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Receptor Expression in Cultured Human Granulosa-Luteal Cells1
Departments of Clinical Chemistry and Obstetrics and Gynecology (A.R.) and the Haartman Institute, Department of Bacteriology and Immunology (R.J., O.R.), University of Helsinki, Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Ari Ristimäki, Research Laboratory, Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, SF-00290 Helsinki, Finland.
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Abstract |
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is a metabolite of arachidonic acid that triggers
regression of the corpus luteum. Recent animal studies have indicated
that PGF2 (FP) receptor messenger ribonucleic acid
(mRNA) is expressed in the corpus luteum. To understand the regulation
of the FP receptor in the ovary we have cloned a partial complementary
DNA (cDNA) sequence of the FP receptor from human granulosa cells
obtained from women undergoing in vitro fertilization.
The sequence of this cDNA is identical to the previously reported FP
receptor sequences obtained from human uterine and placental cDNA
libraries. Low levels of the FP receptor mRNA were observed in freshly
isolated granulosa cells or in cultured granulosa-luteal (GL) cells, as
detected by reverse transcriptase-PCR. hCG and 8-bromo-cAMP increased
the steady state levels of the FP receptor mRNAs after incubation for
24–48 h, as detected by Northern blot hybridization. The stimulatory
effect of hCG was concentration and culture stage dependent. Further,
hCG and 8-bromo-cAMP increased binding of radiolabeled
PGF2 to intact GL cells. In contrast, phorbol
12-myristate 13-acetate inhibited basal as well as hCG- and
8-bromo-cAMP-induced FP receptor mRNA expression and binding of the
radiolabeled ligand. In summary, hCG, 8-bromo-cAMP, and phorbol
12-myristate 13-acetate modulate the expression of the FP receptor in
human GL cells, which may represent a mechanism to regulate the
responsiveness of the ovary to PGF2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGF2 is a prostanoid that triggers the regression
of the corpus luteum in various species, including nonhuman primates
and humans (6, 7). PGF2 signals through an FP receptor
that has been cloned from rodent (8, 9, 10) and ruminant (11, 12) ovarian
and astrocyte complementary DNA (cDNA) libraries. Human FP receptor
cDNAs were also recently cloned from uterine and placental tissues (9, 13). FP receptor contains putatively seven transmembrane domains, and
it signals through G proteins that activate phospholipase C, which
leads to formation of inositol trisphosphate and a subsequent increase
in cytosolic free calcium ions (8, 9, 10, 11, 12, 13).
Human luteal cells bind radiolabeled PGF2 (reviewed in
Refs. 6 and 7), and human ovaries express FP receptor messenger
ribonucleic acid (mRNA) (9). However, the precise cellular localization
of human ovarian FP receptor expression has not been established. The
ovaries of mice and cows express high levels of the FP receptor mRNA,
and expression of the receptor mRNA was localized to large luteal cells
of the corpus luteum (8, 14, 15). Interestingly, levels of the FP
receptor mRNA vary during estrous cycle in sheep and cows (12, 14, 16)
and are high during pregnancy in mice and cows (8, 14). It is not known
whether expression of the FP receptor is regulated in human
ovaries.
To clarify the presence of the FP receptor in human ovaries, we cloned
a partial cDNA sequence of the receptor from granulosa cells obtained
from women undergoing in vitro fertilization. As primary
cultures of human granulosa-luteal (GL) cells provide a well
characterized model to study the hormonal regulation of luteal phase
gene expression (17, 18, 19), we investigated the effects of hCG,
8-bromo-cAMP, and phorbol 12-myristate 13-acetate (PMA) on the steady
state mRNA levels of the FP receptor and the binding of radiolabeled
PGF2 in this cell culture system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reverse transcriptase-PCR (RT-PCR), subcloning, and sequencing
Cytoplasmic RNA (1–2 µg) from granulosa cells was converted
to cDNA using random hexamers, amplified with PCR for 30 cycles of
denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and
extension at 72 C for 1 min with specific primers for human FP receptor
(sense, 5'-CAC AAC CTG CCA GAC GGA AAA C-3'; antisense, 5'-CGA CGC CTG
AAT TTT ATA GTC TCG ATG-3') or human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH; sense, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3';
antisense, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3') as previously
described (21). Amplified products were electrophoresed through an 1%
agarose gel and visualized by ethidium bromide staining, and pGEM
markers (Promega Corp., Madison, WI) were used as mol wt markers. A
490-bp PCR product amplified from reverse transcribed samples was
subcloned into the pGEM-T vector (Promega). Sequencing was performed
with fluorescence-labeled SP6 and T7 primers and the Catalyst 800
Molecular Biology Labstation (Applied Biosystems, Division of
Perkin-Elmer, Foster City, CA). The products were analyzed on an
Applied Biosystems automatic sequencer 373A, and sequences were
compared to the known human FP receptor sequence (13) with MacMolly
Tetra (Soft Gene, Berlin, Germany) on a Macintosh personal computer
(Apple Computer, Cupertino, CA).
RNA isolation and Northern and dot blot analysis
Cytoplasmic RNA was isolated with the modified Nonidet P-40
lysis procedure (22) and quantitated by absorbance at 260 nm. For dot
blots, 1–2 µg RNA were denatured in 7.5% formaldehyde and 6 x
SSC (1 x SSC = 0.15 M NaCl and 0.015
M sodium citrate, pH 7.0) at 50 C for 30 min and spotted
onto Hybond-N nylon membranes (Amersham International, Aylesbury, UK)
with a 96-well Minifold device (Schleicher and Schuell, Keene, NH). For
Northern blots, 10–20 µg RNA were denatured in 1 M
glyoxal, 50% dimethylsulfoxide, and 10 mM phosphate buffer
at 50 C for 60 min, electrophoresed through an 1.2% agarose gel, and
transferred to nylon membranes, which were then UV irradiated for 6 min
with a Reprostar II UV illuminator (Camag, Muttenz, Switzerland).
Linear amplification labeling was performed as previously described
(20) to obtain single stranded human FP receptor cDNA probe. The
labeling mix (10 µl) contained 10 ng cDNA [either a purified 490-bp
restriction enzyme fragment of the cloned human FP receptor cDNA or,
alternatively, a purified 1.6-kilobase (kb) fragment of human FP
receptor cDNA obtained from Merck Frosst Canada, Pointe Claire-Dorval,
Quebec]; 1 µM FP receptor antisense primer; 50
µM each of deoxy (d)-ATP, dGTP, and dTTP; 1.6
µM [-32P]dCTP (3000 Ci/mmol; DuPont-New
England Nuclear, Boston, MA; or Amersham); 1 µl of 10 x PCR
buffer (Perkin-Elmer, Norwalk, CT); and 0.25 U AmpliTaq (Perkin-Elmer).
A purified 0.8-kb cDNA fragment of human GAPDH was labeled using
[-32P]dCTP and the Prime-a-Gene kit (Promega, Madison,
WI). Probes were purified with nick columns (Pharmacia) and used at
1–3 x 106 cpm/ml in hybridization solution
containing 50% formamide, 6 x SSC, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 0.1% BSA, 100 µg/ml herring sperm DNA, 100
µg/ml yeast RNA, and 0.5% SDS at 42 C for 16 h. Filters were
washed three times with 0.1–1 x SSC and 0.1% SDS at 50 C. Dot
blots were quantitated with Fujifilm IP-Reader Bio-Imaging Analyzer BAS
1500 (Fuji Photo Co., Tokyo, Japan) and the MacBas software supplied by
the manufacturer. Northern blots were visualized by
autoradiography.
Binding assays
GL cells were maintained in six-well dishes for 4–5 days and
then treated with or without hCG (30 ng/ml), 8-bromo-cAMP (1
mM), or PMA (10 ng/ml) for 24 h and in the time-course
experiment with hCG for 0, 3, 9, and 30 h. Binding assays were
performed as described by Lake et al. (9) with slight
modifications. Briefly, intact GL cells were incubated in 2 ml binding
buffer [50 mM Tris-HCl (pH 5.8), 2.5 mM
MnCl2, and 1 µM indomethacin (Sigma)]
containing 5 nM [3H]PGF2 (170
Ci/mmol; DuPont-New England Nuclear) at room temperature for 45 min.
Nonspecific binding was determined in the presence of 10
µM PGF2 (Sigma), and the displacement
experiment was performed with 1 nM to 10 µM
PGF2. After the incubation, the cells were washed three
times with ice-cold binding buffer and lysed with 0.5 ml ice-cold 0.5
M NaOH. Radioactivity of the lysate was then quantitated by
liquid scintillation counting. Nonspecific binding was first subtracted
from all values, and the difference between total and nonspecific
binding to untreated cells is shown as 100%.
Statistical analysis
Statistical significance was calculated for a single comparison
with Student’s t test. For multiple comparison, the
t test was used only if one-way ANOVA indicated a
significant difference. All results are shown as the mean ±
SEM, and P < 0.05 was selected as the
statistically significant value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The sequence of the RT-PCR product was (>99%)
identical with the known human FP receptor sequence (13).
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). To study more precisely regulation of FP receptor
mRNA expression, we performed Northern and dot blot hybridization
analyses with cytoplasmic RNA isolated from the GL cells. A 24-h
treatment with hCG did not induce expression of the FP receptor mRNA
during the first or second days of culture, but caused maximal
stimulation after culture for 3–7 days (Fig. 2). In
addition to the 5.2-kb major transcript, minor mRNA species of
approximately 4.5 and 1.6 kb were also detected. Additional experiments
were conducted with cultures grown for 4–6 days.
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. In time-course experiments with hCG (Fig. 4A) and 8-bromo-cAMP (Fig. 4B), expression of the FP
receptor mRNA was inhibited at early (2–8 h) time points and
stimulated when incubated for longer (24–48 h) periods. The effect of
hCG was suppressed by an inhibitor of protein synthesis (CHX; Fig. 5). Interestingly, CHX alone stimulated the expression
of the FP receptor transcript (Fig. 5).
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). Further, it blocked the stimulatory effect of
hCG (Fig. 6B) and 8-bromo-cAMP (Fig. 6C). The effect of PMA as an
inhibitor of 8-bromo-cAMP-induced FP receptor expression was evident at
all time points tested (3, 8, 24, and 48 h; not shown).
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binding
to intact human GL cells and whether this binding can be regulated.
Competition for [3H]PGF2 binding by
nonlabeled PGF2 showed that these cells contain specific
binding sites for PGF2 (Fig. 7A). The
effect of hCG was time dependent (Fig. 7B), and 8-bromo-cAMP increased
the binding of [3H]PGF2 (Fig. 7C). In
contrast, PMA blocked the stimulatory effect of hCG (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4.5 and
1.6 kb), and other investigators have shown the presence of minor FP
receptor mRNAs of various sizes (8, 9, 12, 14). The nature or
biological significance of these transcript isoforms is presently
unknown. As expression of the FP receptor mRNA may be regulated in a cycle- and pregnancy-dependent manner (12, 14, 16), we investigated the effect of hCG on FP receptor mRNA expression. In cultured human GL cells, hCG did not initially regulate FP receptor mRNA expression, but after several days of culture, hCG produced a concentration-dependent stimulatory response. A similar lag period in the responsiveness of several other hormonal parameters to hCG treatment has been observed in previous studies using this cell culture model (17, 18, 19, 20). The initial unresponsiveness of the GL cells may reflect the down-regulation of LH/hCG receptors or its signaling pathways in response to hormone treatment received by the granulosa cell donors. Another possibility is that the granulosa cells luteinize in cell cultures, and the induction of FP receptor expression is present only in luteinized cells. The effect of hCG did not, however, appear until after incubation for 24 h, and it was inhibited by CHX, which suggests that the stimulation of FP receptor expression is at least partially dependent on de novo protein synthesis. However, regulation of FP receptor expression is complex, as CHX alone increased steady state levels of the FP receptor mRNA. This may indicate that the regulatory mechanism is transcriptional activation (23) or posttranscriptional stabilization of the mRNA (24), or both (21).
Compared to hCG, 8-bromo-cAMP stimulated FP receptor mRNA expression with similar kinetics, which suggests that the effect of hCG is mediated via adenylyl cyclase. As the LH/hCG receptor couples to both adenylyl cyclase and phospholipase C, and thus hCG could stimulate protein kinase C (3), we investigated the effect of PMA, a known modulator of protein kinase C activity, on FP receptor expression. PMA inhibited basal FP receptor mRNA expression and blocked the effects of hCG and 8-bromo-cAMP at all time points tested. However, PMA both stimulates and, during prolonged treatments, down-regulates certain protein kinase C isoforms (25). As the effect of PMA was evident as early as after 3 h of incubation, at a point when there should be minimal down-regulation, this may suggest that activation of protein kinase C down-regulates expression of the FP receptor. Further, the stimulation of mRNA expression by hCG and 8-bromo-cAMP and its inhibition by PMA correlated with binding of the radiolabeled ligand. This suggests that the FP receptor mRNA was translated to a functional FP receptor protein.
FP receptor transcript levels are high in the midluteal phase,
variable during the late luteal phase, and low during both natural and
PGF2-induced luteolysis in sheep (12, 26). Similarly,
bovine FP receptor transcripts were expressed at increasing levels from
the early phase to the late phase of the estrous cycle and were reduced
during luteolysis (14). Also, large amounts of the transcript were
found in the corpus luteum during early and middle pregnancy, but the
levels were reduced in late pregnancy (14). Recently, it was reported
that bovine preovulatory follicles contain low concentrations of the FP
receptor transcripts, which were maximally induced 48 h after
ovulation, and these high amounts were maintained in the midluteal
phase (16). Consistent with our observations, the level of expression
of FP receptor mRNA was elevated in response to hCG and forskolin in
bovine GL cell cultures as detected by competitive RT-PCR (16).
However, phorbol didecanoate was without an effect in this model (16).
Similar to our results, ewes injected with PMA showed decreased levels
of the FP receptor mRNA in the corpus luteum, as assessed by slot blot
hybridization (15). It was also reported that LH increased and
PGF2 decreased expression of the receptor (15).
Fluprostenol, a selective FP receptor agonist, also decreased
expression of FP receptor mRNA in rodent osteoblastic cells, as
detected by RT-PCR (27).
These findings suggest that factors released during and after ovulation
and agents produced during the first half of pregnancy induce the
expression of FP receptor mRNA. In contrast, factors associated with
luteolysis may down-regulate expression of the transcript. Our results
suggest that signals transduced through the LH/hCG receptor and
subsequent activation of adenylyl cyclase stimulate FP receptor
expression. It is possible that the cycle-dependent expression is
induced by LH and, during pregnancy, by hCG. As PMA down-regulated FP
receptor mRNA expression, in vivo this down-regulatory
signal could possibly be PGF2 itself, because
PGF2 and PMA share partly similar signal transduction
pathways (4, 5).
The regulation of prostanoid production plays an important role in
several physiological and pathological conditions. Our data suggest
that modulation of FP receptor expression may regulate ovarian
responsiveness to PGF2. To better understand the
biological significance of the present findings, additional studies are
in progress to determine the mechanisms involved in the regulation of
expression of FP receptor mRNA.
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Acknowledgments |
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Footnotes |
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Received June 11, 1997.
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References |
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luteum. Biol Reprod 54:1096–1102[Abstract]
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