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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
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: msoloff{at}marlin.utmb.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
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 up-regulation 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue and cell preparation
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 µg/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 (5'-GGTGGTGGCA/G/C/TGTGTTC/TCAGGT-3' and
5'-TCCAGCAC/TACGATGAAGGCCA-3') 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
5'-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 
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 µg 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 [
-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 amnion cell RNA. The results are expressed as the ratio of OTR mRNA to 18S RNA.
Nuclear run-on assay
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 ß-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 slot-blot apparatus (5 µg 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 deoxyribonuclease-inactivated RNase A (10
µg/ml) in 2 x SSC (standard saline citrate) for 30 min at 37
C.
4-Thioruridine labeling and isolation of thiolated RNA
Amnion cells were incubated either with 100 µM
4-thiouridine or uridine, and [5,6-3H]uridine (0.5
µCi/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 ± 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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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).
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 140-base 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 140-base 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.
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). 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 increase on day 30 precedes the rise in
[125I]OTA binding, which is maximal on day 31 (1).
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). 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).
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). 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).
Cortisol-stimulated 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).
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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).
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
ß-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.
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). 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).
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). 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 µg/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 OTR mRNA is stable after cortisol
stimulation in the presence or absence of actinomycin D or
cycloheximide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 transcription is
stimulated. These mRNAs contain several AUUUA pentamer sequences in the
3'-untranslated region that are associated with rapid mRNA degradation
(for references, see Refs. 22, 23). Although the 3'-untranslated
region of rabbit OTR mRNA has not yet been determined, 3'-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 = 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 5'-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.
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Acknowledgments |
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Footnotes |
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Received February 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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suppresses transcription of the estrogen receptor and oxytocin receptor
genes in the ovine endometrium. Endocrinology 137:1144–1147[Abstract]
-
and ß-tubulin and cytoplasmic ß- and -actin genes using specific
cloned cDNA probes. Cell 20:95–105[CrossRef][Medline]
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),
100 nM cortisol (
), or both agents (•). Each
point is the mean ± 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.
) and cycloheximide (
) increased the half-life of FSK-induced
OTR mRNA from about 3 to about 30 h, but had no effect on OTR mRNA
induced by cortisol treatment.