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School of Surgical and Reproductive Sciences (Obstetrics and Gynecology), University of Newcastle upon Tyne, Faculty of Medical Sciences, Newcastle upon Tyne NE2 4HH, United Kingdom
Address all correspondence and requests for reprints to: Dr. G. Nicholas Europe-Finner, School of Surgical and Reproductive Sciences (Obstetrics and Gynecology), University of Newcastle upon Tyne, 3rd Floor, William Leech Building, Faculty of Medical Sciences, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. E-mail: g.n.europe-finner{at}ncl.ac.uk.
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Abstract |
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Introduction |
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With respect to the maintenance of myometrial quiescence during pregnancy, there is significant evidence indicating that levels of placentally derived human chorionic gonadotropin (hCG) increase dramatically, reaching a peak at 8–10 wk, and then declining slightly thereafter (2). The action of hCG on the myometrium is primarily mediated via coupling of chorionic gonadotropin (CG)/LH receptors to the adenylyl stimulatory G protein, G
s, and the formation of cAMP. Zuo et al. (3), employing Western/ligand blot assays, have shown that the human myometrium during pregnancy expresses CG/LH receptors that are significantly decreased during term and preterm labor. Eta et al. (4) showed that hCG can directly inhibit the amplitude of oxytocin-stimulated contractions in the nonpregnant myometrium, which appears to be modulated by decreasing intracellular calcium levels in myometrial smooth muscle cells. More importantly, Slattery et al. (5) recently demonstrated that hCG exerts a potent inhibitory effect on human myometrial contractility in the third trimester of pregnancy, and this effect is concentration dependent. Furthermore, Kurtzman et al. (6) indicated that hCG blocks prostaglandin-induced preterm delivery in the mouse model. Similarly, Ali et al. (7) described in a pilot study of 100 women with preterm labor that hCG exhibits potent tocolysis, with no fetal side effects. Increased accumulation of cAMP in the myometrium during pregnancy as a result of stimulation of the adenylyl cyclase pathway by hCG may also be potentiated by the increase in levels of Gs- and Gs-coupled adenylyl cyclase activity (8, 9, 10). At the onset of both term and preterm labor, levels of hCG receptors and Gs are significantly decreased. Consequently, the ability to maintain high levels of CG/LH receptor and Gs expression in the myometrium during gestation may aid in precluding the early onset of labor and premature delivery. We recently detailed the molecular processes by which expression of the Gs gene may be regulated in human myometrium during pregnancy. These appear to involve binding of specificity protein (Sp)-like transcription factors to GC boxes within the promoter region of the gene that stimulate expression of the Gs gene via a protein kinase A (PKA)-dependent mechanism (11). However, at present there is little information on the regulatory processes by which the CG/LH receptor gene is regulated in human myometrium during pregnancy and labor.
The CG/LH receptor gene structure has been defined in the human, rat, and mouse (12, 13, 14, 15, 16, 17). Essentially, in various species the gene contains 11 exons, separated by 10 introns. Exons 1–10 encode the 5'-untranslated region and most of the extracellular domain. The primary transcriptional start sites of the LH/CG receptor gene in both humans and rats lie within 180 bp 5' to the translational ATG codon, and deletion analysis has localized the promoter within this region (18, 19, 20). In rat cell lines, expression of the CG/LH receptor gene is constitutively inhibited by upstream sequences –180 to –2678 bp (18, 19), whereas in human cell lines, only small inhibitory effects were caused by this region (20). This region contains seven activator protein-1 (AP1) binding sites as well as a hemipalindromic cAMP response element (hCRE) domain, which have the potential to bind the Jun/Fos and cAMP response element-binding protein (CREB)/modulator protein (CREM)/activating transcription factor (ATF) bZIP transcription factor families, respectively (Fig. 1A
). The human and rat CG/LH receptor gene is TATA-less and contains EREhs direct repeats and two Sp1–4 GC binding domains (I and II) within the proximal promoter (see Fig. 1). The two GC boxes localized within the +1 to –180 bp proximal promoter are bound by Sp1/Sp3 proteins, which may require phosphorylation by PKA (21) for greater DNA binding, and are central to transcription of the gene in the rat and several human cell lines, including JAR (human placental choriocarcinoma) and HeLa cells (18, 19, 20, 22). Transcriptional activation of the CG/LH receptor gene is greatly increased by histone acetylation within the proximal promoter that promotes an accessible chromatin environment for initiation of transcription and is substantially inhibited by histone deacetylases (HDACs), which in most cases promote the formation of a closed promoter and transcriptional repression (23, 24). In this context, HDACs complexed with mSin3A/RbAp48 proteins are thought to be primarily recruited to the Sp1–4 (I) binding domain (see Fig. 1B), resulting in transcriptional silencing of the gene in JAR cells. This effect can be reversed by addition of the HDAC inhibitor trichostatin A (TSA) (23, 24). Moreover, the orphan receptors EAR2/EAR3 and TR4 proteins that bind the EREhs direct repeats located immediately 5' to the two GC boxes (see Fig 1B) may aid in transcriptional repression of the gene (24, 25, 26).
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Materials and Methods |
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Primary adherent cell cultures of human myometrial smooth muscle cells were established from lower segment myometrial tissue samples taken during elective cesarean sections from pregnant nonlaboring women, as described previously (11, 27). Written consent was obtained from all women, and ethical approval was granted by the Newcastle and North Tyneside Health Authority ethics committee. Cultures were incubated in DMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% (vol/vol) fetal bovine serum, 1 U/ml penicillin, and 1 ng/ml streptomycin at 37 C in a humidified atmosphere of 95% air/5% CO2.
Preparation of nuclear extracts
Cultured myometrial cells were harvested by trypsin digestion of extracellular matrix proteins, washed with ice-cold PBS, and resuspended by gentle pipetting in 4 packed cell vol ice-cold hypotonic lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, 100 nM okadaic acid, and 5 nM calyculin A]. After 15-min incubation on ice, 0.25 original vol 10% Nonidet P-40 were added, and cells were vortexed vigorously for 10 sec and centrifuged at 13,000 x g for 1 min. The supernatant, containing cytoplasmic protein, was removed, and the pellet, containing nuclei and cell debris, was resuspended in 0.5 original vol ice-cold, high salt extraction buffer [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonylfluoride, 100 nM okadaic acid, and 5 nM calyculin A]. The suspension was incubated at 4 C with constant agitation for 15 min, then centrifuged at 12,300 x g for 5 min at 4 C. The supernatant, containing nuclear extract, was split into aliquots and stored at –75 C.
Antibodies
The following polyclonal (Pc) and monoclonal (Mc) primary antibodies from Santa Cruz Biotechnology, Inc. (SC; Santa Cruz, CA) and Abcam Ltd. (AB), raised in rabbit (R), mouse (M), or goat (G), were used (at the indicated dilutions for immunoblotting): Sp1 (SC-59, R, Pc, 1:300), Sp2 (SC-643, R, Pc, 1:200), Sp3 (SC-644, R, Pc, 1:300), Sp4 (SC-645, R, Pc, 1:200), HDAC1 (AB-7028, R, Pc, 1:3000), HDAC2 (AB-7029, R, Pc, 1:3000), EAR2 (AB-3889, G, Pc, 1:400), EAR3 (SC-6577, G, Pc, 1:100), TR4 (SC-8619, G, Pc, 1:100), RbAp48 (SC-8270, G, Pc, 1:200), mSin3A (SC-5299, M, Mc, 1:200), c-Jun (SC-1694, R, Pc, 1:200), p-c-Jun (SC-822, M, Mc, 1:100), Jun B (SC-46, R, Pc, 1:100), Jun D (SC-74, R, Pc, 1:200), c-Fos (SC-52, R, Pc, 1:400), FosB (SC-48, R, Pc, 1:200), Fra1 (SC-605, R, Pc, 1:200), Fra2 (SC-171, R, Pc,1:300), hCG/LH receptor (SC-26341, G, Pc, 1:200), and Gß (SC-378, R, Pc 1:200).
EMSAs
All oligonucleotides used for EMSAs/DAPAs and the hCG/LH receptor promoter sequences from which they were derived are detailed in Table 1 and Fig. 2. Oligonucleotides (Invitrogen Life Technologies, Inc.) were 5'-end labeled in 20-pmol aliquots using T4 polynucleotide kinase (Promega Corp., Madison, WI) and 50 µCi [
-32P]ATP (Amersham Biosciences, Arlington Heights, IL), annealed to form double-stranded pairs, and purified using ProbeQuant G50 microcolumns (Amersham Biosciences). Unlabeled competitor oligonucleotide pairs were prepared by annealing 2 nmol of each oligonucleotide to give a final concentration of 10 µM. EMSA reactions were prepared by mixing 50 fmol 32P-labeled double-stranded oligonucleotide probe with 30 µg nuclear extract protein in either buffer A [10 mM Tris (pH 7.5), 200 mM NaCl, 0.5 mM DTT, and 5% glycerol; note that this buffer has been used previously by us to identify low molecular mass Sp-like proteins in the myometrium (11)] or standard EMSA buffer B [25 mM HEPES (pH 7.9), 50 mM KCl, 5 µM ZnSO4, 11 mM ß-mercaptoethanol, 0.05% Nonidet P-40, and 10% glycerol] plus 50 µg/ml polydeoxyinosinic-polydeoxycytidylic acid at room temperature. Where indicated, the myometrial nuclear extract was pretreated for 10 min with 0.1 U potato acid phosphatase (Calbiochem, La Jolla, CA), which was then inactivated by the addition of 2 mM sodium fluoride. For competition, 5 pmol unlabeled oligonucleotides were included. Specificity was also confirmed by employing the unrelated Oct1 oligonucleotide (5'-TGTCGAATGCAAATCACTAGAA-3') described previously (11). For supershift reactions, before the addition of DNA, 0.4 µg polyclonal antibodies (SC) were added to the proteins and incubated at room temperature for 20 min. On addition of DNA, 20 µl of the reactions were incubated for 20 min at room temperature, then loaded onto vertical 4% nondenaturing Tris-borate-EDTA polyacrylamide gels that had been prerun at 100 V for 30 min immediately before loading. Gels were run with cooling water circulation at 200 V, then dried onto 3MM paper (Whatman, Inc., Clifton, NJ) and exposed to X-OMAT LS film (Eastman Kodak, Rochester, NY) before autoradiography.
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Double-stranded 5'-biotin end-labeled oligonucleotides (0.25 µg/reaction; MWG Biotec, Ebersberg, Germany), detailed in Table 1
, were incubated with 50 µg nuclear proteins extracted from cultured myometrial cells in 400 µl binding buffer [12 mM HEPES (pH 7.9), 4 mM Tris-HCl, 60 mM KCl, 5% glycerol, 0.5 mM EDTA, and 1 mM DTT] on ice for 45 min. Forty microliters of Tetralink avidin resin (Promega Corp.) were equilibrated in 1 ml binding buffer for 45 min, then pelleted by centrifugation at 13,000 x g for 3 min. The buffer was removed, and the resin was incubated with the DNA/protein mix for 2 h at 4 C with gentle rotation to allow the formation of DNA/protein/avidin complexes. The resin was then washed five times, each time with 1 ml binding buffer, by centrifugation at 13,000 x g for 3 min, followed by aspiration of supernatant and resuspension in buffer to remove proteins not complexed with DNA and avidin. The resin was resuspended in 36 µl SDS-PAGE loading buffer [62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 25% glycerol, and 0.01% bromophenol blue] and incubated at 100 C for 5 min to dissociate DNA/protein complexes. Proteins were separated by SDS-PAGE and detected by immunoblotting, as described above.
Immunoblotting
For Western blotting, treated myometrial cells were lysed directly by the addition of SDS-PAGE loading buffer [62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 25% glycerol, and 0.01% bromophenol blue]. Proteins were denatured at 100 C for 5 min, separated by SDS-PAGE in 10% gels, and transferred to polyvinylidene difluoride membranes, which were blocked by overnight incubation at 4 C in PBS containing 4% fat-free dried milk. Blocked membranes were washed in PBS and incubated for 2 h at room temperature with the primary antibody diluted in PBS containing 4% fat-free dried milk. Membranes were then washed five times for 5 min/wash with PBS and incubated for 1 h at room temperature with the secondary, horseradish peroxidase-conjugated anti-IgG antibody (DakoCytomation, Carpenteria, CA) at a 1:1000 dilution in PBS containing 4% fat-free dried milk. Membranes were washed in PBS as before, then treated with enhanced chemiluminescence reagent (Amersham Biosciences) and exposed to flash-sensitized Hyperfilm enhanced chemiluminescence reagent (Amersham Biosciences).
Plasmid construction
A set of fragments of the hCG/LH receptor gene promoter, from +1 to –2678 bp, as shown in Fig. 2, was amplified by PCR from human genomic DNA using pfu proofreading thermostable DNA polymerase (Promega Corp.). These fragments were cloned directly into the pCR-Blunt II-TOPO topoisomerase-linked cloning vector (Invitrogen Life Technologies, Inc.), then subcloned by XhoI/KpnI or XhoI/HindIII digestion and ligation into the similarly digested pGL3-E vector (Promega Corp.). The resulting plasmids thus carried hCG/LH receptor gene promoter fragments directing transcription of the firefly luciferase gene, with upstream enhancer elements to increase overall transcription levels.
Transient DNA transfections
Cells were grown in 24-well culture plates to 70% confluence. For each well, 1.5 µl Lipofectamine transfection reagent (Invitrogen Life Technologies, Inc.) was mixed with 25 µl OptiMEM reduced serum medium (Invitrogen Life Technologies, Inc.; not supplemented with antibiotics). Sterile, highly purified plasmid DNA was diluted to 8 µg/ml in OptiMEM medium. For each well, 0.2 µg diluted DNA was mixed with the Lipofectamine/OptiMEM mix and incubated for 30 min before addition of 150 µl serum- and antibiotic-free DMEM. Culture medium was removed from the cells, and they were washed with 200 µl serum- and antibiotic-free DMEM, which was then replaced with the DNA/Lipofectamine/medium mixture. Cultures were incubated at 37 C for 5 h to allow transfection to occur, then 200 µl antibiotic-free DMEM containing 20% serum were added to each well, and cells were incubated for an additional 16 h before treatment.
Luciferase assay
Transfected cell cultures were chemically treated and incubated as indicated. Culture medium was aspirated, and cells were washed with PBS and lysed by addition of 100 µl passive lysis buffer (Promega Corp.) direct to the culture well, followed by incubation for 15 min at room temperature on a rocking platform. Twenty microliters of lysate were transferred to a 1.5-ml microcentrifuge tube containing 100 µl luciferase assay reagent (Promega Corp.) and mixed by brief vortexing, then firefly luciferase activity was measured by a 30-sec integration in a TD 20–20 luminometer (Turner Designs, Palo Alto, CA).
Myometrial cell RNA isolation and RT-PCR
Human myometrial cells were cultured to 75% confluence, followed by treatment with vehicle (ethanol) or 100 ng/ml TSA (330 nM) for 24 h. After treatment, cells were lysed, and total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA), and copurified DNA was removed by employing a ribonuclease-free deoxyribonuclease set (Qiagen).
Duplex RT-PCR employing specific hCG/LH receptor gene and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene primers was carried out using the SuperScript one-step RT-PCR system (Invitrogen Life Technologies, Inc.) in a total volume of 25 µl with 5% (vol/vol) RNaseOUT (Invitrogen Life Technologies, Inc.) ribonuclease inhibitor and 100 ng total RNA template, prepared as described above. Primers for the hCG/LH receptor gene were: forward, 5'-GGAAACCACTCTCTCACAAGT-3'; and reverse, 5'-GGTGGATTGAGAAAGGCTTTATTTG-3', yielding a 474-bp fragment; primers for GAPDH were: forward, 5'-CTGCCGTCTAGAAAAACC-3'; and reverse, 5'-CCACCTTCGTTGTCATACC-3', yielding a 210-bp fragment. Reaction conditions were as follows: RT step: 50 C for 30 min, 94 C for 2 min; amplification step: 32 cycles of 94 C for 30 sec, 59 C for 30 sec, 72 C for 40 sec, then 68 C for 9 min. Five microliters of each reaction were subjected to agarose gel electrophoresis with ethidium bromide staining, followed by densitometric scanning using a UMAX PS 2400 scanner coupled to the Intelligent Quantifier software package (Genomic Solutions, Ann Arbor, MI).
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Results |
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DAPAs in conjunction with Western blotting were initially performed to define the ability of AP1/hCRE, Sp1–4, HDACs, EAR2, EAR3, and TR4 proteins in myometrial cells to bind/complex to their respective sequences within the distal –180 to –2678 bp and the proximal +1 to –180 bp promoter regions of the CG/LH receptor gene. Figure 3A indicates that the biotinylated –62 to –88 bp Sp1–4 (I) and –106 to –127 bp Sp1–4 (II) binding domains within the proximal promoter bind Sp1, Sp3, and Sp4 proteins (no binding was observed using an antibody to Sp2) and also formed protein complexes with HDAC1 and HDAC2 as well as the associated protein RbAp48. This protein has been shown by Zhang and Dufau (27) to complex with Sp1–4/HDACs in JAR cells. mSin3A, which complexes with HDAC1 and HDAC2 (see Fig. 1B), remained undetected in DAPAs and Western blots, possibly indicating low expression levels in this cell type. No binding was observed with CREB/CREM/ATF proteins to the hCRE within the distal promoter, whereas c-Jun, Fra1, and JunD proteins were observed to bind to the biotinylated –1463 to –1487 bp AP1 (I) binding domain within this region (Fig. 3B). The orphan receptor proteins EAR2 and the associated protein TR4 (27) also formed complexes with the biotinylated EREhs direct repeats (Fig. 3C) located immediately 5' to the two Sp1–4 binding domains (see Fig. 1B). However, EAR3 remained undetected in these DAPAs as well as in Western blot analysis, which may indicate the absence or a low level of expression in myometrial cells.
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The binding of Sp1–4, Jun/Fos, and CREB/CREM/ATF proteins to their respective GC boxes, AP1, and hCRE sequences within the proximal and distal promoter regions of the CG/LH receptor was also determined with myometrial cell nuclear extracts and specific oligonucleotides in EMSAs. Employing an oligonucleotide synthesized to the –62 to –88 bp [5'-GGCAGCCAAGGGGCGGGGAGAGGGGTG-3'] Sp1–4 (I) binding domain within the proximal promoter (see Fig. 1), three DNA-protein complexes, I–III, were observed with myometrial cell nuclear extracts (Fig. 4A, lane 1). Greater DNA binding was observed in the presence of forskolin (10 µM; Fig. 4A, lane 2), indicating that these proteins required phosphorylation by PKA for increased activity, as previously described (11, 21). The specificity for binding to this sequence was also determined by competition with excess cold unrelated Oct1 and wild-type/mutated Sp –62 to –88 bp oligonucleotides; no loss in DNA binding was seen with the Oct1 and mutated Sp sequences (Fig. 4B, lanes 2 and 4), but excess wild-type Sp oligonucleotide specifically reduced binding (Fig. 4B, lane 3). Identification of DNA-protein complexes I–III was accomplished by supershift assays with antibodies to Sp1–4 (note that, similar to DAPAs, no supershifts were observed with an antibody raised to Sp2). In each case a substantial reduction in binding to the Sp sequence was seen upon incubation of either individual or combinations of antibodies, as reported by other investigators (23), rather than a shift to a higher molecular mass. Essentially the Sp1 antibody significantly decreased the intensity of complex III (Fig. 4B, lane 5); the Sp3 and Sp4 antibodies abolished complexes II and I, respectively (Fig. 4B, lanes 6 and 7). Identification of complexes I, II, and III as Sp4, Sp3, and Sp1 protein interactions, respectively, was confirmed using combinations of antibodies. In this instance, addition of Sp1and Sp3 antibodies decreased the intensity of complex III and abolished complex II (Fig. 4B, lane 8), addition of Sp1 and Sp4 antibodies decreased the intensity of complex III and abolished complex I (Fig. 4B, lane 9), addition of Sp3 and Sp4 antibodies abolished complexes II and I (Fig. 4B, lane 10), and addition of Sp1, Sp3, and Sp4 antibodies abolished complexes I–III (Fig. 4B, lane 11). We previously reported the binding of novel low molecular weight Sp-like proteins to Sp1–4 GC sequences within the Gs promoter (11). When buffer B, used in the above EMSA reactions, was switched to buffer A, as detailed in Materials and Methods, a similar lower molecular mass DNA-protein complex IV (Fig. 4C) was observed, which was specific, because no loss in DNA binding was seen with the Oct1 and mutated Sp sequences (Fig. 4C, lanes 2 and 4), but excess wild-type Sp oligonucleotide substantially reduced binding (Fig. 4C, lane 3). This result suggests that both the CG/LH receptor and Gs genes are modulated in the myometrium by novel Sp-like proteins as well as the Sp1–4 protein family. Using the other –106 to –127 bp (5'-GAGGGCAGGGGCGGGCCGGCAG-3'; see Fig. 1) Sp1–4 (II) sequence in the proximal promoter in EMSAs with Sp1–4 antibodies gave similar results, indicating that this region also bound Sp1, Sp3, Sp4, and Sp-like proteins (data not shown).
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) in EMSAs, a single DNA-protein complex V was observed. The specificity for binding to this sequence was also determined by competition with excess cold Oct1 and wild-type/mutated AP1 oligonucleotides; where no loss in DNA binding was seen with the Oct1 and mutated AP1 sequences (Fig. 5A, lanes 2 and 4), but excess wild-type AP1 oligonucleotide abolished binding (Fig. 5A, lane 3). To determine the composition of complex V, supershift assays were employed with antibodies to the Jun and Fos families. Only c-Jun/phospho-c-Jun/Fra1/JunD supershift complex VI was observed (Fig. 5B, lanes 2–5), indicating that complex V (Fig. 5B, lane 1) was composed of Jun family members c-Jun, Fra1, and JunD. DNA-protein complex V was also obtained with the three other I, III, and VI AP1 sites (see Fig. 1A), where only c-Jun supershifts were observed (data not shown). In contrast, no binding was observed with AP1 sites II, V, and VII (see Fig. 1A), nor was binding observed using EMSAs and supershift assays with myometrial CREB/CREM/ATF proteins to the hCRE at –1768 to –1792 bp (see Fig. 1A) within the distal promoter region.
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). Incubation with phosphatase abolished Sp1, Sp3, and Sp4 complexes III, II, and I (Fig. 5C, lane 2) and c-Jun/Fra1/Jun D complex V (Fig. 5C, lane 4).
Specific binding of myometrial nuclear proteins to the –147 to –180 bp EREhs direct repeats (see Fig. 1) within the proximal promoter was also observed (Fig. 5D). The specificity for binding to this sequence was again determined by competition with excess cold Oct1 and wild-type EREhs oligonucleotides; no loss in DNA-protein complex VII binding was seen with the Oct1 (Fig. 5D, lane 2), but excess wild-type EREhs oligonucleotide abolished binding (Fig. 5D, lane 3).
Myometrial CG/LH receptor promoter reporter gene luciferase assays
CG/LH receptor promoter reporter luciferase constructs, detailed in Fig. 2, were employed in transient transfection of cultures of human myometrial cells to determine the functional activity of the AP1, Sp1 to -4, and EREhs sites within the distal and proximal promoter regions in regulating transcription of the CG/LH receptor gene in the myometrium. Initially, the basal luciferase activity of individual constructs with sequential deletions of regions of the CG/LH receptor gene from –2678 to –156 bp were determined and are shown in Fig. 6A. Essentially sequential deletions of the promoter had a biphasic effect on basal luciferase activity. For example, deletions from –2678 to –1919 bp resulted in a small decrease in activity, deletions from –1919 to –1458 bp resulted in a significant increase in activity, deletions from –1458 to –828 bp caused a decrease in activity, whereas deletions from –828 to –176 bp caused an increase in activity. This pattern of activity is similar to that observed by Hu et al. (28) using CG/LH receptor promoter luciferase constructs in human choriocarcinoma JEG-3 cells and correlates with the presence of negative control regions within the distal –1458 to –2678 bp promoter, which contains the seven AP1 binding sites that have been implicated in negative regulation of the gene in the rat (18, 19). The increase in activity from –176 to +1 bp also conforms with the promoter region regulating transcriptional activity observed in JEG-3 (28), JAR, and HeLa cells (18, 19, 20, 22). The possible central role of this region in regulating expression of the gene in the myometrium was confirmed employing the CG/LH receptor deletion luciferase constructs in transfected myometrial cells treated in the presence or absence of the HDAC inhibitor TSA, forskolin, and hCG, as described in Fig. 6B, where data are presented as the fold increase over basal activity in untreated cells. In each instance, myometrial cells transfected with individual luciferase constructs were treated for 24 h after transfection with either TSA (100 ng/ml) to inhibit HDACs; with forskolin (10 µM)/hCG (10 nM) to increase DNA binding activity of Sp1, Sp3, Sp4, and Sp-like proteins; or with their combination to observe additive effects on luciferase activity. In each instance, the addition of TSA to transfected cells resulted in a substantial increase in luciferase activity compared with the effect of similar treatment in empty vector controls. Sequential deletions from the distal –2678 bp to the proximal –176 bp promoter had no statistical effect on TSA-stimulated luciferase activity on individual constructs, indicating that the distal negative control regions were not inhibiting TSA-driven transcription and, more importantly, that this activity was controlled via the proximal –176 to +1 bp promoter. Interestingly in contrast to previous reports (24, 25, 26), deletion of the EREhs direct repeats at –176 to –156 bp appeared to have little effect in modulating the transcriptional activity of the proximal promoter in myometrial cells, because no increase in activity was seen when employing the p156/1 construct compared with the p176/1 construct upon administration of TSA. Incubation of cells with either forskolin or hCG had no significant effect on the luciferase activity of individual constructs compared with that of empty vector controls. However, incubation of myometrial cells with TSA in the presence of forskolin inhibited the ability of TSA to stimulate luciferase activity in all constructs. This effect of forskolin on TSA action did not appear to be mediated via activation of PKA, because transfection of myometrial cells with the full-length p2678/1 luciferase construct (note that all constructs responded similarly in the presence of TSA alone or TSA plus forskolin) in the presence of TSA, forskolin, plus the specific PKA inhibitor KT5720 (1 µM) did not reverse the inhibitory effect of forskolin on luciferase activity (see Fig. 6C), which may indicate a nonspecific effect on TSA per se.
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To determine whether TSA could affect endogenous expression of the hCG/LH receptor gene in vivo, myometrial cells were incubated in either the presence or the absence of TSA (100 ng/ml) for 24 h, and total RNA and protein were extracted and subjected to either duplex RT-PCR with specific hCG/LH receptor gene and GAPDH control primers or Western blotting with a specific hCG/LH receptor antibody and a Gß antibody as a loading control. Figure 7 indicates that administration of TSA to myometrial cell cultures results in a substantial 2- to 3-fold increase in both CG/LH receptor mRNA and protein levels compared with untreated cells (note that treatment for 12 h with TSA produced similar increases in mRNA and protein levels; data not shown) and also supports the data obtained with hCG/LH receptor gene promoter reporter luciferase constructs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, with the CG/LH receptor gene constructs indicated that sequential deletion of AP1-containing regions did not increase luciferase activity in the presence of TSA, which removes the inhibitory effect of HDACs on expression of the gene. Consequently, it seems unlikely that these domains play a major role in regulating the expression of this receptor in vivo in the myometrium. Furthermore, because no binding of myometrial proteins to the hCRE site within the distal promoter was seen employing either DAPAs or EMSAs, this domain also appears to have no role in regulating the expression of the gene.
In contrast, data from DAPAs, EMSAs, and luciferase assays with the CG/LH receptor gene constructs all indicate that the proximal +1 to –180 bp promoter containing the two Sp1–4 GC binding domains is central to regulating the expression of the gene in myometrial cells. This is in agreement with observations made in several human cell lines (20, 23, 24). However, these studies only indicated that Sp1 and Sp3 proteins are involved in transcriptional modulation of the gene, whereas we show here, using DAPAs/EMSAs, that myometrial Sp4 proteins as well as a novel putative low molecular weight Sp-like protein also form complexes with these sites. Moreover, greater DNA binding activity of all Sp proteins was seen in the presence of forskolin, suggesting that Sp1, Sp3, Sp4, and Sp-like proteins require phosphorylation by PKA for increased activity to occur. In this context, Rohlff et al. (21) have also shown that Sp1 proteins are dependent on PKA phosphorylation for greater activity, and we have recently described low molecular weight Sp-like proteins binding to the multiple Sp1–4 GC boxes within the human Gs promoter (11). Zhang and Dufau (23, 24) provided convincing evidence that the Sp1–4 (I) binding site is the primary site for binding Sp1/Sp3 proteins that subsequently recruit HDAC1/HDAC2/RbAp48/mSin3A complexes to silence the expression of the gene in JAR cells. Although we could not detect mSin3A proteins in myometrial cells with Western blotting; DAPAs indicated that HDAC1, HDAC2, and RbAp48 proteins all formed complexes with both Sp1–4 (I) and (II) binding domains within the proximal promoter. However, because we did not use mutational analysis of the proximal promoter to define which site is the critical site, as described by Zhang and Dufau (23), it remains unclear whether either or both sites are essential for transcriptional modulation in myometrial cells. DAPAs also indicated that the myometrial orphan receptor proteins EAR2 and TR4 formed complexes with the EREhs direct repeats located immediately 5' to the two GC boxes. Even though EAR3 remained undetected in myometrial cells, these EREhs direct repeats may not play a role in transcriptional inhibition of the gene as has been observed in other cell types (24, 25, 26). This is evidenced by data from luciferase assays presented in Fig. 6B, where deletion of this inhibitory region should have caused an increase in luciferase activity in the presence of TSA. The importance of HDACs in silencing the gene in the myometrium is also highlighted by these luciferase assays in which incubations with TSA resulted in a severalfold increase in activity with each construct tested. It is noteworthy that the addition of either forskolin or hCG, which should increase the phosphorylation state of the Sp protein via activation of PKA, had no effect on luciferase activity in either instance. However, the administration of both TSA and forskolin completely negated the effect of TSA alone in increasing luciferase activity. This effect appeared not to be mediated via activation of PKA, because similar incubations in the presence of the specific PKA inhibitor KT5720 did not reverse the inhibitory effect of forskolin on TSA activation of the luciferase construct and thus may indicate a nonspecific effect of forskolin on TSA in these experiments.
In conclusion, we provide strong evidence indicating that inhibition of HDAC1/2 activities by TSA results in a substantial induction in hCG/LH receptor gene promoter activity, and perhaps more importantly, we show that this agent can cause a substantial increase in levels of endogenous mRNA and protein in cultures of human myometrial cells. Consequently, our studies may implicate the use of TSA and similar agents as tocolytics in increasing CG/LH receptor levels in the myometrium in vivo, resulting in the subsequent maintenance of uterine relaxation during gestation. In this respect, Condon et al. (29), using the mouse model, have shown that administration of TSA delayed the onset of parturition by 24–48 h.
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Acknowledgments |
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Footnotes |
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First Published Online March 22, 2005
Abbreviations: AP1, Activator protein-1; ATF, activating transcription factor; CG, chorionic gonadotropin; CREB, cAMP response element-binding protein; CREM, cAMP response element modulator protein; DAPA, DNA affinity purification assay; DTT, dithiothreitol; EAR, nuclear orphan receptor; EREhs, estrogen response element half-site; G, goat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; hCRE, hemipalindromic cAMP response element; HDAC, histone deacetylase; M, mouse; Mc, monoclonal; Pc, polyclonal; PKA, protein kinase A; R, rabbit; Sp, specificity protein; TR4, testicular orphan receptor 4; TSA, trichostatin A.
Received October 5, 2004.
Accepted March 4, 2005.
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s in pregnancy. Endocrinology 132:2484–2490s in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 79:1835–1839[Abstract]
s in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic AMP and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter. J Clin Endocrinol Metabl 87:5675–5685
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