Molecular Human Reproduction, Vol. 8, No. 7, 597-605,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
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It is well known that the smooth muscle of the human myometrium is a target for the steroid hormones progesterone (P4) and estrogen. Progesterone is believed to participate in the maintenance of pregnancy, while estrogen is possibly involved in the process of parturition by promoting cervical dilatation. We examined the combined effects of P4 and 17ß-estradiol (E2) on components of signalling pathways in human myometrial cells in vitro by immunoblotting. Long-term treatment of myometrial cells with a series of concentrations of P4 and E2 in combination caused a change in the phosphorylation status of p42/44 mitogen-activated protein kinase and of c-Jun N-terminal kinase (SAPK/JNK). P4 and E2 caused a decrease in protein expression of Gq
, Gz, Gi1/2 and, to a lesser extent, G0. The two steroids caused a decrease in the expression of the two small GS isoforms. Cyclo-oxygenase-2 expression was increased by 2.5-fold after steroid treatment, while proliferating cell nuclear antigen expression levels remained unchanged. These observations show that the combination of P4 and E2 influences intracellular and membrane-bound components of signal transduction pathways in human myometrial cells. The implications of the two steroid hormones on intracellular signalling pathways in the human myometrium merits further investigation. G-proteins/kinases/myometrium/steroids
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Introduction |
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The human uterine smooth muscle and the uterine vasculature are targets for estrogen and progesterone (P4), two known modulators of the myometrial contractile state (Maggi et al., 1992
; Vagnoni et al., 1998; Rupnow et al., 2001). P4 is considered to be necessary for pregnancy maintenance and its withdrawal in many species is a key event in parturition, whereas 17ß-estradiol (E2) augments the capacity for uterine contraction and cervical dilatation and therefore promotes parturition by causing myometrial contractions (Mesiano, 2001). Existing evidence on the effects of P4 and E2 on processes such as formation of gap junctions supports the concept that these steroids play a role in human parturition. While the involvement of estrogens and P4 in pregnancy is widely acknowledged, little is known about the steroid-mediated mechanisms that control the expression of key proteins regulating the excitability and contractility of the human myometrium. Previous work has shown that P4 increases intracellular calcium concentrations in myometrial cells in vitro (Fomin et al., 1999) and that estrogen promotes calcium influx into myometrial cells (Wehling, 1997).
Mitogen-activated protein (MAP) kinases are ubiquitous serine/threonine kinases that are activated by a wide variety of extracellular signals and are essential in triggering cell cycle progression (Seger and Krebs, 1995). It has been demonstrated that oxytocin acutely activates MAP kinase through an islet-activating protein-sensitive G-protein in human uterine myometrial cells, suggesting that MAP kinase may be involved in the modulation of human and rat myometrial contractility by oxytocin (Ohmichi et al., 1995, 1997; Nohara et al., 1996). Another well characterized subfamily of the MAP kinase superfamily are the stress-activated protein kinases (SAPK), also referred to as Jun N-terminal kinases (JNK), that function in a protein kinase cascade transducing cellular stress signals (Kyriakis and Avruch, 1990; Hibi et al., 1993; Derijard et al., 1994).
The presence of the rate-limiting enzyme in prostaglandin synthesis, prostaglandin endoperoxide-H synthase (cyclo-oxygenase; COX), in the non-pregnant uterus has been previously reported (Moonen et al., 1984). Two isoforms of COX exist, namely COX-1 and COX-2 (Hla et al., 1992). COX-2 is localized in human myometrial cells and it has been shown that human labour is associated with the up-regulation of prostaglandins within the uterus, synthesised via COX-2 (Moonen et al., 1985; Slater et al., 1999a,b; Sparey et al., 1999). COX-2 expression can be induced through multiple signalling pathways involving protein kinase A, protein kinase C, tyrosine kinases and lipopolysaccharides (Xie and Hershman, 1995). It is known that E2 and P4 regulate prostaglandin synthesis in bovine endometrium during the estrus cycle (Xiao et al., 1998), but the effects of these steroids on human myometrial COX-2 are unknown.
Similarly, very little is known about the effects of estrogen and P4 on G-proteins in the human myometrium. In rat myometrium, it has been demonstrated that Gi1/2 and Gq subunits are physiological targets for both steroids in vivo (Cohen-Tannoudji et al., 1995). It is known that estrogen and P4 modulate the expression of G proteins in lactotropes (Livingstone et al., 1998).
We have previously reported on the synergistic effects of P4 and E2 on nitric oxide synthase expression in isolated myometrial cells (Zervou et al., 1999a). These observations suggest that ovarian steroids may have a profound influence on the myometrial intracellular microenvironment with important physiological implications.
In our attempt to make progress in identifying further actions of P4 and E2 upon the human myometrium, we sought to investigate their combined action on cultured myometrial cells by determining their effects on the phosphorylation status of the family of p42/44 MAP kinases and SAPK/JNK kinases, on the expression of G-proteins, and on the expression of the enzyme COX-2. Unlike most other animal species including sheep, `P4 withdrawal' does not occur in human or primate pregnancies (Csapo and Pinto-Dantas, 1965; Mazor et al., 1993). P4 and E4 are present in high concentrations throughout human gestation, and no dramatic changes are seen towards the end of term. P4 production by the placenta reaches ~300 mg per day at the end of pregnancy (Perusquia, 2001), while E2 increases by almost 100-fold during pregnancy compared with non-pregnant levels (Mesiano, 2001). Therefore, we hypothesized that the two steroid hormones modulate the expression of key proteins in the myometrial smooth muscle cells, causing an alteration to their profile, with an impact on uterine physiology and activity.
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Materials and methods |
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Materials
Hanks' Balanced Salt Solution (HBSS) with and without Ca2+/Mg2+, Dulbecco's Modified Eagle's Medium (DMEM), penicillin G, streptomycin, L-glutamine, fatty acid-free bovine serum albumin, P4, calcium ionophore A23187 and E2 were provided by Sigma (Poole, UK). Fetal calf serum (FCS) and non-essential amino acids were products from Labtech (East Sussex, UK) and Gibco Brl (Paisley, UK) respectively. Anti-phosphorylated p42/44 and anti-total MAP kinase antibodies were raised in rabbits, with a synthetic peptide corresponding to residues 345–358 of rat p42 MAP kinase (New England Biolabs, Beverly, MA, USA). The antibody against human proliferating cell nuclear antigen (PCNA) was provided by Zymed (San Francisco, CA, USA).
Antisera against the Gz -subunit were purchased from Calbiochem (Nottingham, UK). Anti-G protein antibodies GC/2, AS/7, RM/1 and QL directed against the -subunits, were obtained from New England Nuclear-DuPont (Boston, MA, USA). All primary antibodies were raised in rabbits. AS/7, RM/1 and QL were polyclonal, whereas GC/2 and Gz antibodies were monoclonal. The anti-rabbit IgG antibodies were obtained from Sigma. All electrophoretic reagents were obtained from BioRad (Richmond, CA, USA). Anti-COX-2 polyclonal antibody was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Polyclonal antibodies against human SAPK/JNK were produced by immunizing rabbits with a full length p54 SAPK/JNK2 fusion protein. The antibody was raised against a recombinant protein corresponding to amino acids 50–111 mapping near the C-terminus of COX-2 of human origin, non-cross-reactive with COX-1.
Selection of cells and experimental subjects
All tissue samples were collected from women undergoing a gynaecological operation at Women's Hospital, University Hospitals of Coventry and Warwickshire, NHS Trust, Coventry, UK. The study was approved by the local ethics committee, and informed consent was obtained from each patient prior to operation. Myometrial biopsies were taken from the upper third of the uterine body ~5 mm away from endometrial or serosal surfaces, immediately after hysterectomy. All women recruited to the study were premenopausal and had not been exposed to steroid treatment for at least 3 months prior to the operation, and did not have either an intrauterine contraceptive device (IUD) in situ or evidence of uterine pathology, such as fibroids or polyps. Human adenocarcinoma breast cancer MCF-7 cells were obtained from the European Collection of Animal Cell Cultures (CAMR, Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK). Although no positive controls were available for SAPK/JNK, COX-2, G protein subunits or PCNA, MCF-7 cells were used as a positive control for MAP kinase experiments. This cell line was chosen because it expresses both estrogen and P4 receptors. MCF-7 cells have been used in the past to characterize steroid effects on p42/44 MAP kinase. No such controls are available for the rest of the proteins studied here.
Establishment of primary myometrial cell cultures and maintenance of MCF-7 cells
Myometrial biopsies weighing ~3 g were collected from women undergoing hysterectomy for menorrhagia. Primary myometrial cell cultures were established as previously described (Phaneuf et al., 1993). Cells were re-suspended in DMEM, containing 10% FCS, 0.2% L-glutamine, 10 000 IU/ml penicillin G and 7610 IU/ml streptomycin, supplemented with P4 and E2. A `no supplement' culture served as a negative control. Myometrial cells were plated into 25 cm2 culture flasks at a density of 0.5–2x104 cells/cm2 and stored at 37°C in a humidified atmosphere (95% air and 5% CO2) for up to 72 h. The purity of the myocyte cultures was assessed as previously described (Zervou et al., 1999a) using the ratio of the number of cells stained for -actin to the total cell nuclei present. Analysis of large numbers of cells indicated that >95% of the cultured cells were identified as smooth muscle cells. All primary cultures were maintained for up to 4 days, and incubated with charcoal-stripped FCS and phenol red-free media for 24 h prior to steroid treatments. In this way, endogenous and exogenous steroids were eliminated. MCF-7 cells were maintained in phenol red-free minimum essential medium (Sigma) with 1% non-essential amino acids, 10% charcoal-stripped fetal bovine serum, 10 000 IU/ml penicillin (Sigma) and 7610 IU/ml streptomycin (Sigma).
Protein extraction from cultured cells
Media were removed and cell monolayers were washed with phosphate-buffered saline (PBS) at room temperature. A buffer containing PBS, 1% NP40 (Sigma), 0.5% sodium deoxycholate (Gibco), 0.1% sodium dodecyl sulphate (SDS; Gibco), 10 µg/ml polymethyl-sulphonyl fluoride, 30 µl/ml aprotinin and 10 µl/ml sodium orthovanadate (100 mmol/l) was added to the cultures. The cells were scraped off the tissue culture surface with cell scrapers. The lysate was then centrifuged for 10 min at 13 000 g, at 4°C. The supernatant was removed and stored as the total cell lysate. The protein concentration was determined in all tissue extracts using the BioRad Reagent, according to the manufacturer's instructions.
Immunoblotting
Myometrial cell lysates (80 µg) were solubilized with Laemmli buffer (5 mol/l urea, 0.17 mol/l SDS, 0.4 mol/l dithiothreitol and 50 mmol/l Tris–HCl, pH 8.0), mixed and placed in a boiling water bath for 5 min and allowed to cool at room temperature. Samples were separated on an SDS–10% polyacrylamide gel and the proteins were electrophoretically transferred to a nitrocellulose filter at 250 mA for 16–18 h in a transfer buffer containing 20 mmol/l Tris, 150 mmol/l glycine and 20% methanol. The filter was then blocked in PBS containing 0.1% Tween-20 and 5% dried milk powder (w/v) for 2 h at room temperature. After three washes with PBS–0.1% Tween, the nitrocellulose filters were incubated with each primary antibody against MAP kinase and JNK total and phospho-forms, PCNA, COX-2 and G-protein -subunits as described previously (Karteris et al., 2000). All primary antisera were used at a 1:1000 dilution in PBS–0.1% Tween for 1 h at room temperature. The filters were washed thoroughly for 30 min with PBS–0.1% Tween before incubation with the secondary anti-rabbit HRP-conjugated Ig (1:2000) for 1 h at room temperature and further washing for 30 min with PBS–0.1% Tween. In order to detect the antibody complexes, solution A containing 100 mmol/l Tris pH 8.0 and 30% H2O2 was mixed with solution B containing 100 mmol/l Tris pH 8.0, 90 mmol/l coumaric acid and 250 mmol/l luminol, and applied to immunoblots for 2 min at room temperature. Immunoblots were visualized by exposure on X-ray film (Fuji Photo Film Co. Ltd, Tokyo, Japan), and were replicated at least twice on each tissue sample.
Statistical analysis
All experiments were performed in triplicate, independently, on myometrial biopsies from four individuals. The intensities of immunoreactive staining were measured using a scanning densitometer coupled to a scanning software package (Image Quant, Molecular Dymanics, Pharmacia Amersham Life Sciences, Little Chalfont, UK). Data are shown as the mean ± SEM of each measurement. In each case results were evaluated between groups by using the two-tailed Student's t-test. A P-value of <0.05 was considered significant.
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Results |
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Effect of steroids on p42144 MAP kinase in myometrial and MCF-7 cells
Primary myocyte cultures were treated for up to 24 h with P4 and E2 combined, at a series of concentrations, optimized at 5 µmol/l or with the calcium ionophore A23187 at 10 µmol/l for 15 min. SDS–polyacrylamide gel electrophoresis was carried out using myometrial cell protein extracts from treated and untreated myocyte cultures, followed by immunodetection with antibodies against total (Figure 1
) and phosphorylated p42/44 MAP kinase (Figure 2). The treatment decreased the phosphorylated form of p42 and p44 MAP kinase (both at P < 0.05), while total p42/44 MAP kinase remained unchanged. The ionophore A23187 caused an increase in p44 MAP kinase (P < 0.05).
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The effect of steroids on p42/44 MAP kinase in vitro was also tested in the human adenocarcinoma MCF-7 cells (Figure 1a,c
and Figure 2a,c), which endogenously express estrogen and P4 receptors and were used as a positive control. P4 and E2 in combination, for 15 min, each at 5 µ mol/l, caused a slight increase in the expression of phosphorylated p44 and a more marked increase in phosphorylated p42 MAP kinase protein levels (P < 0.01), while total p42/44 MAP kinase remained unchanged.
Effects of steroids on SAPK/JNK expression
In an attempt to investigate any changes in the expression of SAPK/JNK in cultured myocytes, primary myometrial cultures were treated for up to 24 h with P4 and E2 combined, at a series of concentrations, optimized at 5 µmol/l. Protein amounts for total SAPK/JNK were shown to be equal in both untreated and steroid-treated myometrial cell extracts, as demonstrated with immunoblotting using an antibody recognizing total SAPK/JNK. Two protein bands were detected, at 46 and 54 kDa, corresponding to p46 and p54 SAPK/JNK (Figure 3). An antibody recognizing the phosphorylated form of SAPK/JNK was also used. The phospho-SAPK/JNK (Thr183/Tyr185) antibody detects the phosphorylated isoforms of all SAPKs/JNKs. This antibody did not cross-react with endogenous levels of the corresponding phosphorylated forms of p42/44 MAP kinase or p38 MAP kinase. Combined steroidal treatment caused a decrease in phosphorylated p46 SAPK/JNK (P < 0.01; Figure 3b,d). The phosphorylated form of p54 was detected in neither the untreated, or the hormone-treated myometrial protein extracts with this antibody.
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Effect of steroids on COX-2
Immunoblotting experiments were performed, following treatment of myocyte cultures for up to 24 h with the combination of P4 and E2, at a series of concentrations, optimized at 5 µmol/l. A single protein band of 62 kDa was obtained in all samples, corresponding to the expected size band of human COX-2 (Figure 4
). This experiment was carried out using an anti-COX-2 antibody, which does not recognize COX-1. The COX-2 protein levels were substantially increased by 2.5-fold in the steroid-treated samples compared with the untreated control myocyte cultures (P < 0.01).
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Effect of steroids on PCNA
Cultured myometrial cells were treated with the combination of P4 and E2. Immunoblotting of protein samples showed that cultured myometrial cells contained immunoreactive PCNA, with the expected molecular mass of ~36 kDa. Treatment for up to 24 h with P4 and E2 combined, at a series of concentrations, optimized at 5 µmol/l did not cause a noticeable change in PCNA protein levels (Figure 5
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P4 and E2 effects on G-protein
-subunitsExposure of myometrial cell cultures for up to 24 h with the combination of P4 and E2, at a series of concentrations, optimized at 5 µmol/l, had profound effects on the expression of the
subunits of G-proteins (Figure 6).
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0.01.Detection of Gq
Probing with QL, a specific antibody for
q, 11 (C-terminus), we detected a single band at 42.5 kDa in myometrial cells. Estrogen and P4 treatment reduced the expression of Gq by 3-fold compared with the control (P < 0.01; Figure 6a,b).
Detection of Gz
A similar effect was found on the subunit of Gz. Probing with a specific antibody that does not cross-react with any of the other G-proteins, we were able to detect z as a 39 kDa protein, which was down-regulated by almost 3-fold following treatment with P4 and estrogen (P < 0.01; Figure 6c,d).
Detection of Gs
Immunoblotting experiments, using a specific antibody against the RM1 (C-terminus) region of s, detected four s species of apparent molecular weight 45, 47, 54 and 67 kDa in both treated and untreated myometrial cells. P4 and estrogen treatment had no apparent effect on the two large isoforms (54 and 67 kDa), whereas it reduced the expression of the small isoforms (47 and 44 kDa) by 2.5- (P = 0.01) and 3-fold (P < 0.01) respectively (Figure 6e,f).
Detection of Gi1/2
For this study we used an antibody (AS/7) which recognizes both i1 and i2. This antibody detected a band of 41 kDa in myometrial cells, indicating the presence of the G i1/2 subunit. P4 and estrogen treatment reduced its expression by 2-fold when compared with the control (P < 0.01; Figure 6g,h).
Detection of G0
Immunoblotting with a specific 0 antibody (GC/2) (N-terminus) detected a band of 40.5 kDa in the myometrial cell extract. P4 and estrogen treatment caused a slight reduction in G0 expression when compared with the control (Figure 6i,j).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the present study, we demonstrate that P4 and E2 influence the expression of important intracellular and membrane-bound signalling molecules in human myometrial cells in culture. During our experimental design, the authors agreed to use P4 and E2, in combination, to mimic the hormonal milieu during pregnancy, since the roles of the two steroids in human pregnancy are not fully explained. Due to limited evidence on the exact role of E2 in human pregnancy and on the extensive cross-talk between P4 and E2 signalling pathways in all reproductive tissues, the steroids were used in combination, in this preliminary survey of potential responses, in an attempt to mimic the steroid hormone milieu of pregnancy.
Estrogen is required for synthesis of P4 receptors (Aronica and Katzenellenbogen, 1991) and estrogen receptor expression is estrogen-dependent in reproductive tissues (Ing and Ott, 1999). Cross-talk between estrogen and P4 receptors has been reported, suggesting interactions between the estrogen and P4 intracellular pathways (Migliaccio et al., 1998; Katzenellenbogen, 2000).
In our study, P4 and E2 were used at relatively high concentrations to mimic the hormonal milieu of pregnancy. P4 production by the placenta reaches ~300 mg per day at term (Perusquia, 2001), while E2 increases almost by 100-fold during pregnancy (Mesiano, 2001). The concentrations of P4 and E2 were optimized at 5 µmol/l after a series of dose–response experiments (data not shown). Similar effects were exerted by P4 and E2 at concentrations up to 10 µmol/l, whereas higher amounts of the two steroids appeared to have toxic effects.
A number of time-point experiments were also performed as part of this study (data not shown). Myometrial cells were treated with P4 and E2 for up to 16 h. The effects of these steroids on protein kinases remained unchanged over the 16 h period, although some effects were evident before this time-point. A short-term incubation of up to 20 min could be part of a study on rapid, non-transcriptional events of steroids. Although myocytes were treated with steroids for up to 24 h, it was decided that steroid treatment for >16 h was not necessary for the purpose of our study. The change in the phosphorylation state of p42/44 MAP kinase in MCF-7 cells is rapid (Migliaccio et al., 1996), and therefore the cell line was treated with steroids for only 15 min. Similarly, 15 min of treatment with A23187 is the time required to initiate membrane-linked, Ca2+-associated signalling (Chen et al., 1999).
Here we demonstrate that P4 and E2 treatment of cultured myometrial cells causes a down-regulation in the phosphorylation status of p42/44 MAP kinase. This was in direct contrast to the calcium ionophore A23187, which caused an increase of the two phosphorylated protein kinases, demonstrating the activation of MAP kinase in the presence of increased Ca2+ concentrations. The effect of P4 and E2 is surprising in view of the fact that P4 increases intracellular Ca2+ concentrations in myometrial cells in vitro (Fomin et al., 1999) and that E2 promotes Ca2+ influx into myometrial cells (Wehling et al., 1997) as part of the non-transcriptional events initiated by the two steroid hormones in myometrial cells. This suggests that the effects of P4 and estrogen on MAP kinase phosphorylation status are not due to direct effects on Ca2+ mobilization. MAP kinase can also be activated in myometrial cells by G-protein-coupled receptor ligands such as oxytocin, endothelin and urocortin (Kimura et al., 1999; Molnar et al., 1999; Grammatopoulos et al., 2000).
We used MCF-7 cells only for MAP kinase experiments due to the fact that the cell line provides a very appropriate positive control, as demonstrated in a plethora of studies (Migliaccio et al., 1996; Castoria et al., 1999,Mougdil et al., 2001). The use of MCF-7 cells also shows the considerable variety of effects steroids could cause in different cell types. The same cell line could not be used as a positive control for the study of either SAPK/JNK (Caristi et al., 2001) or G protein -subunit expression. MCF-7 cells express COX-2 (Liu and Rose, 1996). However, these cells would not be an appropriate positive control for the study of steroid effects on COX-2 expression.
In previous studies, we have shown a steroid-mediated increase of COX-2 mRNA levels in human myometrial cells in culture (Zervou et al., 1999b). In this study we confirm that this increase in mRNA is translated into increased protein expression. Our findings support the pivotal role that COX-2 has in myometrial physiology, as already demonstrated by others (Slater et al., 1999a,b; Allport et al., 2001).
In our studies the combination of E2 and P4 did not cause a noticeable change in the proliferation events of isolated human myometrial cells, as revealed by PCNA immunodetection. Our findings complement the work by Matsuo et al. and Maruo et al. in terms of showing the combined effect of P4 and E2 on PCNA (Matsuo et al., 1999; Maruo et al., 2000).
Protein kinases such as SAPK/JNK and MAP kinase are believed to interact with a wide range of G protein subunits. Examples are the activation of SAPK/JNK by Gi in HEK293 cells (Yamauchi et al., 2000). MAP kinase is also activated by Gi (Jo et al., 1997). Gi and Gs are believed to interact with Src pathways, which are closely related to MAP kinase pathways (Ram and Lyengar, 2001). Some G protein-coupled receptors are able to activate JNK in certain cell types (Naor et al., 2000). The Gi-coupled m2 muscarinic acetylcholine receptor activates JNK in COS-7 cells (Coso et al., 1996). G13 has also been shown to stimulate the COX-2 promoter in NIH3T3 cells (Slice et al., 1999), but no data are available on the link between G subunits and COX-2 expression.
In the human myometrium, a steroid-mediated expression of cytoplasmic and membrane-linked components would cause a `shift' in G protein coupling to certain G protein-coupled receptors as part of tissue remodelling. A number of signal transduction pathways can be either activated or inactivated, leading to a change in smooth muscle contractile state.
This study demonstrates for the first time that ovarian steroids regulate expression of the subunits of G-proteins in primary human myocyte cultures. Treatment with P4 and E2 markedly decreased expression of Gi1/2, Gz, Gq and Gs and to a lesser extent G0. Interestingly, of the four isoforms of the Gs -subunit, only the smaller isoforms appeared to be down-regulated by P4 and E2. It is attractive to speculate that one of the mechanisms that might contribute to their down-regulation would be the effects of P4 and E2. It is known that the levels of Gs fall at the onset of parturition (Europe-Finner et al., 1994). Moreover the fact that short isoforms are affected provides further evidence for the importance of Gs alternative splicing. Europe-Finner and colleagues have shown that alternative splicing of Gs precursor mRNA has a primary role in regulating expression of Gs protein isoforms during pregnancy and labour (Europe-Finner et al., 1997).
It appears, therefore, that the -subunits of G-proteins in human myometrial cells are physiological targets for P4 and E2 in vitro. Our data are in agreement with previous findings on steroid-mediated expression of G-protein -subunits in other tissues. In lactotropes, combined treatment with P4 and E2 leads to decreased Gi/G0 amounts (Livingstone et al., 1998). Moreover, in-vivo administration of P4 in rat myometrium has been shown to significantly reduce the amounts of Gq (Cohen-Tannoudji et al., 1995).
Classically, steroids exert their effects transcriptionally through nuclear receptors. However, recent evidence shows that steroids can influence membrane physicochemical properties (Wehling, 1997). P4 treatment has been shown to influence calcium signalling evoked by ligand stimulation of G-protein coupled receptors expressed in several cell lines (Burger et al., 1999). Many signalling mechanisms initiated by peptide hormone receptors can also be activated by membrane actions of steroid hormones (Watson and Gametchu, 1999). Such membrane-initiated responses on G-protein activity with subsequent effects on a number of signal transduction pathways could eventually influence transcriptional events, critical for the physiological and biochemical responses of myocytes during pregnancy.
Collectively, our findings indicate a number of novel P4- and E2-mediated effects on signal transduction pathways involving intracellular as well as membrane-bound components of human myometrial cells. These two steroid hormones could potentially modify the protein levels of these components and may be linked to myometrial tissue remodelling due to pregnancy.
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Acknowledgements |
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The authors would like to thank consultant gynaecologists and theatre staff at the University Hospitals of Coventry and Warwickshire, NHS Trust, West Midlands. This work was funded by the Sir Jules Thorn Charitable Trust (96/02A) and by the Wellcome Trust. E.W.H. is the WPH Charitable Trust Chair of Medicine.
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Notes |
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1 To whom correspondence should be addressed. E-mail: szervou{at}cell.bio.warwick.ac.uk
* These authors have contributed equally to this work
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References |
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Allport, V.C., Pieber, D., Slater, D.M., Newton, R., White, J.O. and Bennett, P.R. (2001) Human labour is associated with nuclear-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the `functional progesterone withdrawal'. Mol. Hum. Reprod., 7, 581–586.
Aronica, S.M. and Katzenellenbogen, B.S. (1991) Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology, 128, 2045–2052.
Burger, K., Fahrenholz, F. and Gimpl, G. (1999) Non-genomic effects of progesterone on the signaling function of G protein-coupled receptors. FEBS Lett., 464, 25–29.[Web of Science][Medline]
Caristi, S., Galera, J.L., Matarese, F., Imai, M., Caporali, S., Cancemi, M., Altucci, L., Cicatiello, L, Teti, D., Bresciani, F. et al. (2001) Estrogens do not modify MAP kinase-dependent nuclear signaling during stimulation of early G(1) progression in human breast cancer cells. Cancer Res., 61, 6360–6366.
Castoria, G., Barone, M.V., Di Domenico, M., Bilancio, A., Ametrano, D., Migliaccio, A. and Auricchio, F. (1999) Non-transcriptional action of estradiol and progestin triggers DNA synthesis. EMBO J., 18, 2500–2510.[Web of Science][Medline]
Chen, Z., Yuhanna, I.S., Galcheva-Gargova, Z., Karas, R.H., Mendelsohn, M.E. and Shaul, P.W. (1999) Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J. Clin. Invest., 103, 401–406.[Web of Science][Medline]
Cohen-Tannoudji, J., Mhaouty, S., Elwardy-Merezak, J., Lecrivain, J.L., Robin, M.T., Legrand, C. and Maltier, J.P. (1995) Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy. Effects of progesterone and estradiol. Biol. Reprod., 53, 55–64.[Abstract]
Coso, O.A., Teramoto, H., Simonds, W.F. and Gutkind, J.S. (1996) Signaling from G protein-coupled receptors to c-Jun kinase involves beta gamma subunits of heterotrimeric G proteins acting on a Ras and Rac1-dependent pathway. J. Biol. Chem., 271, 3963–3966.
Csapo, A.M. and Pinto-Dantas, C.A. (1965) The effect of progesterone on the human uterus. Proc. Natl Acad. Sci. USA, 54, 1069–1076.
Derijard, B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T., Karin, M. and Davis, R.J. (1994) JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell, 76, 1025–1037.[Web of Science][Medline]
Europe-Finner, G.N., Phaneuf, S., Watson, S.P. and Lopez Bernal, A. (1993) Identification and expression of G proteins in human myometrium: up-regulation of G alpha s in pregnancy. Endocrinology, 132, 2484–2490.
Europe-Finner, G.N., Phaneuf, S., Tolkovsky, A.M., Watson, S.P. and Lopez Bernal, A. (1994) Down-regulation of G alpha s in human myometrium in term and preterm labour: a mechanism for parturition. J. Clin. Endocrinol. Metab., 79, 1835–1839.[Abstract]
Europe-Finner, G.N., Phaneuf, S., Cartwright, E. Mardon, H.J. and Lopez Bernal, A. (1997) Expression of human myometrial G alpha s messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways. J. Mol. Endocrinol., 18, 15–25.
Fomin, V.P., Cox, B.E. and Word, R.A. (1999) Effect of progesterone on intracellular calcium homeostasis in human myometrial cells. Am. J. Physiol., 276, C379–C385.[Medline]
Grammatopoulos, D.K., Randeva, H.S., Levine, M.A., Katsanou, E.S. and Hillhouse, E.W. (2000) Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activates protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1 and R2ß CRH receptor subtypes and stimulation of Gq-proteins. Mol. Endocrinol., 14, 2076–2091.
Hibi, M., Lin, A., Smeal, T., Minden, A. and Karin, M. (1993) Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev., 7, 2135–2148.
Hla, T. and Neilson, K. (1992) Human cyclooxygenase-2 cDNA. Proc. Natl Acad. Sci. USA, 89, 7384–7388.
Ing, N.H. and Ott, T.L. (1999) Estradiol up-regulates estrogen receptor-alpha messenger ribonucleic acid in sheep endometrium by increasing its stability. Biol. Reprod., 60, 134–139.
Jo, H., Sipos, K., Go, Y.M., Law, R., Rong, J. and McDonald, J.M. (1997) Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and Gbeta/gamma-dependent signaling pathways. J. Biol. Chem., 272, 1395–1401.
Karteris, E., Grammatopoulos, D., Randeva, H. and Hillhouse, E.W. (2000) Signal transduction characteristics of the corticotropin-releasing hormone receptors in the feto-placental unit. J. Clin. Endocrinol. Metab., 85, 1989–1996.
Katzenellenbogen, B.S. (2000) Mechanisms of action and cross-talk between estrogen receptor and progesterone receptor pathways. J. Soc. Gynecol. Investig., 7, S33–S37.[Web of Science][Medline]
Kimura, A., Ohmichi, M., Takeda, T., Kurachi, H., Ikegami, H., Koike, K., Masuhara, K., Hayakawa, J., Kanzaki, T., Kobayashi, M. et al. (1999) Mitogen-activated protein kinase cascade is involved in endothelin-1-induced rat puerperal uterine contraction. Endocrinology, 140, 722–731.
Kyriakis, J.M. and Avruch, J. (1990) pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine. J. Biol. Chem., 265, 17355–17363.
Liu, X.H. and Rose, D.P. (1996) Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res., 56, 5125–5127.
Livingstone, J.D., Lerant, A. and Freeman, M.E. (1998) Ovarian steroids modulate responsiveness to dopamine and expression of G-proteins in lactotropes. Neuroendocrinology, 68, 172–179.[Web of Science][Medline]
Maggi, M., Magini, A., Fiscella, A., Giannini, S., Fantoni, G., Toffoletti, F., Massi, G. and Serio, M. (1992) Sex steroid modulation of neurophysial hormone receptors in human nonpregnant myometrium. J. Clin. Endocrinol. Metab., 74, 385–392.[Abstract]
Maruo, T., Matsuo, H., Samoto, T., Shimomura, Y., Kurachi, O., Gao, Z., Wang, Y., Spitz, I.M. and Johansson, E. (2000) Effects of progesterone on uterine leiomyoma growth and apoptosis. Steroids, 65, 585–592.[Web of Science][Medline]
Matsuo, H., Kurachi, O., Shimomura, Y., Samoto, T. and Maruo, T. (1999) Molecular bases for the actions of ovarian sex steroids in the regulation of proliferation and apoptosis of human uterine leiomyoma. Oncology, 57 (Suppl. 2), 49–58.[Medline]
Mazor, M., Wiznitzer, A., Levy, J., Sharoni, Y., Meril, Z., Minster, A. and Glezerman, M. (1993) The relationship between estrogen/progesterone ratio and term human parturition. Isr. J. Med. Sci., 29, 97–99.[Web of Science][Medline]
Mesiano, S. (2001) Roles of estrogen and progesterone in human parturition. In Smith, R. (Ed.) The Endocrinology of Parturition. Basic Science and Clinical Application. Front. Horm. Res., Basel, Karger.
Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E. and Auricchio, F. (1996) Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J., 15, 1292–1300.[Web of Science][Medline]
Migliaccio, A., Piccolo, D., Castoria, G., Di Domenico, M., Bilancio, A., Lombardi, M., Gong, W., Beato, M. and Auricchio, F. (1998) Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J., 17, 2008–2018.[Web of Science][Medline]
Molnar, M., Rigo, J. Jr, Romero, R. and Hertelendy, F. (1999) Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells. Am. J. Obstet. Gynecol., 181, 42–49.[Web of Science][Medline]
Moonen, P., Klok, G. and Keirse, M.J. (1984) Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy. Prostaglandins, 28, 309–321.[Web of Science][Medline]
Moonen, P., Klok, G. and Keirse, M.J. (1985) Immunohistochemical localisation of prostaglandin endoperoxide synthase and prostacyclin synthase in pregnant human myometrium. Eur. J. Obstet. Gynecol. Reprod. Biol., 19, 151–158.[Web of Science][Medline]
Moudgil, V.K., Dinda, S., Khattree, N., Jhanwar, S., Alban, P. and Hurd, C. (2001) Hormonal regulation of tumor suppressor proteins in breast cancer cells. J. Steroid Biochem. Mol. Biol., 76, 105–117.[Web of Science][Medline]
Naor, Z., Benard, O. and Seger, R. (2000) Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor. Trends Endocrinol. Metab., 11, 91–99.[Web of Science][Medline]
Nohara, A., Ohmichi, M., Koike, K., Masumoto, N., Kobayashi, M., Akahane, M., Ikegami, H., Hirota, K., Miyake, A. and Murata, Y. (1996) The role of mitogen-activated protein kinase in oxytocin-induced contraction of uterine smooth muscle in pregnant rat. Biochem. Biophys Res. Commun., 229, 938–944.[Web of Science][Medline]
Ohmichi, M., Koike, K., Nohara, A., Kanda, Y., Sakamoto, Y., Zhang, Z.X., Hirota, K. and Miyake, A. (1995) Oxytocin stimulates mitogen-activated protein kinase activity in cultured human puerperal uterine myometrial cells. Endocrinology, 136, 2082–2087.[Abstract]
Ohmichi, M., Koike, K., Kimura, A. Masuhara, K., Ikegami, H., Ikebuchi, Y., Kanzaki, T., Touhara, K., Sakaue, M., Kobayashi, Y. et al. (1997) Role of mitogen-activated protein kinase pathway in prostaglandin F2alpha-induced rat puerperal uterine contraction. Endocrinology, 138, 3103–3111.
Perusquia, M. (2001) Nongenomic action of steroids in myometrial contractility. Endocrine, 15, 63–72.[Web of Science][Medline]
Phaneuf, S., Europe-Finner, G.N., Varney, M., MacKenzie, I.Z., Watson, S.P. and Lopez Bernal, A. (1993) Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G proteins. J. Endocrinol., 136, 497–509.
Ram, P.T. and Iyengar, R. (2001) G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation. Oncogene, 20, 1601–1606.[Web of Science][Medline]
Rupnow, H.L., Phernetton, T.M., Shaw, C.E., Modrick, M.L., Bird, I.M. and Magness, R.R. (2001) Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS. Am. J. Physiol. Heart Circ. Physiol., 280, H1699–H1705.
Seger, R. and Krebs E.G. (1995) The MAPK signaling cascade. FASEB J., 9, 726–735.[Abstract]
Slater, D.M., Dennes, W., Sawdy, R., Allport, V. and Bennett, P. (1999a) Expression of cyclo-oxygenase types-1 and –2 in human fetal membranes throughout pregnancy. J. Mol. Endocrinol., 22, 125–130.[Abstract]
Slater, D.M., Dennes, W.J., Campa, J.S., Poston, L. and Bennett, P.R. (1999b) Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy. Mol. Hum. Reprod., 5, 880–884.
Slice, L.W., Walsh, J.H. and Rozengurt, E. (1999) Galpha(13) stimulates Rho-dependent activation of the cyclooxygenase-2 promoter. J. Biol. Chem., 274, 27562–27566.
Sparey C., Robson, S.C., Bailey, J., Lyall, F. and Europe-Finner, G.N. (1999) 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 labour. J. Clin. Endocrinol. Metab., 84, 1705–1710.
Vagnoni, K.E., Shaw, C.E., Phernetton, T.M., Meglin, B.M., Bird, I.M. and Magness, R.R. (1998) Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase. Am. J. Physiol., 275, H1845–H1856.[Medline]
Watson, C.S. and Gametchu, B. (1999) Membrane-initiated steroid actions and the proteins that mediate them. Proc. Soc. Exp. Biol. Med., 220, 9–19.[Medline]
Wehling, M. (1997) Specific, nongenomic actions of steroid hormones. Ann. Rev. Physiol., 59, 365–393.[Web of Science][Medline]
Xiao, C.W., Liu, J.M., Sirois, J. and Goff, A.K. (1998) Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-
in bovine endometrial cells. Endocrinology, 139, 2293–2299.
Xie, W. and Hershman, H.R. (1995) v-src induces prostaglandin synthase-2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. J. Biol. Chem., 270, 27622–27628.
Yamauchi, J., Kaziro, Y. and Itoh, H. (1995) Carboxyl terminal of G protein beta subunit is required for association with gamma subunit. Biochem. Biophys Res. Commun., 214, 694–700.[Web of Science][Medline]
Zervou, S., Klentzeris, L.D. and Old, R.W. (1999a) Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia. Mol. Hum. Reprod., 5, 1048–1054.
Zervou, S., Klentzeris, L.D. and Old, R.W. (1999b) Steroid effects of myometrial prostaglandin endoperoxideH-synthase-2 and oxytocin receptor. Abstract, 81st Annual Meeting of the Endocrine Society, The Endocrine Society, San Diego, USA. p192.
Submitted on June 25, 2001; resubmitted on December 28, 2001; accepted on April 17, 2002.
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