parathyroid hormone [PTH]) plus IBMX, PTH-rP1–34 amide (10-7 M; a potent analogue of PTH-rP) plus IBMX, or forskolin (100 µM) (Fig. 7). The levels of TSP-1 mRNA were not decreased in response to treatment with Asn10Leu11-PTH-rP7–34 amide (10-7 M; a potent antagonist of PTH-rP), which does not cause an increase in cAMP levels (Fig. 7). Another PTH analogue, D-Tyr12,Tyr34PTH7–34 amide (10-7 M), plus IBMX also caused a decrease in the level of TSP-1 mRNA.



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  		FIG. 7. TSP-1 mRNA in myometrial cells in culture. Confluent cells in serum-free medium were treated with IBMX (I; 50 µM), agonists or antagonists of PTH/PTH-rP plus IBMX, or forskolin plus IBMX for 8 h. The specific PTH/PTH-rP agonists and antagonist are described in Results. The ethidium bromide-stained 28S rRNA subunits are presented for reference. Total RNA (15 µg) was applied to each lane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The distribution of TSP in myometrial tissues of pregnant and ovulatory women differs markedly. TSP is localized principally in vascular smooth muscle in myometrium of ovulatory women and in myometrial smooth muscle cells in myometrium of pregnancy. The levels of TSP-1 mRNA and TSP protein in myometrial tissues of pregnancy are increased during labor and after the administration of oxytocin. Importantly, however, the levels of TSP-1 mRNA and TSP protein in myometrial tissues from approximately one-half of the term pregnancies that were evaluated before labor (15 of 29) also were high. This finding strongly suggests that the increase in TSP-1 gene expression occurs during phase 1 of parturition, i.e., the time of uterine preparedness for labor. Conversely, and in support of this possibility, the levels and distribution of TSP-1 mRNA and protein in myometrium in the other approximately one-half of these pregnancies (term before labor) were much lower and similar to those in myometrial tissue from ovulatory women. Nonetheless, the findings of this study do not permit a precise determination of the timing of increased TSP-1 expression in myometrium during pregnancy. It cannot be determined from the findings of this study when the increase in expression of TSP-1 in myometrium occurred relative to the onset of labor, since it is not known when labor will begin in the course of a given pregnancy. Similarly, the functional status of the uterus with respect to modifications in preparation for labor cannot be defined from clinical history, physical examinations, or laboratory findings. Consequently, it is not possible to state whether the higher levels of TSP-1 mRNA in myometrial tissues obtained during labor are indicative of increased TSP-1 expression in preparation for labor or else are the consequence, even in part, of the labor process. This issue is not resolved by the finding of increased levels of TSP-1 mRNA in myometrial tissues from women who were in oxytocin-induced labor. The very fact that labor was induced in these women indicates that phase 1 of parturition was in place at the time of oxytocin administration. Otherwise, oxytocin induction would not have been successful.

It is noteworthy that there was tremendous variation in the levels of TSP-1 mRNA and TSP protein within a given section of many myometrial tissues evaluated, including most of those obtained during labor. There was little or no TSP-1 mRNA or irTSP in some muscle bundles, moderate TSP expression in some, and strong TSP expression in others. This extreme range of TSP-1 mRNA and TSP protein was common and is suggestive of local regulation of expression, possibly in a paracrine or autocrine fashion.

The mechanism(s) through which myometrial quiescence is maintained during phase 0 of human pregnancy is not clear. In many mammalian species, progesterone is believed to serve a crucial role in this process. Indeed, progesterone withdrawal precedes the initiation of parturition in the majority of mammalian species studied; and in these species, progesterone administration late in pregnancy forestalls the timely onset of labor. But in primate pregnancy, including that of the human, progesterone withdrawal does not occur before the initiation of parturition and progesterone administration does not prolong gestation. TGFß acts to attenuate the action of progesterone on selected progesterone-responsive genes [20]. These selective antiprogestin effects of TGFß are mediated through progesterone receptor-independent mechanisms and may be important in the processes that precede the initiation of human parturition. The distribution of TSP in myocytes of myometrial tissue appeared to progress from intracellular, perinuclear localization to extracellular or surface deposition, consistent with the matricellular nature of TSP and the possibility that it may function in this site to effect activation of TGFß. An increase in TSP localization on myometrial smooth muscle cells could facilitate the activation of L-TGFß, and TGFß may act to modify the functional phenotype of myometrial smooth muscle cells.

It also is reasonable to hypothesize that processes other than those affected by progesterone are important in the maintenance of myometrial quiescence during human pregnancy. Multiple hepathelical receptors that nominally act by linkage to the G{alpha}s-subunit of the G-proteins have been identified in human myometrium. Activation of these receptors effects myometrial relaxation by activation of K+ channels and adenylyl cyclase, and hence, increased intracellular levels of cAMP.

TGFß acts in some cells to decrease the number of selected hepathelical receptors that may serve in myometrium to activate the G{alpha}s-subunit of the G-proteins. For example, TGFß treatment causes a reduction in ß-adrenergic-induced increases in cAMP [50], and TGFß effects a reduction in PTH-rP/PTH receptors in renal epithelial cells [21], rat osteoblasts [51], and MC3T3-E1 pro-osteoblasts [52]. In addition, opposing actions of TGFß and cAMP have been demonstrated in several systems, e.g., the regulation of plasminogen activator inhibitor expression [28], c-sis expression in endothelial cells [26], heparin-binding factor-induced increase in adenylyl cyclase activity in human arterial endothelial cells [27], cAMP-induced inhibition of collagen lattice formation [30], and adenylyl cyclase signaling elements in cardiomyocytes [31]. TGFß also acts in a number of systems to inhibit NO formation [32–36], another potential mechanism for the maintenance of uterine quiescence during human pregnancy.

Consequently, the actions of TGFß on myometrium near term could facilitate the transition of the uterus from a state of quiescence to one of contractile responsiveness by a variety of mechanisms. The increased expression of TSP in myometrium may constitute a mechanism for increased activation of latent TGFßs secreted by myometrial smooth muscle cells.

The finding of TSP protein, but not TSP-1 mRNA, in the muscle walls of arterial vessels is intriguing. There are several possible explanations for these observations: It is possible that TSP detected in the vessels is bound to TSP receptors. Alternatively, it is possible that the half-life of TSP-1 mRNA in these cells is short, relative to that of TSP. And it also is possible that the TSP antibodies recognize an isoform other than TSP-1 since cross-reactivity with specific isoforms of TSP other than TSP-1 has not been evaluated. On the other hand, the cDNA used for in situ hybridization is not highly homologous with TSPs 2, 3, or 4, and hence, it is unlikely that mRNAs for these isoforms would be detected with the TSP-1 cRNA or TSP-1 cDNA probes used in this study.

The expression of TSP in a variety of cell types in culture, including vascular smooth muscle cells, is increased in response to mitogenic stimuli [1, 5–7, 10]. Majack and colleagues demonstrated an immediate early gene response of TSP-1 in vascular smooth muscle cells treated with PDGF [6]. The increase in TSP-1 mRNA identified by these investigators is consistent with the conclusion that replicating smooth muscle cells express TSP-1. PDGF acts as a mitogen in a variety of smooth muscle cells, including human myometrial cells as demonstrated here and by others [53]. In this study, treatment of myometrial cells with PDGF or TPA for 24 h, as well as with analogues of PTH and PTH-rP for 8 h, which activate adenylyl cyclase through interaction with hepathelical receptors linked to G{alpha}s, acted to reduce the levels of TSP-1 mRNA. Treatment of myometrial cells with PDGF for shorter times (1–4 h) had little effect on the level of TSP-1 mRNA. In this context, it may be important that the levels of TSP-1 mRNA in confluent myometrial cells in culture, which are not contact inhibited, were relatively high.

During human pregnancy, there is marked hypertrophy but little hyperplasia of myometrial smooth muscle cells. Stretch of smooth muscle cells, which is massive in myometrium during pregnancy, leads to activation of protein kinase C. It is tempting to speculate that persistent stretch of the myometrium leads to down-regulation of protein kinase C, a process that may be mimicked by treatment of myometrial cells in culture with TPA for long times. The latter process has been demonstrated previously in these cells for parathyroid hormone-related protein [54]. The findings of the present study are supportive of the conclusion that additional mechanisms are involved in the regulation of TSP-1 gene expression, including a mechanism by which the levels of TSP-1 mRNA is suppressed. Myometrial cells in monolayer culture are a useful model in which to investigate the regulation of TSP-1 gene expression.


   ACKNOWLEDGMENTS
 
The authors thank Sheila Brandon, L.V.N., and Valencia Hoffman and the house staff in Obstetrics-Gynecology at Parkland Memorial Hospital for assistance in obtaining myometrial tissues; Patrick Keller, Charles Kresge, Bobbie Mayhew, Barbara Murry, and Jess Smith for skilled technical assistance; and Kathy Loppnow for expert editorial assistance.


   FOOTNOTES
 
1 This research was supported, in part, by USPHS Grant 5-P50-HD11149. Back

2 Correspondence: M. Linette Casey, Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235–9051. FAX: 214 648 8683; casey{at}grnctr.swmed.edu Back

Accepted: May 26, 1998.

Received: February 5, 1998.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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