| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Departments of Surgical Sciences (J.I.G.) and Obstetrics and Gynecology, The Medical School, University of Newcastle upon Tyne, United Kingdom NE2 4HH
Address all correspondence and requests for reprints to: Dr. James I. Gillespie, Departments of Physiological Sciences and Obstetrics and Gynecology, The Medical School, The University, United Kingdom NE2 4HH. E-mail: j.i.gillespie{at}ncl.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The expression of the gap junction protein connexin 43 (Cx43) has been studied in detail (9, 10, 11, 12, 13, 14). Treatment of isolated human myometrial tissue with media containing high estrogen and low progesterone concentrations resulted in an increase in Cx43 expression (11). In addition, activation of human myometrial cells with phorbol ester increased the messenger ribonucleic acid levels of c-fos and c-jun, followed by an increased expression of Cx43 messenger ribonucleic acid, which resulted in a significant increase in Cx43 protein levels 2–4 h later (13). This work has been interpreted to suggest that Cx43 gene expression is regulated by changes in the estrogen/progesterone ratio working through the activation of protein kinase C and the production of specific transcription factors (11, 12, 13). Less is known about the activation of other CAP genes. We have reported that the expression of the RyR2 isoform of the ryanodine-sensitive intracellular Ca2+ release channel on the sarcoplasmic reticulum is up-regulated in the human myometrium as term approaches and that this increase in expression can be mimicked in vitro by the cytokine transforming growth factor-ß1 (TGFß1) (8). This leads to the idea that TGFß1 could be playing a similar role in activation of the RyR2 gene in the processes of normal preparation of the myometrium in vivo. These observations raise the possibility that cytokines may form part of a signaling system, different from that activating Cx43, responsible for the activation of specific CAP genes in vivo.
TGFß is a multifunctional cytokine that acts on many cells and cellular processes. The TGFßs are a family of five related peptides, three of which are found in mammalian tissues (TGFß1, TGFß2, and TGFß3) (15). They exert their actions via three receptors: type I, type II, and type III (16, 17). The intracellular processes linking TGFß and TGFß receptor activation to a cellular response can be complex depending on the machinery available within the cell, but ultimately this leads to the activation/inhibition of gene expression (18).
Indirectly, there may be some evidence for the involvement of cytokines
in the modulation of uterine function in pregnancy. In premature labors
involving infection, activated leukocytes release of a number of
cytokines and chemokines, including interleukin-1 (IL-1), IL-6, IL-8,
IL-10, tumor necrosis factor-
, and TGFß (reviewed in Ref. 19).
These agents have been reported to have a number of complex actions and
interactions, including remodeling of the cervix and promotion of the
synthesis of cyclooxygenase enzyme in the fetal membranes (20, 21, 22). The
resulting increased levels of PGE2 can result in myometrial
activation. These strong premature contractions along with cervical
softening can initiate labor and lead to delivery (20, 21).
If cytokines play a role in the activation of specific CAP genes in the pregnant myometrium, it is essential to demonstrate the presence of receptors to cytokines in myometrial cells in vivo and show that the levels of these cytokines change. As there is some evidence linking RyR2 gene expression to TGFß, the present report focuses specifically on TGFß1 and TGFß receptors, types I, II, and III, in an attempt to assess their contributions to the events that prepare the myometrium for normal delivery.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Polyclonal antibodies to TGFß receptors type I (TßRI), type II (TßRII), and type III (TßRIII) were obtained from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK). The TßRI antibody, R-20, corresponds to amino acids 482–501 at the carboxyl-terminus of human TßRI. The TßRII antibody, C-16, was raised against the peptide corresponding to amino acids 550–565 within the carboxyl-terminus of human TßRII. The TßRIII antibody, C-20, corresponds to amino acids 830–849 within the carboxyl-terminus of human TßRIII. The cytokine TGFß1 and goat antimouse IgG were purchased from Sigma Chemical Co. (Dorset, UK). Goat antirabbit IgG-linked horseradish peroxidase was purchased from Dako (High Wycombe, UK). The protein assay kit and high grade electrophoretic reagents were purchased from Bio-Rad (Hemel Hempstead, UK). Nitro-cellulose membrane was obtained from Schleicher and Schuell (London, UK). The enhanced chemiluminescence assay and TGFß1 ELISA system were obtained from Amersham International (Buckinghamshire, UK).
Tissue collection
Samples of myometrium were taken from the lower uterine segment of patients undergoing hysterectomy (nonpregnant; n = 11), elective caesarean section (pregnant nonlaboring; n = 11), or emergency cesarean section (spontaneous laboring; n = 11). This investigation had the ethical approval of Newcastle Area Health Authority, and informed consent was obtained from all patients. The clinical indication for hysterectomy in nonpregnant women was benign gynecological disorder such as menorrhagia, dysmenorrhea, and myoma uteri. Cesarean section at term (38–40 weeks gestation) was performed due to previous section or cephalopelvic disproportion. For emergency cases where labor had progressed to the active stage with the cervix 7–10 cm dilated, the diagnosis was fetal distress. In this group amniotic membranes were intact, and no history or presence of infection was noted. The samples were excised about 5 mm away from the decidua, serosal layer, or tumor, washed in phosphate-buffered saline, cut into small pieces, and frozen at -70 C until required.
Preparation of tissue lysates
Three groups of samples (nonpregnant, pregnant nonlaboring, and spontaneous laboring samples) were used. Frozen tissues were homogenized in 3 vol cold homogenizing buffer containing 25 mmol/L Tris base (pH 7.6), 0.25 mol/L sucrose, 1 mmol/L ethylenediamine tetraacetate (23), 5 µg/mL pepstatin A, 5 µg/mL leupeptin, and 5 µg/mL aprotinin. The homogenate was then centrifuged at 1500 x g for 30 min at 4 C. The supernatant was removed and recentrifuged to obtain a clear lysate. The protein content in tissue lysates was determined by the method of Bradford (24) using the Bio-Rad protein assay kit. Aliquots of samples were rapidly frozen and kept at -70 C until required. All procedures were carried out at 4 C.
Immunohistochemistry
Paraffin-embedded sections of myometrium (4 µm thick) were mounted onto poly-L-lysine-coated slides. Sections were deparaffinized in xylene for 2 min and rehydrated in a series of ethanol and water for 1 min for each step. The following incubations were performed at room temperature. Blocking of endogenous activity was achieved by a 10-min incubation in 3% hydrogen peroxide with subsequent washing in running tap water and phosphate-buffered saline, pH 7.3, for 2 min. For TßRI and TßRII antibody incubations, sections were incubated with normal goat serum, and excess blocking serum was removed before incubation in primary antibodies at a concentration of 3 µg/mL for 2–3 h in a humidified chamber and then washed with phosphate-buffered saline; sections were incubated for 45 min with horseradish peroxidase-conjugated anti-rabbit IgG (1:100). For sections incubated with TßRIII antibody, 2% BSA was used for blocking nonspecific binding, and horseradish peroxidase-conjugated donkey antigoat IgG (1:150) was used as the secondary antibody. After two washes, sections were visualized after 5- to 10-min incubation in freshly prepared diaminobenzidine solution and subsequent washing in running tap water. Sections were counterstained with hematoxylin, washed in water, cleared in xylene, dehydrated in graded alcohol, and mounted in DPX (Merck, Leicestershire, UK). Controls were processed in an identical manner, but in the absence of primary antibody.
Western blot and immunoblot analysis
Equal amounts of myometrial tissue lysates (40 µg) were solubilized in Laemmli (25) sample buffer containing 4% SDS with 10% ß-mercaptoethanol and boiled for 5 min. All samples were then electrophoresed on 7.5% SDS-PAGE at 40–45 mA until complete elution of the dye front in a Protean II xi cell (Bio-Rad). Proteins from the gel were electrotransferred onto a nitro-cellulose membrane (0.45 µm) using a Bio-Rad semidry blotting apparatus with 25 mmol/L Tris, 0.195 mol/L glycine, and 20% (vol/vol) methanol transfer buffer, pH 8.6 (23), at 20 V for 2 h. The nitro-cellulose strips were blocked overnight in Tris-buffered saline with Tween [TBST; 10 mmol/L Tris-HCl, 150 mmol/L NaCl (pH 8), and 0.05% Tween-20] containing 5% nonfat dried milk. Thereafter, the blots were washed three times with TBST and incubated for 3 h at room temperature with primary antibodies specific to TßRI, TßRII, and TßRIII at 0.5 µg/mL (1:200 dilution) in TBST with 3% milk. After three 15-min washings, goat antirabbit and donkey antigoat horseradish peroxidase-conjugated secondary antibody in the same incubation buffer were applied to the blots at a dilution of 1:2500 for 1 h. The blots were washed three times, and the immunoreactive bands were visualized by enhanced chemiluminescence with Fuji x-ray film (Fuji, Tokyo, Japan). To ensure that the immunoreactive bands detected were specific, the blots were incubated with preneutralized receptor antibodies using the peptides to which they were raised. Antibody neutralization was performed using the protocol of Santa Cruz Technologies, and blots were then reprobed with nonneutralized antibodies to check for immunoreactive staining as a control. Immunoblottings were repeated at least twice on each tissue with similar results. The molecular weights of the immunoreactive bands were determined using molecular weight standards from Bio-Rad.
Quantification of immunoreactive bands
The intensities of immunoreactive staining were measured with a GS 300 Laser Scanning Densitometer. As levels of Gß-subunit protein do not change during pregnancy and labor (26), this Gß-subunit was used as an internal control for the amount of protein loaded onto the gels. For each blot, normalization was performed by dividing the densitometric value of each receptor by that of the Gß-subunit protein in the same lane. Data for the expression of both receptor types in each group represent the mean and SEM from six or seven independent determinations. The values are expressed as a percentage of the relative value obtained from the nonpregnant control myometrium.
TGFß1 immunoassay
For assessment of the concentration of TGFß1 in the human myometrium, tissue lysates from nonpregnant, pregnant nonlaboring, and spontaneous laboring myometrium were prepared as previously described and stored at -70 C. A TGFß1 (Biotrak) ELISA system was used according to the manufacturer’s protocol. This involved activation of tissue lysates by acid hydrolysis so as to convert latent TGFß to the active form; thus, total TGFß1 levels in the tissues were determined. This assay had a sensitivity of 4 pg/mL. Samples reading as blanks were given a value of 0 pg/mL for the analysis. The concentration of cytokine was calculated from the linear regression analysis obtained from the log of TGFß1 concentrations vs. the log of the optical density standard curve. The mean total TGFß1 concentration is reported as picograms per g tissue wet wt.
Statistical analysis
Data are expressed as the mean ± SEM. The levels of expression of both receptor types between groups were analyzed using ANOVA followed by the Bonferroni test. Statistic analysis for the TGFß1 immunoassay was performed using Kruskal-Wallis test. P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, A–C, shows photomicrographs of tissue sections of human myometrium
from nonpregnant, pregnant nonlaboring, and spontaneous laboring women,
respectively. Each section illustrates control sections (a), sections
stained for type I TGFß receptor (b), and staining for type II TGFß
receptor (c). In each of the three patient groups, TGFß receptors for
types I and II could be detected in the myometrial smooth muscle cells.
No expression of type III TGFß receptor was detected in any of the
tissues studied (data not shown).
|
shows nitro-cellulose blots stained
with antibodies to TGFß receptor types I, II, and III in nonpregnant,
pregnant nonlaboring, and spontaneous laboring tissues, respectively.
Blots were also stained with an antibody to the G protein subunit,
Gß, as a loading control because the expression of this G protein
subunit remains constant in the human myometrium during pregnancy and
labor (26). As in Fig. 1
, the receptor proteins for TGFß receptor
types I and II were detected in all samples, but no protein
corresponding to TGFß receptor type III was found. Incubation of
nitro-cellulose blots with neutralized antibody (see Materials
and Methods) resulted in the absence of any immunoreactive
staining, thus indicating the specificity of the antibodies for TGFß
receptor subtypes (data not shown). The relative expression of
receptors in each tissue was assessed using a scanning densitometer.
The data for receptor type I and II expression in the myometrial
tissues after normalizing for loading (using the Gß antibody) are
shown in Fig. 2B. The levels of TGFß receptor type I and II
expression in nonpregnant samples were arbitrarily assigned a value of
100%. The amounts of receptors in pregnant nonlaboring tissues and
spontaneous laboring myometrium are expressed relative to this value.
The expression levels of TGFß receptor types I and II were
significantly increased by 168 ± 19% (n = 6) and 162
± 22% (n = 7) in pregnant nonlaboring tissues (P
< 0.05) compared to those in nonpregnant myometrium. The expressions
of both receptor types obtained from spontaneous laboring myometrium
were 93 ± 12% (n = 6) and 85 ± 11% (n = 7),
respectively. These values were significantly lower than those in
pregnant nonlaboring tissues (P < 0.01), which
suggests that there is less protein for both receptor types I and II in
spontaneously laboring myometrium.
|
illustrates measurements of the tissue
concentrations of the total TGFß1 (latent plus active) in
nonpregnant, pregnant nonlaboring, and spontaneous laboring myometrium
using ELISA. From Fig. 3, the amount of TGFß1 was increased from
334 ± 10 pg/g tissue wet wt in nonpregnant myometrium to 534
± 73 and 674 ± 106 pg/g tissue wet wt in pregnant nonlaboring
and spontaneous laboring myometrium (n = 3 in each group),
respectively. However, these values were just outside the range of
statistically significant difference between groups because of the
small sample size (P = 0.06).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this context, the numbers of oxytocin receptors and endothelin receptors are also increased in the myometrium at term (27, 28). Conversely, the number of angiotensin receptors is decreased as term approaches (29). Thus, it would appear that a common mechanism that could influence myometrial function would be to up- or down-regulate the expression of key surface membrane receptors. In this way, the myometrium can alter its function from a quiescent weakly contractile and poorly excitable tissue to one capable of powerful coordinated contractions.
Human myometrial cells in vitro have been shown to synthesize and release TGFß (30, 31). In turn, it has been shown that the amount of TGFß synthesized can be increased by GnRH, 17ß-estradiol, and medroxyprogesterone acetate (30). Taken together, these observations indicate that the TGFß signaling system is under hormonal regulation. This may also be occurring in late pregnancy. TGFß receptors are unlikely to have a role in the direct activation of contractions. It is more likely that they are associated with activation of myometrial cell growth and differentiation in preparation for parturition. Thus, it can be speculated that the myometrium is prepared for term as a result of a cascade of events involving the sequential expression of signals, receptors, and CAPs.
Growth factors and cytokines have been postulated as being potential regulators of many functions in the nonpregnant uterus and in early pregnancy. TGFß has been detected in amniotic fluid isolated from the late first trimester, where it may be involved in immune responses (32, 33), and has also been detected during the second trimester, when it may stimulate the secretion of other bioactive substances. It has been reported that TGFß receptors and TGFß are present in the nonpregnant human uterus and that the levels of expression of both change during the menstrual cycle (34). TGFß has also been reported in the murine uterus at implantation, where it may be involved in synchronizing embryonic development (35, 36). In the nonpregnant ovariectomized mouse uterus, the expression of TGFß can be transiently increased by the injection of estrogen (35). Roles for estrogen and progesterone have been suggested to account for the variations in TGFß receptors and TGFß isoforms in the human uterus during the menstrual cycle (34). In other cell types, the expression of TGFß and TGFß receptors has been reported to be influenced by several hormones (37, 38). For example, ACTH up-regulates TGFß receptors in bovine adrenocortical cells (37), and androgens affect receptor expression in rat ventral prostate (38).
A rise in the concentrations of other cytokines has been noted in amniotic fluid during normal and preterm deliveries. These include IL-6, IL-8, and granulocyte colony-stimulating factor (39). The source of the cytokines in late pregnancy is controversial, and the myometrium itself may be one site of production (4, 40). In addition, leukocytes invading the decidua and the decidual cells themselves have been implicated (39, 41, 42). Interestingly, the expression of IL-8 is enhanced by pretreatment with IL-1 and progestin (41), suggesting that a cascade of events may be acting to prepare the uterus for parturition.
As the levels of TGFß receptor types I and II were significantly reduced compared to those in pregnant nonlaboring tissues and the levels of total TGFß1 remained elevated at parturition, this may indicate the down-regulation of TGFß receptors after exposure to elevated levels of TGFß1. If this is the case, then, the decrease in TGFß receptor expression may occur due to increased levels of active TGFß1. However, this can only be surmised, as only total levels of TGFß1 (latent plus active) were determined in this investigation. A recent report indicates that the IL-8 receptor is down-regulated upon prolonged exposure to its ligand (43). There are also data showing that a number of other receptor types are down-regulated when exposed to their ligands, e.g. the angiotensin (44), somatostatin (45), and opioid receptors (46). A related phenomenon has been noted in the human myometrium with respect to the oxytocin receptor (47). In human myometrial cells, oxytocin receptors appear to be down-regulated upon prolonged exposure to oxytocin, with the effect that the myometrial cells become less sensitive to the hormone (48, 49). Thus, receptor down-regulation may be a common feature in the human myometrium to limit that action of specific hormones and agents.
Several investigations have shown that the activation of the TGFß signaling pathway involves binding of TGFß to the type II receptor, resulting in the formation of a complex between type I and type II receptors with the consequent phosphorylation of the type I receptor (reviewed in Refs. 17, 18). However, in some instances TGFß can activate cells that lack detectable levels of type II receptor (50) via activation of type I receptors alone (51). It has also been shown that a group of vertebrate homologs of Sma and Mad (mother against dpp), designated SMADs, in the cytoplasm is involved in the TGFß signaling pathway from the receptor to the nucleus (18, 52). In addition to SMADs, other proteins using the type I receptor, such as FK506-binding immunophilin FKBP12, may also modulate receptor signaling (17, 18). Members of the small GTP-binding protein family, Ras or Rac, have also been implicated in the signaling cascade (18). However, at the present time, it is not known how TGFß exerts its action on the human myometrium.
|
Footnotes |
|---|
Received December 3, 1997.
Revised January 29, 1998.
Revised April 3, 1998.
Accepted April 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
selectively stimulates prostaglandin
F2 production by macrophages in human term decidua. Am J Obstet Gynecol. 167:815–820.[Medline]
, and epidermal growth factor on prostaglandin production in
human chorion cells. Am J Reprod Immunol. 30:146–153.
s in pregnancy. Endocrinology. 132:2484–2490.s in human
myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab. 79:1835–1839.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  G. Helguera, M. Eghbali, D. Sforza, T. Y. Minosyan, L. Toro, and E. Stefani Changes in global gene expression in rat myometrium in transition from late pregnancy to parturition Physiol Genomics, January 8, 2009; 36(2): 89 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 M. S. Soloff, Y.-J. Jeng, M. Ilies, S. L. Soloff, M. G. Izban, T. G. Wood, N. I. Panova, G. V.N. Velagaleti, and G. D. Anderson Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector Mol. Hum. Reprod., September 1, 2004; 10(9): 685 - 695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bailey, C. Sparey, R. J. Phillips, K. Gilmore, S. C. Robson, W. Dunlop, and G.N. Europe-Finner Expression of the cyclic AMP-dependent transcription factors, CREB, CREM and ATF2, in the human myometrium during pregnancy and labour Mol. Hum. Reprod., July 1, 2000; 6(7): 648 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hatthachote and J. I. Gillespie Complex Interactions Between Sex Steroids and Cytokines in the Human Pregnant Myometrium: Evidence for an Autocrine Signaling System at Term Endocrinology, June 1, 1999; 140(6): 2533 - 2540. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Sparey, S. C. Robson, J. Bailey, F. Lyall, and G. Nicholas Europe-Finner The Differential Expression of Myometrial Connexin-43, Cyclooxygenase-1 and -2, and Gs{alpha} Proteins in the Upper and Lower Segments of the Human Uterus during Pregnancy and Labor J. Clin. Endocrinol. Metab., May 1, 1999; 84(5): 1705 - 1710. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
