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Division of Clinical Science (E.K.), Warwick Medical School and Biomedical Research Institute (S.Z., H.S.R.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Marine Science Institute (Y.P., J.D., P.T.), University of Texas at Austin, Port Aransas, Texas 78373; and The Office of the Dean (E.W.H.), The Medical School, University of Leeds, Leeds LS2 9NL, United Kingdom
Address all correspondence and requests for reprints to: Professor Peter Thomas, The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373. E-mail: thomas{at}utmsi.utexas.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and mPRß, in human myometrium that are differentially modulated during labor and by steroids in vitro. The mPRs are coupled to inhibitory G proteins, resulting in a decline in cAMP levels and increased phosphorylation of myosin light chain, both of which facilitate myometrial contraction. Activation of mPRs leads to transactivation of PR-B, the first evidence for cross-talk between membrane and nuclear PRs. Progesterone activation of the mPRs leads also to a decrease of the steroid receptor coactivator 2. Our data indicate the presence of a novel signaling pathway mediated by mPRs that may result in a functional progestin withdrawal, shifting the balance from a quiescent state to one of contraction. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recently, a novel cDNA was discovered in spotted sea trout ovaries that has all the characteristics of an mPR (8) and is structurally unrelated to nuclear steroid receptors, but instead has features typical of G protein-coupled receptors. Subsequently, three closely related cDNAs (mPR, mPRß, and mPR
) were identified in human tissues that encode G Protein-coupled receptor-like proteins with several characteristics of membrane-bound progestin receptors (9).
This manuscript provides the first description of a functional role for mPRs in mammalian cells. Our results indicate the presence of functional mPR and mPRß on human myometrial cell membranes and provide evidence for a cross-talk between nuclear and membrane PRs in human myometrium that implicates them in events leading toward a progestin withdrawal at term.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and mPRß in Human Pregnant Myometrium and mPRß mRNA as revealed by RT-PCR (Fig. 1A
). Real-time PCR shows that the predominant isoform in myometrial cells is mPR (Fig. 1B). In addition, there is no apparent difference in the expression of both mPRs in myometrial cells compared with snap-frozen myometrial tissue (Fig. 1C). Immunofluorescent analysis revealed expression of mPR and mPRß proteins in tissue sections from the lower segment of laboring myometria where staining is detectable across the outer longitudinal muscle layer (Fig. 1D), and on myometrial cells as granular staining across the plasma membrane of the smooth muscle spindle (Fig. 1E). Preabsorption with blocking peptides for mPR and mPRß confirmed the specificity of the antibody reactions (Fig. 1D).
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and mPRß and mPRß polyclonal antibodies demonstrated the presence of both receptor proteins on plasma membranes of human pregnant myometrial cells with molecular masses of approximately 40 kDa. Interestingly, an additional 80-kDa band was also detected in myometrial plasma membranes for both mPRs (Fig. 2, A and B). An 80-kDa band can also be detected in plasma membranes of breast cancer cells (MDA-MB-231) stably transfected with human mPR (Fig. 2C), or with mPRß (Fig. 2E). This molecular weight is double that predicted from the deduced amino acid sequences of the mPRs and is likely indicative of the formation of sodium dodecyl sulfate (SDS)-resistant homodimers. In both preparations, neither of these bands was detected after preabsorption of the antibodies with their blocking peptides, nor in untransfected breast cancer cells, thus indicating the bands are specific for each of the mPRs (Fig. 2).
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and mPRß are differentially regulated by estrogen (E2) and progestin (P4) in myometrial cells, both at the mRNA and protein levels. P4 and E2 (100 nM) significantly induced mPR mRNA expression. P4 causes greater up-regulation after 4 h treatment, and together they exert an additive effect: significant induction occurs after 4 h and 16 h as assessed by real-time PCR (Fig. 3A). The mPRß gene, however, was regulated solely by E2, showing significant up-regulation after 8 h of treatment (Fig. 3B) and returning back to basal levels at 16 h (results not shown). Semiquantitative Western blot analysis showed that this mRNA up-regulation was associated with subsequent increases in mPR and mPRß protein levels after 18 h treatment (Fig. 3, C and D). This differential regulation by P4 suggests that the two receptors may have divergent functions.
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, panel III), whereas E2 and 11-deoxycortisone were ineffective (Fig. 4A, panels V and VII, respectively).
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and mPRß caused a decrease in mRNA (Fig. 4B
) and protein expression (Fig. 4C) for the two receptors, as shown by real-time RT-PCR and Western blot analysis, respectively. This decrease in mPR protein abundance was associated with greater than a 50% decrease in specific [3H]P4 binding to myocyte membranes compared with that measured after transfection with the control siRNA oligos (P < 0.05, Student’s t test; Fig. 4D). Competitive binding studies of [3H]P4 binding to myocyte membranes in the presence of single high concentrations (200 nM) of P4, R5020, or dexamethasone showed that binding was specific for P4. P4 (200 nM) displaced approximately 90% of the receptor-bound [3H]P4, whereas neither R5020 nor dexamethasone significantly displaced [3H]P4 from the membrane preparations at this concentration (Fig. 4E).
Coupling of mPRs to Pertussis Toxin (PTX)-Sensitive G Proteins in Human Myometria
Specific binding of [35S]GTPS to myometrial membranes was increased after treatment with 200 nM P4, but not with 200 nM R5020 or dexamethasone (Fig. 4F). A lower P4 concentration (100 nM) also significantly increased specific [35S]GTPS binding, whereas 100 nM cortisol was ineffective (Fig. 4G). These results suggest that the receptors are localized on the membrane and are capable of activating G proteins specifically in response to P4. Immunoprecipitation of the membrane-bound [35S]GTPS with specific -subunit G protein antibodies showed that these receptors activate the family of Gi -subunits (Fig. 4H).
Using the nonhydrolysable GTP analog, [32P]GTP-azidoanilide, we demonstrated for the first time that myometrial mPRs can activate multiple PTX-sensitive Gi proteins when challenged with P4 (100 nM; Fig. 5, B and C). The activation of the Gi proteins by P4 was concentration dependent over the range of 1–100 nM (Fig. 5A). Treatment of membranes prepared from snap-frozen human pregnant myometrial tissue with P4 caused activation of Gi1/2, Gi3, and Go, but not Gq/11 (Fig. 5B). Moreover, when membranes prepared from cultured myometrial cells were treated with P4 (100 nM), a very similar pattern of activation was observed (Fig. 5C).
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and mPRß are directly coupled to Gi proteins. Coimmunoprecipitation of solubilized myocyte membrane fractions first with a goat anti-Gi -subunit antibody, and subsequent Western blotting with the mPR and mPRß antibody, showed strong immunoreactive bands (Fig. 5D), whereas no bands were detected with control goat serum (results not shown). Hormonal activation and dissociation of a portion of the receptor/G protein complexes by pretreatment with 100 nM P4 resulted in decreased amounts of intact mPR/Gi/o complexes that could be immunoprecipitated and detected on the Western blots, as predicted (Fig. 5D). Coupling of the mPRs to a Gi/o protein was also observed using an alternative procedure in which, after treatment of the myocyte membranes with 100 nM P4, the solubilized mPR/Gi/o -subunit complex is immunoprecipitated with the mPR and mPRß antibodies and subsequently detected on the Western blots with antibodies to the Gi/o -subunit. (Fig. 5E). Thus, these two different experimental approaches provide conclusive evidence that both mPRs can couple to inhibitory (PTX sensitive) G protein -subunits. Free P4 was used in all the G protein activation and coupling experiments, because they were conducted with isolated myometrial cell membranes devoid of intracellular components. Experiments using P4-BSA (100 nM) showed similar activation of Gi/o in human myometrial biopsy tissue (supplemental Fig. 1A published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).
Inhibition of Adenylyl Cyclase by mPRs
Acute inhibition of adenylyl cyclase was observed after P4 treatment, confirming the activation of inhibitory G protein-dependent pathways. Treatment of myometrial cells with 100 nM P4-BSA for 10 min significantly reduced both basal and isoproterenol-induced cAMP levels (Fig. 5F). This decrease was concentration dependent over the concentration range of 10 nM to 100 nM (supplemental Fig. 1B). The inhibitory effect of P4-BSA on isoproterenol stimulation of adenylyl cyclase activity was completely reversed upon preincubation with PTX (200 ng/ml; Fig. 5F). This inhibition of adenylyl cyclase activity by P4 is consistent with the notion that these receptors couple to the Gi-protein family and are in agreement with previous studies (8, 10).
Similar results were obtained with membranes obtained from pregnant myometrial biopsies. Treatment of these myometrial membranes with P4-BSA for 15 min caused a similar reduction in both basal and isoproterenol-induced cAMP levels as observed in the cells, whereas pretreatment of the membranes with preactivated PTX (200 ng/ml) reversed the P4-BSA effects (supplemental Fig. 1C).
P4-Induced Phosphorylation of Myosin Light Chain (MLC) in Human Myometrial Cells
We hypothesized that activation of the mPRs might affect myometrial tone, because cyclic nucleotides are the major second messengers that modulate the contractile status of smooth muscle cells. Smooth muscle cell contraction and the rates of myosin-filament formation and actin-activated myosin ATPase activity are primarily regulated by phosphorylation at position Ser19 of the MLC by MLC kinase (11, 12). cAMP interferes with MLC phosphorylation through multiple mechanisms resulting in smooth muscle relaxation. Therefore, we assessed the effect of P4 on the phosphorylation of MLC, using myometrial cells obtained from the lower uterine segment at term.
P4-BSA induced phosphorylation of MLC at Ser19 in both a concentration- and time-dependent manner (supplemental Fig. 1D), with maximal effects at 100 nM and 15 min, respectively, whereas the total amount of MLC remained unchanged (Fig. 6A). The effect of P4-BSA on MLC phosphorylation in myometrial cells was abolished by pretreatment with PTX (200 ng/ml) (Fig. 6A), demonstrating the involvement of Gi subunits in mediating mPR actions. Interestingly, siRNA experiments showed that this effect is mediated exclusively via mPR and not mPRß, because P4-induced MLC phosphorylation was only inhibited in myometrial cells transfected with siRNA for mPR (Fig. 6, B and C).
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can promote ERK1/2 phosphorylation (8). However, treatment of human myometrial cells with P4 or P4-BSA did not increase phosphorylation of ERK1/2, even at concentrations as high as 1 µM (data not shown). However, p38 MAPK was activated by P4-BSA (Fig. 6D). Treatment of myocytes with P4-BSA for 15 min caused phosphorylation of p38 MAPK, which was inhibited by prior treatment with PTX (200 ng/ml), whereas the total amount of p38 MAPK was unchanged (Fig. 6D).
A potent and specific p38 MAPK inhibitor, SC-68376, with no cross-reactivity toward ERK or c-Jun N-terminal kinase activity, was used to assess the involvement of this pathway in the phosphorylation of MLC by P4. Treatment of myometrial cells with SC-68376 totally inhibited MLC phosphorylation induced by P4-BSA (Fig. 6E). Collectively, these data suggest that the P4-induced phosphorylation of MLC is modulated via a Gi/p38 pathway in human pregnant myometrial cells.
A membrane-initiated action of P4 in human myometrium was clearly demonstrated in these in vitro experiments, because P4 covalently linked to BSA cannot enter the cells, and its binding is restricted to cell surface PRs. Identical acute progestin effects on phosphorylation of p38 and MLC were observed after exposure to free P4. These significant responses occur within 15 min, too rapid for a genomic mechanism of action (supplemental Fig. 1, panels E and F).
Transactivation of Nuclear PRs by mPRs
Cross-talk between nuclear and membrane progestin receptors was demonstrated by assessing the binding of the nuclear PR to a glucocorticoid-responsive element (GRE), linked to a luciferase reporter vector. Treatment of pregnant myometrial cells with 100 nM P4 in the presence or absence of PTX (200 ng/ml) did not alter the GRE-luc activity, which is likely due to the repressor effects of the dominant-negative PR-A (13), because high amounts of PR-A transcripts are present in the myocyte culture system (Fig. 7; columns i and j compared with column b). However, when myometrial cells were transfected with PR-B, thus resembling myometrium in early pregnancy, shifting the PR ratio in favor of the functional nuclear receptor, P4 (100 nM) induced a 5-fold increase in GRE activity (Fig. 7; column f compared with column d). Interestingly, PTX treatment followed by the same P4 treatment resulted in decreased activation above basal (column g compared with column f), whereas PTX treatment alone (column h) had no effect. The PTX effects are specific to P4, because PTX pretreatment does not inhibit the effect of dexamethasone that was used as a positive control (compare column k vs. column e), or the effect of R5020 (compare column m vs. column l), a progestin agonist that does not interact with mPRs. Interestingly, there was no apparent effect of P4-BSA on GRE-luc activity in the presence or absence of PTX (compare columns n and o with column d). Experiments using siRNA revealed that when either mPR was silenced, there was a significant down-regulation of GRE activity induced by P4 (columns p and q). Collectively, our data suggest that activation of mPR signaling via Gi proteins serves to potentiate hormone-activated nuclear PR-B (Fig. 7).
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). The down-regulation of SRC2 by P4-BSA was transient and mRNA expression had returned to pretreatment levels by 48 h (Fig. 8B). Using a specific antibody for SRC2, we reveal that treatment of myometrial cells with P4-BSA for 16 h resulted in a decrease in SRC2 protein levels, in agreement with our real-time RT-PCR data (Fig. 8A, inset). However, this effect was transient, with no apparent decrease at 3 h, and SRC2 had returned to basal levels by 24 h (result not shown).
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but not mPRß, compared with that observed in cells transfected with the siRNA control or untransfected cells (Fig. 8C). Collectively, these data suggest that changes observed in the SRC2 gene in myometrial cells after P4 treatment are mediated via mPR and a dedicated Gi pathway.
Future studies should determine the exact mechanisms of the involvement of SRC2 in the transactivation of PR-B. However, recent evidence suggests a putative role for SRC2 in reproductive tissues. In human uterine smooth muscle cells, TNF- diminished P4-stimulated PR-B transactivation by reducing expression of SCR1 and SRC2 (14). Moreover, acute disruption of SRCs (including SRC2), prevented steroid-dependent reproductive behavior in a rodent and mouse model (15).
SRC2 mRNA levels were also significantly decreased (2-fold) in laboring myometria, when compared with nonlaboring ones, as assessed by real-time RT-PCR (supplemental Fig. 1G), in agreement with previous studies where only a semiquantitative approach was used (4). In addition, in laboring myometria the mean relative abundance of mRNA encoding for mPRß was significantly increased compared with levels in nonlaboring tissue, whereas mPR mRNA was not different between the two groups (supplemental Fig. 1G). These changes were also reflected at the protein level (supplemental Fig. 1H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The present results clearly show that the newly described mammalian mPRs, mPR and mPRß, are expressed in human pregnant myometria and in pregnant myometrial cells in vitro, both at the mRNA and protein levels. Moreover, we show that these receptors couple exclusively to PTX-sensitive G protein -subunits in these cells to inhibit adenylyl cyclase, and increase phosphorylation of MLC via a pathway involving activation of p38 MAPK. Previous studies indicate an involvement of the p38 MAPK cascade in IL-1B-stimulated prostaglandin synthesis in myometrium (17), suggesting that this pathway may contribute to increased contractile frequency and strength during labor.
We also obtained the first evidence for cross-talk between nuclear PRs and these novel mPRs, with mPR activation leading to regulation of nuclear PR activity. The partial inhibition of P4-dependent induction of a GRE-luc reporter by PTX suggests that Gi signaling is involved in the regulation of PR transactivation activity. Experiments using siRNA strongly suggest that both mPR and mPRß are involved in this process. With regard to the residual activity after P4 and PTX treatment, it is probably due to activation of two distinct mechanisms by P4 treatment: the mPRs (mPR and mPRß) and the nuclear PR-B. PTX treatment blocks only the membrane-initiated response, but not the nuclear one, as it is evident with the use of the specific nuclear PR agonist R5020, or dexamethasone.
These findings have prompted us to propose a model for the actions of progesterone in human myometrium before and during the onset of labor. Early in pregnancy, PR-B predominates in the human myometrium and, as a result, the mPR acts synergistically. It transactivates the PR-B via a mechanism involving activation of Gi subunits, and this results in an amplification of the PR effect (i.e. relaxation, etc.). During labor, there is a shift of the PR balance, with PR-A acting as a repressor. At the same time, circulating levels of sex steroids up-regulate both mPR and mPRß. Subsequent activation of the mPRs at the end of pregnancy, in turn, down-regulates SRC2, which in combination with an altered nuclear PR-B/A ratio, leads to a decrease in the transcription activity of the nuclear PR-B in the human myometrium. As a result, the mPRs cannot transactivate the PR-B any longer. This cascade of events then allows P4 to act preferentially on the mPRs during labor, to evoke responses such as inhibition of adenylyl cyclase and phosphorylation of MLC. We propose that these events, mediated by the mPRs, can promote a shift from a quiescent phase and sensitize the human myometrium toward a contractile state at term (Fig. 9A). These effects can take place within the same cell, because there is colocalization of nuclear with membrane PRs (Fig. 9B).
Our findings, potentially, have several very important human health implications, because they provide detailed information on a previously unrecognized progesterone-signaling cascade in human myometrial smooth muscle cells. A more comprehensive understanding of progesterone actions during pregnancy will provide a theoretical basis for the development of new hypotheses and treatments. Indeed, a few studies have suggested unexpected stimulatory effects of P4 on human myometrial contractile activity in vitro, when the myometrium is not deprived of P4 (18, 19, 20).
In addition, this research may lead to the development of other potential medical benefits such as hormonal regulation of the mPRs in myometria of women who have abnormal receptor expression levels, diagnosis of women at risk for preterm birth, and detection of genetic defects in mPRs, ultimately resulting in a reduction in the number of preterm births. Due to ethical restrictions, we used biopsy material obtained from the lower uterine segment, an area with relatively minor involvement in uterine contractility during labor. Future studies should determine where there are any differences in the expression and signaling characteristics of these receptors between the lower uterine segment and the uterine fundus.
Finally our data provide the first clear evidence for reciprocal regulation of classical steroid actions and alternative cell surface-initiated ones mediated by two structurally distinct classes of steroid receptors. The intimate and complex interactions between the two steroid mechanisms in myometrial cells suggest that those mediated by mPRs are obligatory for the overall cellular response to P4. Perhaps cell surface-initiated steroid actions are not alternative ones after all.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture, Transient Transfection, and Reporter Gene Analysis
Primary cultures of myometrial cells were used at passages 2–4, and maintained as described previously (21). Cells were plated in six-well plates (36 mm) 24 h before transient transfection, at approximately 70% confluency. Transient transfection was performed with pGRE (Mercury Pathway Profiling System; BD Biosciences, Palo Alto, CA) in the presence or absence of human PRB (kind gift of Professor P. Chambon, Institut National de la Santé et de la Recherche Médicale, France). Myometrial cells were transfected using the calcium phosphate method (Calphos Transfection Kit; BD Biosciences), according to the manufacturer’s manual.
All cultures were cotransfected with 0.5 µg of the pRL-TK vector (Renilla luciferase) (Promega, Southampton, UK), to correct for transfection efficiency, as described previously (22). Media were replaced, after 5 h, with fresh culture medium containing various stimuli. Where suitable, cultures were pretreated with PTX (Calbiochem, San Diego, CA) for 16 h before addition of stimuli. The cells were grown until 90% confluent. Cell extracts were assayed using a dual-luciferase reporter assay system according to manufacturer’s instructions (Promega). Luciferase activity was measured for 10 sec using a Luminoscan RS Luminometer (Labsystems, Helsinki, Finland).
siRNA Transfection of Myocytes
The myocytes were cultured as described above until they were 60% confluent. The culture medium was replaced with serum-free medium 30 min before transfection. The transfection mixture was produced by incubating 1 ml of Opti-mem solution supplemented with 3% lipofectamine 2000 (Invitrogen, Paisley, UK) at room temperature for 5 min, 50 µl of siRNA oligo solution (20 µM, Invitrogen) was added, and the mixture was incubated for an additional 30 min. The transfection mixture was added to 10 ml cell culture medium to make a final concentration of 100 nM siRNA, and the cells were transfected for 24 h. The cells were retransfected with the same amount of siRNA after the first transfection for another 24 h. Samples were taken for RT-PCR analysis after the second transfection and the cells were incubated for an additional 12–24 h before they were harvested for measurement of P4 binding activity. The sequences of the oligonucleotides used in this study are given in Table 1.
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(GenBank accession no. AF313620), mPRß (GenBank accession no. AF313619). For mPR and mPRß, 33 cycles were performed consisting of an initial denaturing step at 94 C for 30 sec, followed by extension at 60 C for 1 min, and elongation at 72 C for 1 min. The PCR would be expected to yield a product, at 200 bp for mPR and at 289 bp for mPRß.
Real-Time RT-PCR
Quantitative PCR was performed on a Roche Light Cycler system (Roche Molecular Biochemicals, Manheim, Germany). PCRs were carried out in a reaction mixture consisting of 5.0 µl reaction buffer and 2.0 mM MgCl2 (Biogene, Kimbolton, UK), 1.0 µl of each primer (1 ng/µl), 2.5 ml of cDNA, and 0.5 µl of Light Cycler DNA Master SYBR Green I (Roche).
Protocol conditions consisted of denaturation at 95 C for 15 sec, followed by 40 cycles of 94 C for 1 sec, 58 C for 5 sec, and 72 C for 12 sec, followed by melting curve analysis. For analysis, quantitative amounts of genes of interest were standardized against the housekeeping gene ß-actin (Table 1). As a negative control for all the reactions, preparations lacking RNA or reverse transcriptase were used in place of the cDNA. RNAs were assayed from two to three independent biological replicates. The RNA levels were expressed as a ratio, using Delta-delta method for comparing relative expression results between treatments in real-time PCR (23).
Cloning and Sequencing of PCR Products
To confirm the specificity of the RT-PCR products, they were electrophoresed, purified by using the QIAquick Gel Extraction Kit (QIAGEN, Crawley, UK), and ligated into the EcoRI restriction site of the pGEM-T Easy vector (Promega, Southampton, UK) using T4 DNA ligase. The vector was transformed into the DH5 strain of Escherichia coli, and bacterial plasmid preparations were obtained by the QIAprep Spin Plasmid Kit (QIAGEN). The identity of each of the PCR products and hence the specificity of the PCR were confirmed by DNA sequencing.
Immunofluorescent Analysis
Fixed myometrial cells were washed in PBS and incubated with 10% BSA for 1 h before incubation with antibodies raised against either mPR or mPRß at a 1:100 dilution. All dilutions were made in 1% BSA in PBS. Cells were incubated with primary antibody for 1 h, and were then washed with PBS, before incubation with antirabbit IgG-FITC-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 45 min. The slides were then thoroughly rinsed with PBS, and the cell nuclei were visualized by applying the DNA-specific dye 4,6-diamido-2-phenylindole at a final concentration of 1 µg/ml.
For the colocalization study, the same mPR antibodies were used in conjunction with an antimouse PR-B (Santa Cruz Biotechnology). The secondary antibodies used for detection were an antirabbit IgG-tetramethylrhodamine isothiocyanate-conjugated antibody for mPR and mPRß, and an antimouse IgG-FITC-conjugated antibody (Santa Cruz Biotechnology).
Localization of P4-Binding Sites Using P4-BSA-FITC
Human pregnant myometrial cells were exposed to P4-BSA-FITC (Sigma Chemical Co., St Louis, MO) for 30 min in serum-depleted media, in chamber slides, in the presence or absence of 10 µg unlabeled P4 (Sigma), 17ß-estradiol (Sigma), and deoxycortisone (Sigma). The cell monolayers were then washed with PBS and incubated for an additional 15 min. The cells were observed under a fluorescent microscope using oil immersion x100 objective with excitation at 488 nm.
P4 Membrane Receptor-Binding Assay
P4 binding to plasma membrane preparations of myocytes was assayed by a single-point procedure (24). One set of tubes (total binding) contained 2.0 nM [2,4,6,7-3H]P4 (Amersham Pharmacia Biotech, Piscataway, NJ; specific activity, 102.1 Ci/mmol), and another set also contained 100-fold excess (200 nM) nonradiolabeled P4 (nonspecific binding). After a 30-min incubation at 4 C with the membrane fractions, the reaction was stopped by filtration [Whatman (Clifton, NJ) GF/B filters, presoaked in assay buffer], the filters were washed, and bound radioactivity was measured by scintillation counting. Each assay point was run in triplicate, and the assays were repeated utilizing different batches of myocytes.
Protein Extraction from Cultured Cells
Myometrial cells were cultured in six-well dishes until 80% confluency and then maintained overnight in serum-free media, before addition of agonists/antagonists 24 h later. Cells were then lysed by the addition of 300 ml SDS-PAGE sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM dithiothreitol. The solubilized material was then removed from the dishes, sonicated for 20 sec, heated for 5 min at 100 C, and cooled on ice. The protein concentration was determined in all tissue extracts using the Bio-Rad Reagent (Bio-Rad Laboratories, Inc., Hercules, CA), according to the manufacturer’s instructions.
Immunoblotting Analysis
Samples were separated on an SDS-12.5% polyacrylamide gel, and the proteins were electrophoretically transferred onto a nitrocellulose filter at 250 mA for 16–18 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. The filter was then blocked in PBS containing 0.1% Tween 20 and 5% dried milk powder (wt/vol), for 2 h at room temperature. After three washes with PBS-0.1% Tween, the nitrocellulose filters were incubated with primary antibody for the phospho- and total ERK1/2, p38 (Cell Signaling Technology, Beverly, MA); MLC, (Santa Cruz Biotechnology); SRC2 (Calbiochem) and G proteins (Calbiochem). The primary antisera were used at a 1:1000 dilution in PBS-0.1% Tween overnight at 4 C. The filters were washed thoroughly for 30 min with PBS-0.1% Tween 20, before incubation with the horseradish peroxidase-conjugated immunoglobulins (1:2000) for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween 20. Antibody complexes were visualized as previously described (21).
Immunoblotting for mPRs
Membrane proteins (20 µg) in 20 µl buffer were loaded onto each lane of a 12% SDS gels, subjected to PAGE, and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% nonfat milk in a TBST (50 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.4) buffer for 1 h, incubated overnight with the human (h) mPR polyclonal antibodies at a dilution of 1:3000, followed by three 5-min washes with TBST buffer, and a secondary incubation for 1 h at room temperature with horseradish peroxidase conjugated to goat antirabbit antibody (1:5000, Cell Signaling Technology), and final washing three times for 15 min with TBST buffer. Blots were then treated with enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to x-ray film.
Specific polyclonal antibodies were generated commercially (Sigma and Genosys, Woodlands, TX) in rabbits against synthetic 15-amino acid peptides derived from the N-terminal domains of hmPR (TVDRAEVPPLFWKPC) and hmPRß (KILEDGLPK MPCTVC) and conjugated to keyhole limpet hemocyanin. Animals were bled after seven to eight intradermal injections in Freud’s complete adjuvant. The specificity of the antibody reaction was confirmed by blocking 10 µl of antiserum, diluted 1:45 in PBS, with the peptide (final concentration of 2 µg/ml), and incubating the mixture at room temperature for 1.5 h. The mixture was subsequently diluted 1:66 in TBST for Western blotting. Protein levels on the Western blots were estimated by densitometry (Image J, version 1.13, NIH).
Photoaffinity Labeling of -Subunits
Human myometrial membranes (150–200 µg) were incubated for 3 min at 30 C with P4 (100 nM) in buffer C (50 mM HEPES, 30 mM KCl, 10 mM MgCl2, 1 mM Benzamidine, 0.1 mM EDTA), followed by the addition of 5 µM GDP and 3 µCi of GTP-AA. After incubation for 3 min at 30 C in a darkened room, membranes were placed on ice and collected by centrifugation at 13,000 rpm for 15 min at 4 C. The supernatant was carefully removed, and the membrane pellet was resuspended in 120 µl of buffer C containing 1.6 mg/ 5 ml dithiothreitol. Samples were vortexed and irradiated for 5–10 min at 4 C with an ultraviolet light (254 nm) from a distance of 5 cm, to cross-link the GTP-AA to the G proteins. Immunoprecipitation using 10 µl of undiluted G protein antisera was then carried out as previously described (25).
Coimmunoprecipitation of Gi with mPRs
Two different procedures were used for the coimmunoprecipitation studies. Incubated human myocytes were treated with 0 nM (vehicle) or 100 nM P4 for 10 min. Cells were rinsed twice with ice-cold PBS, and triethanolamine buffer, pH 7.5 (50 mM triethanolamine, 25 mM KCl, 5 mM MgCl2) containing 0.25 M sucrose, 0.1% of protease inhibitor cocktail (Sigma) was added to the cells, which were subsequently frozen at –80 C. Plasma membranes were prepared as described previously and resuspended in immunoprecipitation buffer (IP buffer; 0.1 mM EDTA, 1% Triton X-100 in Ca2+- and Mg2+-free PBS, pH 7.5) to a final volume of 300 µl and concentration of 2 mg/ml membrane protein. The membrane suspension was incubated overnight at 4 C with 1:100 of goat anti-Gi antibody and control goat IgG (Santa Cruz Biotechnology). Plasma membranes were then incubated for an additional 2 h at 4 C with 20 µl protein A agarose beads (Santa Cruz) added in the IP buffer. Beads were washed twice with 1 ml IP buffer, and immunoprecipitates were eluted by boiling for 5 min in SDS sample buffer. Samples were run on a 12% Tris-glycine SDS-polyacrylamide gel, and proteins were transferred to polyvinylidine difluoride membranes. Membranes were treated with antibody to mPR and mPRß (1:3000), and immunoreactivity was visualized by chemiluminescence.
In the second procedure, solubilized myometrial membranes treated with P4 (100 nM) and no supplement were incubated with 3 µl of undiluted antisera of mPR and mPRß, and the coupling to Gi was assessed using a commercially available G protein immunoprecipitation kit (Sigma), according to manufacturer’s protocol.
cAMP Studies
Human pregnant myometrial cells were seeded in 12-well dishes and cultured until 95% confluency. Before treatments, cells were washed once with phenol red-free DMEM, containing 0.1% BSA, followed by preincubation with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) for 30 min. Cells were then stimulated with P4-BSA (0.1–1000 nM) in the presence or absence of isoproterenol (100 nM) for 15 min at 37 C; reactions were terminated by addition of 0.1 M HCl. After an overnight freeze-thaw cycle, the cAMP levels were measured in the supernatants using RIA.
Myometrial cell membrane preparations (50 µg protein) were treated with P4-BSA (100 nM) in the presence of isoproterenol (100 nM) in 50 µl of extraction buffer for 30 min at 22 C, before the addition of 100 µl of 50 mM Tris-HCl containing 10 mM MgCl2, 1 mM EGTA, 1 g/liter BSA, 1 mM ATP, ATP regeneration system (7.4 mg/ml creatine phosphate, 1 mg/ml creatine phosphokinase), 100 µm 3-isobutyl-1-methylxanthine, 0.15 mM bacitracin, pH 7.4, at 37 C. The reaction was terminated after 10 min by the addition of 1 ml of 0.1 M imidazole buffer, pH 7, followed by heating of the tubes in boiling water for 5 min. The amount of cAMP in the supernatants was determined by RIA.
G Protein Activation
Specific binding of [35S]GTPS to myometrium plasma membranes was determined by the procedure of Liu and Dillon (26) with few modifications (27). Membranes (200 µg protein) were incubated with 10 µM GDP and 0.5 nM [35S]GTPS (
12,000 cpm; 1.03 Ci/µmol) in 350 µl Tris buffer (100 nM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.6 mM EDTA, 0.1% BSA, 50 mM Tris-HCl, pH 7.4) at 25 C for 30 min in the presence of 100 nM P4 or cortisol. Nonspecific binding was determined by addition of 100 µM GTPS. Aliquots (100 µl) of the incubates were subsequently filtered through Whatman GF/B glass fiber filters, followed by several washes and subsequent scintillation counting.
Immunodetection of G Protein -Subunits
The [35S]GTPS-labeled G protein -subunits were identified by immunoprecipitation using specific -subunit antibodies (28). Membranes were incubated with 1 µM P4 for 30 min at 25 C in 250 µl Tris buffer containing 4 nM [35 S]GTPS, 10 µM GDP, and protease inhibitors. Buffer at 4 C containing 100 µM GDP and 100 µM unlabeled GTPS was added to stop the reaction. The mixture was centrifuged (19,000 rpm for 15 min), and the resulting pellet was resuspended in immunoprecipitation buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 25 mM Tris-HCl, with protease inhibitors, pH 7.4). The samples were incubated for 6 h at 4 C with specific antisera to the Gi and Gs (Sigma; dilution, 1:500). Protein A-Sepharose was added, and the mixture was incubated overnight. The immunoprecipitates were subsequently collected by centrifugation (9000 rpm for 2 min) and washed in buffer (50 mM HEPES,100 µM NaF, 50 mM sodium phosphate, 100 mM NaCl, 1% Triton X-100, and 1% SDS). The pellets were boiled in 0.5% SDS, and the radioactivity in the immunoprecipitated [35S]GTPS-labeled G protein -subunits was counted in a scintillation counter.
Statistical Analysis
Data are shown as the mean ± SD of each measurement. For the real-time PCR measurements and Western immunoblotting, results were evaluated between groups by using two-tailed Student’s t test, with significance determined at the level of P < 0.05. For Western immunoblotting experiments, the densities were measured using a scanning densitometer coupled to scanning software ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA; and Amersham Pharmacia, Little Chalfont, UK). For the second messenger measurements, a one-way ANOVA was used, followed by Dunnett’s test, to compare each treatment dose. For luciferase assays, ±SD values were calculated for each experiment, and data were analyzed using one-way ANOVA. When a significant effect was found, one-way ANOVA was followed by post hoc test (Tukey procedure) to locate differences between groups.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Summary of Disclosure Forms for Potential Conflict of Interest: E.K., S.Z., Y.P., J.D., E.W.H., H.S.R., and P.T. have nothing to declare.
First Published Online February 16, 2006
1 E.K. and S.Z. should be considered first joint coauthors. 
Abbreviations: CBP, cAMP response element-binding protein; E2, estrogen; FITC, fluorescein isothiocyanate; GRE, glucocorticoid-responsive element; IP buffer, immunoprecipitation buffer; MLC, myosin light chain; mPR, membrane progestin receptor; P4, progesterone; PTX, pertussis toxin; SDS, sodium dodecyl sulfate; siRNA, short interfering RNA; SRC, steroid receptor coactivator.
Received for publication July 5, 2005. Accepted for publication January 17, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, P < 0.05 comparing ISO+P4-BSA+PTX with ISO+P4-BSA. Ab-IP, Antibody used for immunoprecipitation; Abs-IP, antibodies used for immunoprecipitation.
