Induction of Oxytocin Receptor Gene Expression in Rabbit Amnion Cells

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 8 Printed in U.S.A.

Induction of Oxytocin Receptor Gene Expression in Rabbit Amnion Cells* YOW-JIUN JENG, STEPHEN J. LOLAIT,

AND

MELVYN S. SOLOFF

Department of Obstetrics and Gynecology (Y.J.J., M.S.S.) and the Sealy Center for Molecular Science (M.S.S.), University of Texas Medical Branch, Galveston, Texas 77555-1062; and the Dorothy Crowfoot Hodgkin Laboratories, Department of Medicine, University of Bristol, Bristol, United Kingdom BS2 8HW ABSTRACT Oxytocin (OT)-stimulated PGE2 release by rabbit amnion is enhanced by the up-regulation of oxytocin receptors (OTR), which increase about 200-fold at the end of pregnancy. As recent studies have shown that PGs are essential for parturition, the rise in amnion OTR and associated PGE2 synthesis are probably essential for labor initiation. The present work was directed toward understanding the mechanisms of OTR up-regulation. Levels of agents that stimulate adenylyl cyclase activity and cortisol are increased in amniotic fluid at the end of pregnancy. Addition of either forskolin or cortisol to cultured amnion cells caused an increase in OTR ligand-binding sites and steady state OTR messenger RNA (mRNA) levels. Forskolin treatment elevated OTR mRNA levels rapidly, but transiently,

O

XYTOCIN (OT) stimulates PGE2 release by rabbit amnion cells (1). This effect is mediated by oxytocin receptors (OTR), which increase about 200-fold in rabbit amnion at the end of pregnancy (1). In view of the recent demonstrations that transgenic mice lacking either cytoplasmic phospholipase A2, a key enzyme involved in PG synthesis (2), or PG receptors (3) fail to deliver their offspring at term, amnion PGE2 might play a critical role in the initiation of parturition in other species as well. The rise in OTR concentration in amnion is accompanied by increases in both cortisol and agents that are capable of activating adenylyl cyclase activity in amniotic fluid (see Ref. 4 for references). Administration of a glucocorticoid (GC) to pregnant rabbits caused a substantial increase in amnion OTR concentrations (5) and resulted in preterm labor (6). The up-regulation of OTRs can be mimicked in amnion cells maintained in primary culture by the addition of forskolin (FSK), or agents that elevate intracellular cAMP concentrations, and GC (4, 5). We showed previously that the effects of FSK and cortisol on OTR concentrations in cultured rabbit amnion cells were synergistic (5). Thus, the actions of hormones that elevate amnion cAMP and of cortisol in up-regulating OTR concentrations in the amnion appear to be of physiological importance. The molecular basis for the increase in OTR-binding sites in amnion cells, however, has not been reported preReceived February 19, 1998. Address all correspondence and requests for reprints to: Dr. Melvyn S. Soloff, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1062. E-mail: [email protected]. * This work was supported by NIH Grant HD-26168 (to M.S.S.) and a grant from the Welcome Trust, UK (to S.J.L.).

whereas cortisol’s effects were slower and sustained. Actinomycin or cycloheximide, added 3 h after forskolin, led to a sustained elevation in OTR mRNA levels, suggesting that forskolin increases the activities of OTR mRNA-destabilizing factors along with increasing OTR mRNA concentration. Cortisol did not appear to affect OTR mRNA stability. Measurement of OTR mRNA transcription rates showed that forskolin’s effects were maximal within 1 h of treatment. In contrast, cortisol-induced transcription was not apparent until 8 h. The effects of forskolin and cortisol on OTR gene transcription were synergistic. Thus, the increase in OTR mRNA levels occurring after either forskolin or cortisol treatments is the result of induction of OTR gene expression, but the effects of the two agents appear to occur at separate sites. (Endocrinology 139: 3449 –3455, 1998)

viously. Alternative regulatory possibilities include transcriptional and/or posttranscriptional control, posttranslational modifications (unmasking of cryptic receptor sites; activation of existing sites by mechanisms such as phosphorylation/dephosphorylation, palmitoylation, or other covalent modifications; and conversion of a precursor to an active product), and the appearance/disappearance of activating/ inhibitory substances. In the myometrium, the rise in OTR ligand-binding activity (7, 8) is a reflection of increases in steady state OTR messenger RNA (mRNA) concentrations (9, 10). Except for a single study of OTR transcription rates in ewe endometrium, in which there was a 2-fold reduction after interferon-t treatment (11), there have been no other reported studies of OTR transcription rates. In the present studies, we have sought to determine whether the up-regulation of OTR in amnion cells is the result of increases in OTR mRNA, and whether these increases are the result of transcriptional or posttranscriptional activity. We also examined the basis for the synergistic effects of FSK and cortisol in OTR up-regulation. This work involved cloning a partial complementary DNA (cDNA) fragment of the rabbit OTR, using RT-PCR. Our findings show that increased OTR mRNA levels account for the upregulation of OTR-binding activity. Furthermore, the elevated mRNA levels are the result of transcriptional activation of the OTR gene by both FSK and cortisol. The locus of synergy between the two agents is also at the transcriptional level. Materials and Methods Chemicals FSK, cortisol, actinomycin D, cycloheximide, uridine, 4-thiouridine, collagenase, and other chemicals were purchased from Sigma Chemical

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Co. (St. Louis, MO). DMEM (high glucose), FBS, and penicillin/streptomycin were purchased from Life Technologies (Grand Island, NY). [5,6-3H]Uridine was purchased from DuPont-New England Nuclear (Boston, MA).

amnion cell RNA. The results are expressed as the ratio of OTR mRNA to 18S RNA.

Tissue and cell preparation

Cultured amnion cells were rinsed twice in ice-cold PBS, dissociated from culture plates by scraping with a rubber policeman, and collected by centrifugation. The cells were resuspended in 4 ml ice-cold sucrose buffer I (0.32 m sucrose, 3 mm CaCl2, 2 mm MgOAc, 0.1 mm EDTA, 1 mm dithiothreitol, 0.5% Nonidet P-40, and 10 mm Tris-Cl, pH 8.0) and homogenized with 15 strokes of a Dounce homogenizer (Kontes Co., Vineland, NJ) (15). Nuclei were isolated by sucrose gradient centrifugation according to the method of Greenberg and Bender (15). DNA plasmids for hybridization included the vector pUC18, 7.6 kb of the rabbit OTR gene in pUC18 that was used for DNA sequence analysis, full-length chicken b-actin cDNA cloned into pBR322 (16), and a 461-bp fragment of a rat cyclophilin cDNA (17) cloned into pSP65. The DNA samples were linearized with the appropriate restriction endonucleases, alkali denatured, and filtered through nitrocellulose membranes using a slotblot apparatus (5 mg DNA/slot). Run-on transcription and RNA hybridization were carried out as described previously (15). Background labeling of the filters was reduced by treatment with deoxyribonucleaseinactivated RNase A (10 mg/ml) in 2 3 SSC (standard saline citrate) for 30 min at 37 C.

Timed pregnant New Zealand rabbits (Ray Nichols Rabbitry, Lumberton, TX) were received on day 16 of pregnancy and killed on day 27, unless otherwise noted. The rabbits were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The research protocol was approved by the institutional committee on animal care and use, University of Texas Medical Branch. Amnion cells were cultured as described previously (1). About 8 million cells were plated onto 10-cm tissue culture plates, and the cells were maintained for up to 1 month in DMEM containing 5% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37 C (95% humidity) in the presence of 5% CO2.

[125I]OT antagonist ([125I]OTA) binding The concentration of OT-binding sites on intact cells was measured using an iodinated OT antagonist (OTA), as described previously (12).

RNA extraction Total RNA from treated amnion cells and from amnions on different days of pregnancy was isolated using the method of Chomczynski and Sacchi (13).

RT-PCR, cloning, and sequencing of rabbit OTR Two degenerate primers (59-GGTGGTGGCA/G/C/TGTGTTC/ TCAGGT-39 and 59-TCCAGCAC/TACGATGAAGGCCA-39) based on cDNA sequences in the second and sixth transmembrane regions of the human, rat, and pig OTR were used to amplify DNA from rabbit amnion cDNA. cDNA was synthesized by random priming, and PCR was performed for 35 cycles of 30 sec at 95 C, 1 min at 58 C, and 1 min at 72 C, using reagents in the GeneAmp PCR kit (Perkin-Elmer, Foster City, CA). The amplified DNA was cloned using pCRII (Invitrogen, Carlsbad, CA). Using this probe, a 7.5-kb fragment of rabbit OTR genomic DNA extending from about 1.5 kb of the 59-flanking sequence to the intron located between the sixth and seventh transmembrane regions (cloned into pUC18) was derived from a genomic clone in EMBL3 SP6/T7 l bacteriophage (Clontech Laboratories, Palo Alto, CA). DNA sequencing was performed using a cycle-sequencing protocol and AmpliTaq DNA polymerase (Perkin-Elmer). Sequence analysis was performed using an Applied Biosystems model 373A DNA sequence analyzer (Perkin-Elmer).

Ribonuclease (RNase) protection assay (RPA) The PCR-generated rabbit cDNA clone in pCRII was linearized at an internal site with RsaI, and T7 RNA polymerase was used to transcribe the probe from the linearized template, using a MAXIscript kit (Ambion, Austin TX). The probe, which is comprised of about 490 bases of OTR and about 100 bases of vector sequence, was labeled with [32P]CTP (800 Ci/mmol) and purified by denaturing PAGE (5% polyacrylamide and 8 m urea). Solution hybridization of the labeled RNA probe with 20 mg total RNA and subsequent RNase digestion were performed using a RPA II kit, according to the manufacturer’s instructions (Ambion). Protected fragments were isolated by denaturing electrophoresis as described above. DNA markers were generated by AluI digestion of a pML plasmid (14) and end labeled using [g-32P]ATP and polynucleotide kinase after dephosphorylation with calf intestinal alkaline phosphatase. Gels were dried, quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and exposed to Kodak X-Omat AR films (Eastman Kodak, Rochester, NY) with intensifying screens. Riboprobe to 18S RNA was also generated from template (Ambion) and used simultaneously with the OTR probe. The protected 18S fragment was used to normalize for variations in recovery of OTR protected fragments after the various procedural steps. The completeness of digestion with RNase was verified using yeast transfer RNA instead of

Nuclear run-on assay

4-Thioruridine labeling and isolation of thiolated RNA Amnion cells were incubated either with 100 mm 4-thiouridine or uridine, and [5,6-3H]uridine (0.5 mCi/ml) for 1 h. Thiolated RNA (newly synthesized RNA) was isolated by a modification of the procedure of Johnson et al. (18). Total RNA was extracted as described above and dissolved in 50 mm sodium acetate, pH 5.5, containing 0.1% SDS, 0.15 m NaCl, and 4 mm EDTA (buffer A). The amount of tritium in each sample was determined by liquid scintillation counting. Equal amounts of tritiated samples were denatured by heating at 65 C for 5 min, cooled rapidly on ice, and adsorbed to slurries of phenyl mercury agarose (Affi-Gel 501, Bio-Rad, Richmond, CA) for 2 h at 4 C. The gels were packed individually into sterile tuberculin syringes and rinsed with 10 vol buffer A, followed by 10 vol buffer A containing 0.5 m NaCl. Thiolated RNA was eluted with 2 ml buffer A containing 10 mm 2-mercaptoethanol and concentrated by ethanol precipitation. The amount of RNA eluted was determined by RPA and reflected newly transcribed OTR mRNA.

Statistical methods Assays were performed in triplicate, and the results are expressed as the mean 6 se. Experiments were performed on cells pooled from two animals, using at least three separate pools. Student’s t test was used to compare treated and control groups. All tests were made at the 0.05 level of significance.

Results Generation of a rabbit OTR cDNA probe for RPA

OTR mRNA concentrations in rabbit amnion were too low to be quantified by Northern blotting, except on the day of labor. We, therefore, found it necessary to quantify OTR mRNA levels by RPA. By using RT-PCR, we obtained about a 600-nucleotide fragment of OTR cDNA composed of the area between the second and sixth transmembrane regions. Comparison of the nucleotide sequence of the fragment with OTR cDNAs from other species indicated about 92% homology. A notable region of dissimilarity corresponds to the center of the third intracellular loop (Fig. 1). Subsequent analysis of rabbit genomic DNA clones (Jeng, Y.-J., and M. S. Soloff, unpublished) allowed us to obtain cDNA sequence corresponding to the amino-terminal end of the rabbit OTR (Fig. 1).

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FIG. 1. Comparison of the deduced amino acid sequences among species in which OTR cDNAs have been cloned. Transmembrane regions 1–7 (TM1TM7) are underlined. Residues differing from the majority in each position are indicated by a boxed border. The greatest dissimilarity in sequence between species occurs in the area between TM5 and TM6, corresponding to the third intracellular loop. The rabbit OTR sequence was derived from a genomic clone lacking TM7. The probe used for RPA was generated by RTPCR, using amplimers to TM2 and TM6. References for the cDNAs are as follows: human myometrium (9), pig kidney (30), sheep endometrium (31), bovine endometrium (32), and rat RIN cells (33).

Validation of the identity of rabbit OTR receptor cDNA by RNase protection

Using RPA, we showed that rabbit RNA protected the probe based on the PCR-cloned sequence from RNase digestion. RNA from other species did not afford protection (Fig. 2). RNase treatment of rabbit amnion samples yielded three fragments: one corresponding to the expected size (490 bases), and two others of about 350 and 140 bases. The 140base fragment comigrated with minor fragments generated by the 18S RNA probe and is not shown in Fig. 2. The same three fragments were obtained when the positive strand transcribed from the cDNA plasmid was analyzed by RPA (data not shown). These findings indicate that there is sufficient secondary structure in some of the transcripts to leave an unprotected site, digestion of which yields the 350- and 140base fragments. As changes in the intensities of the 490- and 350-base bands occurred in a parallel fashion, we added the two values for quantitative purposes. OTR mRNA levels in amnion tissue on different days of pregnancy

In comparing OTR mRNA levels, as measured by RPA, in amnion tissue taken on days 25, 27, 30, and 31 (end of gestation) days of pregnancy, we found that mRNA was not detectable on day 25, was barely seen on day 27, and was greatly elevated on days 30 and 31 (Fig. 3). The increase in OTR mRNA between days 27 and 31 was about 35-fold. This pattern generally corresponds with that of OTR concentrations measured by [125I]OTA binding, except that the mRNA

FIG. 2. RPA of labeled antisense rabbit OTR RNA with RNAs from cells containing OTR from different species. Yeast transfer RNA was used as a negative control. Two fragments, 490 and 350 nucleotides, were protected by rabbit RNA, but not by RNA from the other species indicated. A third fragment of 140 bases (350 1 140 5 490) was also observed (not shown), but it was not used for analytical purposes because it comigrated with fragments generated by the 18S RNA probe. Rabbit amnion cells were either untreated or treated with FSK (25 mM) and cortisol (100 nM) for 4 h. The amount of 18S RNA protected in each sample with an 18S RNA probe was determined to normalize the data.

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FIG. 3. OTR mRNA levels in rabbit amnion tissue during pregnancy, as measured by RPA. The 490- and 350-base protected fragments are shown as described in Fig. 2. P, Unprotected 590 RNA probe. FIG. 5. Effects of 25 mM FSK (f), 100 nM cortisol (M), and both agents (F) on rabbit OTR mRNA levels in cultured amnion cells as determined by RPA. The intensity of labeling of the protected fragments was quantified with a PhosphorImager, and the amount of protected fragment in each lane was normalized relative to the concentration of 18S RNA. Each point is the mean 6 SE of at least three replicates. All treatments significantly (P , 0.05) increased OTR mRNA levels at all time points compared with those in untreated cells.

FIG. 4. Synergy between FSK and cortisol in elevating OTR concentrations, as measured by [125I]OTR binding to amnion cells. Cells were treated with 25 mM FSK (f), 100 nM cortisol (M), or both agents (F). Each point is the mean 6 SE of at least three replicates. All treatments significantly (P , 0.05) increased [125I]OTR binding at all time points compared with that in untreated cells.

increase on day 30 precedes the rise in [125I]OTA binding, which is maximal on day 31 (1). Regulation of specific [125I]OTA-binding sites and OTR mRNA levels in cultured rabbit amnion cells

As reported previously (5), treatment of amnion cells with either FSK (25 mm) or cortisol (100 nm) up-regulated [125I]OTA binding, and the effects of the two were markedly synergetic (Fig. 4). FSK caused about an 8-fold increase in OTR-binding sites by 8 h of treatment, and the increased level was maintained for up to 48 h (Fig. 4). Cortisol increased the concentration of OTR-binding sites by about 4-fold after 8 h of treatment, and binding was increased by 20-fold after 48 h (Fig. 4). The concentration of OTR-binding sites was increased by the addition of FSK and cortisol together by about 50- and 90-fold after 8 and 48 h, respectively (Fig. 4). RPA was used to quantify OTR mRNA levels in rabbit

amnion cells after treatment with FSK, cortisol, and a combination of the two. FSK (25 mm) treatment of amnion cells taken on day 27 of pregnancy caused a transient, 100-fold increase in OTR mRNA at 4 h (Fig. 5). Cortisol (100 nm) treatment caused a progressive rise in OTR mRNA levels, about 7-fold by 4 h and about 20-fold by 24 h (Fig. 5). Cortisolstimulated increases did not peak even after 48 h (data not shown). Combination of the two agents produced a synergistic response, resulting in about a 200-fold increase in OTR mRNA concentration by 4 h, followed by a decline to generally about 50% of the peak level to 24 h (Fig. 5). The effects of either FSK or cortisol were blocked by pretreatment of amnion cells for 1 h with actinomycin D (1 mg/ml; data not shown). Pretreatment of rabbit amnion cells with cycloheximide (5 mg/ml) had no significant effect on OTR mRNA levels in unstimulated or FSK-stimulated cells, but increased OTR mRNA levels severalfold after cortisol stimulation (data not shown). These findings indicate that the up-regulation of OTR mRNA levels requires transcriptional activity, but do not discern whether the effects on the OTR gene are direct. The results also indicate that protein synthesis is not required for either FSK or cortisol up-regulation of OTR mRNA. In fact, inhibition of protein synthesis enhanced the effects of cortisol on OTR mRNA steady state levels. Effects of FSK and cortisol on OTR mRNA transcription

To determine whether FSK and cortisol stimulate transcription of the OTR gene, nuclei from treated cells were isolated and used for nuclear run-on assays. Both FSK and cortisol treatments increased transcription rates from the OTR gene (Fig. 6). FSK treatment resulted in a 10-fold increase in OTR transcription after 1 h or treatment (Fig. 6).

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FIG. 6. Effects of FSK (25 mM) and cortisol (100 nM) on transcription rates of the OTR gene, as measured by nuclear run-on analysis. Nuclei were isolated 1 and 8 h after FSK and cortisol treatments, respectively, or 1 h after treatment with both agents together. B, Basal (no treatment).

FIG. 7. RPA of thiolated OTR transcripts in rabbit amnion cells after FSK (25 mM) and cortisol (100 nM) treatments. Cells were pulse labeled with 4-thiouridine (100 mM) for 1 h at increasing times after FSK and/or cortisol treatments and harvested at the times indicated, and the thiolated transcripts were isolated by affinity chromatography. The concentrations of transcript were assessed by RPA.

Cortisol treatment caused a 3-fold increase in OTR transcription after 8 h (Fig. 6). A 1-h treatment with both FSK and cortisol caused about a 13-fold increase in OTR transcription (Fig. 6). In contrast, neither FSK nor cortisol treatment alone affected transcription from b-actin or cyclophilin genes (Fig. 6). The combined treatments resulted in elevated expression of both control genes, but the increment was considerably less than that seen with the OTR gene. In addition to nuclear run-on assays, transcription rates were determined in cells that were pulse labeled with 4-thiouridine (100 mm) for 1 h at different time points after the addition of FSK, cortisol, or both, and newly synthesized thiolated RNA was isolated by affinity chromatography. Maximal induction of OTR mRNA levels occurred within the first hour of FSK treatment (Fig. 7). Transcription continued at a reduced rate at 2 and 9 h (Fig. 7). Transcription was also induced by cortisol, but only after 8 and 17 h of treatment. In addition, transcription rates were less than those induced by FSK. In general, steady state levels of OTR mRNA after cortisol stimulation (Fig. 5) paralleled transcriptional activity (Fig. 7). The effects of FSK and cortisol on OTR transcriptional rates were synergistic at both 9 and 17 h after treatment, but particularly at 17 h after treatment (Fig. 7). OTR mRNA stability

As the sharp decline in steady state OTR mRNA levels occurring after 4 h of FSK treatment did not appear to be the result of a reduction in transcription rate, we examined the effects of FSK on OTR mRNA stability. When actinomycin D (1 mg/ml) was added to cells that were pretreated with FSK

FIG. 8. Effects of actinomycin (AD; 1 mg/ml) and cycloheximide (C6; 5 mg/ml) on OTR mRNA stability. A control group (M) was not treated with either agent. Cells were pretreated with FSK (25 mM) for 3 h (A) or cortisol (100 nM) for 16 h (B) before addition of the inhibitors. Both actinomycin (‚) and cycloheximide (E) increased the half-life of FSKinduced OTR mRNA from about 3 to about 30 h, but had no effect on OTR mRNA induced by cortisol treatment.

for 3 h, OTR mRNA levels did not decline appreciably over 6 h (as occurs in the absence of actinomycin; Fig. 8A). The half-life of OTR mRNA was increased from about 3 h in the untreated cells to about 30 h with actinomycin treatment. Similar results were obtained by treatment of the cells with 5 mg/ml cycloheximide (Fig. 8A). These results indicate that inhibition of RNA or protein synthesis prevents the synthesis of factors that destabilize OTR mRNA. When actinomycin or cycloheximide was added after 16 h of cortisol treatment, there was no effect on OTR mRNA levels for up to an additional 6 h (Fig. 8B). The half-life of OTR mRNA after cortisol treatment was comparable to that seen after the addition of FSK plus actinomycin D (;30 h). These results suggest that

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OTR mRNA is stable after cortisol stimulation in the presence or absence of actinomycin D or cycloheximide. Discussion

As has been shown previously in the myometrium (9, 10), our findings demonstrate that up-regulation of OTR in rabbit amnion tissue at the end of pregnancy is associated with an increase in OTR mRNA levels. We showed previously that the up-regulation could be induced in vivo by administration of a synthetic GC to pregnant rabbits or in vitro by the addition of cortisol to rabbit amnion cells in culture (5). In the present and previous (5) studies, we showed that the effects of cortisol on OTR-binding sites in vitro were greatly potentiated by FSK. Both cortisol and FSK treatments increased steady state OTR mRNA levels in cultured rabbit amnion cells, but the response times were different. FSK caused a rapid, transient increase in OTR mRNA levels compared with cortisol, which caused a slower, progressive increase. The effects of both agents on steady state OTR mRNA levels were synergistic. We used two different approaches to quantify nascent transcript levels: run-on assays using isolated nuclei, and incorporation of 4-thiouridine into mRNA in whole cells treated with FSK and/or cortisol. The latter assay is more physiologically relevant than run-on assays, in which isolated nuclei are used under conditions where neither initiation nor termination of transcription is measured. Our findings indicate that the same conclusions can be drawn from the results of both methods. Both FSK and cortisol increased OTR gene transcription rates. In agreement with the results of studies of OTR mRNA steady state levels, the effects of FSK on OTR mRNA synthesis were rapid compared with those of cortisol. OTR transcriptional activity was very low initially after cortisol stimulation, but the rate increased after longer treatment times (to 16 –17 h, the last time point examined). The transience of the OTR mRNA response to FSK was not due to a short lived effect of FSK on OTR transcription, because FSK-stimulated OTR transcription rates remained elevated for at least 9 h. Inhibition of RNA or protein synthesis using actinomycin or cycloheximide, respectively, converted the FSK-induced transient increase in steady state OTR mRNA levels to a more permanent increase. These findings indicate that in addition to activating OTR gene expression, FSK stimulates the synthesis of factors that destabilize OTR mRNA. Our findings are similar to the results of experiments showing that cAMP destabilizes LH/hCG receptor mRNA in porcine Leydig cells (19). hCG-induced decreases in LH/hCG receptor mRNA (mediated by cAMP) were also inhibited by actinomycin (19). Like FSK-stimulated increases in amnion OTR mRNA levels, c-fos mRNA and mRNAs of other members of the immediate early gene family are induced rapidly and transiently in different cell types. However, unlike transcription of the OTR gene, transcription of immediate early genes ceases completely within 30 – 60 min after induction (20, 21). The degradation of c-fos mRNA and mRNAs of other immediate early genes is very rapid and is largely responsible for the transient nature of mRNA accumulation after tran-

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scription is stimulated. These mRNAs contain several AUUUA pentamer sequences in the 39-untranslated region that are associated with rapid mRNA degradation (for references, see Refs. 22 and 23). Although the 39-untranslated region of rabbit OTR mRNA has not yet been determined, 39-untranslated sequences of the closely related human and rat sequences have been shown to contain AU-rich elements (9, 24). If these elements were involved in destabilizing rabbit OTR mRNA, degradation could involve their association with factors that are induced by FSK. Steady state levels of OTR mRNA remained elevated for some time after cortisol treatment (t1/2 5 ;30 h). It is difficult to determine whether cortisol affects the OTR mRNA half-life, however, because intracellular cortisol levels or GC activity might remain elevated for some time after removal of the steroid from the medium. The synergistic actions of cAMP and GCs have been reported in other systems. In rabbit fetal lung in vitro, both (Bu)2cAMP and dexamethasone (DEX) increased surfactant protein B (SP-B) mRNA levels (25). The (Bu)2cAMP-dependent increase in SP-B mRNA levels resulted from elevated SP-B gene transcription, whereas the DEX-dependent increase resulted from the increases in both SP-B gene transcription and SP-B mRNA stability (25). DEX also had an additive effect on cAMP-induced somatostatin gene transcription when a somatostatin promoter/chloramphenicol acetyltransferase (CAT) reporter construct was transfected into PC12 rat pheochromocytoma cells (26). The effects of DEX were attributed to a sequence upstream from a cAMP response element (CRE) site, as deletion of this upstream region abolished the stimulatory effects of DEX without affecting cAMP responsiveness. On the other hand, mutation of the CRE abrogated both DEX- and cAMP-dependent transcriptional activities (26). These findings suggest that GC receptors might form trimeric complexes with CRE-binding proteins and DNA. Protein kinase activation was also shown to stimulate expression of the rat serine dehydratase promoter fused to CAT, and induction could be enhanced by DEX (27). DEX alone had no effect on CAT activity (27). Deletion analysis allowed demonstration of two distinct regions, one containing a CRE site and another that was essential for the enhancement of cAMP induction by DEX (27). Other studies have shown that the synergistic interactions between GC and cAMP could occur by other complex mechanisms (28, 29). Our findings suggest that both cAMP and cortisol affect OTR gene transcription, but by separate mechanisms, as reflected by distinct time courses and by the synergistic effects of the two agents. It remains to be determined whether there are functional GC and CREs in the rabbit OTR gene. No typical GRE or CRE sites have been demonstrated in the 59-flanking sequences of OTR genes in humans (34), rats (35, 36), or cows (32). Therefore, it is not clear from the present studies whether either forskolin or cortisol directly affects the interactions of transcription factors with separate, atypical recognition sites in the OTR promoter or whether the effects are mediated by other gene products preceding the interaction of regulatory factors with the OTR promoter. Our findings will serve as the basis for more detailed studies of the mechanisms of FSK and cortisol activation of OTR gene expression.

OXYTOCIN RECEPTORS Acknowledgments DNA sequence analysis was carried out in the Recombinant DNA Laboratory of the Sealy Center for Molecular Science. We thank Dr. Miriam Falzon for advice in setting up the nuclear run-on assays, and Solweig Soloff for screening the genomic library.

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