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Subunit Gene Expression
Department of Biochemistry, Molecular Biology, and Cell Biology
(A.M., K.E.M.) Northwestern University Evanston, Illinois
60208
Department of Physiology and Biophysics (J.U.)
Chicago Medical School North Chicago, Illinois 60064
Institut de Genetique et de Biologie Moleculaire et
Cellulaire (P.S.-C.) Centre National de la Recherche
Scientifique-INSERM-ULP BP163, 67404 Ilkirch, Strasbourg,
France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit gene
expression that occurs just before ovulation. We found that the
predominant CREM mRNAs in the ovary correspond to previously described
internal transcripts of the CREM gene that encode the inducible cAMP
early repressor (ICER). ICER mRNAs are strongly induced in the ovary by
exogenous gonadotropins in immature rats and are transiently expressed
in the ovary immediately after the preovulatory LH surge in adult
cycling rats. Although ICER is expressed in multiple ovarian cell
types, expression in granulosa cells is observed only in response to LH
stimulation. ICER mRNAs are also induced by the activation of
cAMP-signaling pathways in cultured primary granulosa cells. To
determine whether ICER can act as a functional repressor to modulate
potential target genes such as the inhibin -subunit gene, an ICER
expression construct was transiently cotransfected into a granulosa
cell line along with an inhibin -subunit promoter-luciferase
reporter gene. Both basal and cAMP-induced expression of the inhibin
-subunit promoter were suppressed by ICER. These studies reveal that
CREM, a tissue-specific factor, is expressed and regulated by
gonadotropins in the ovary, that the predominant CREM transcripts
encode the repressor protein ICER, and that ICER is capable of
inhibiting cAMP-induced expression of the inhibin -subunit gene. Our
findings are consistent with a role for repressors such as ICER in
mediating the suppression of inhibin -subunit gene expression that
occurs in the ovary at the time of the preovulatory LH surge. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit of inhibin, a dimeric gonadal hormone that, in
turn, acts to suppress the synthesis and secretion of pituitary FSH
(1). Another FSH target gene is that encoding the LH receptor (2, 3),
and once the mature ovarian follicle acquires functional LH receptors,
the preovulatory LH surge on proestrus afternoon triggers the
morphological and biochemical changes associated with ovulation of the
oocyte and luteinization of the remaining follicle cells. In response
to the LH surge, the inhibin -subunit gene is rapidly
down-regulated, leading to decreased inhibin secretion and providing an
environment permissive to the prolonged elevation (the secondary surge)
of FSH secretion that occurs on estrus morning and is critical for the
recruitment of a new cohort of ovarian follicles (for reviews see Refs.
1, 4, and 5).
This basic pattern of FSH stimulation and LH repression during the
estrous cycle is common to a number of genes expressed in the ovary,
including aromatase (6), the LH receptor (2), the FSH receptor (7), and
the inhibin - and ß-subunits (8). Other genes are similarly
induced by FSH in ovarian granulosa cells, but their expression remains
elevated after the LH surge and during the process of luteinization
(9). Thus, determining the molecular mechanisms through which
gonadotropins exert these effects on gene expression is important for
understanding the normal control of the ovarian cycle and promises to
lend insight into disruptions of this control that might be associated
with reproductive dysfunction, such as infertility.
The gonadotropins FSH and LH act through stimulatory G protein-coupled receptors expressed on target cells (10, 11) and transduce their signal, at least in part, by the activation of adenylyl cyclase and the production of the second messenger cAMP (12, 13). Recent findings suggest that the gonadotropins can also act through signaling pathways that lead to the activation of protein kinase C (14, 15) or intracellular tyrosine kinases (16). While it is not known how FSH and LH might differentially regulate gene expression, the number of LH receptors far exceeds the number of FSH receptors on the granulosa cells of preovulatory follicles (17), and the levels of intracellular cAMP generated by FSH in small antral follicles are much lower than those generated by LH in preovulatory follicles (18, 19, 20), suggesting that the magnitude of the go-nadotropin-induced cAMP signal might regulate the resultant transcriptional responses.
Many of the transcriptional responses to cAMP are thought to be mediated by a family of cAMP-responsive factors that belong to the larger superfamily of bZip proteins, so named for the basic region and leucine zipper motifs that form the DNA binding and dimerization domains of these proteins (21, 22, 23). Perhaps the best characterized of these factors are the cAMP-response element (CRE)-binding protein (CREB) (24, 25, 26) and the CRE-modulatory protein (CREM) (27, 28, 29). These proteins have a very similar domain structure, and both CREB and CREM are expressed as a family of isoforms that can act as cAMP-regulated stimulators or repressors of gene transcription (26, 27, 30, 31, 32). CREB is thought to be fairly ubiquitously expressed, whereas CREM expression is tissue-specific and is highly regulated in neuroendocrine tissues and cell types (27).
Alternative RNA processing leads to the formation of mRNAs that encode
diverse isoforms of the CREB and CREM proteins. The full-length forms
of these proteins, which include a kinase-inducible (phosphorylation)
domain and one or more glutamine-rich domains with transactivation
function, act as transcriptional activators. For example, CREB 341 and
CREB 327 are strong activators of transcription (33, 34), and CREM 
can augment CREB-mediated activation of cAMP-responsive genes (30).
Shorter isoforms of these proteins that include the DNA-binding and
dimerization domains but lack the kinase-inducible or transactivation
domains can act as transcriptional repressors. The proteins I-CREB
(35), CREM , ß, 
, and S-CREM (27, 32) are examples of such
repressor isoforms. These repressor proteins might act either by
occupying CRE sites in target gene promoters as transcriptionally
inactive homodimers or by forming inactive heterodimers with CREB or
other transcriptional activators (32, 36, 37, 38). One of the best
characterized of these repressor isoforms, and the focus of this study,
is the inducible cAMP early repressor (ICER) (39, 40). The ICER mRNA is
transcribed from an intronic promoter of the CREM gene and encodes a
protein that includes DNA-binding and dimerization domains but lacks
the kinase-inducible and transactivation domains, and thus acts as a
potent repressor. The intronic promoter that regulates ICER expression
is itself cAMP responsive, and ICER therefore autoregulates its
expression. ICER has been proposed to be a key signal that is
transiently induced to attenuate cAMP-dependent signaling pathways
(39).
We reported previously that the full-length activator isoform of CREB
is expressed in the rat ovary and that the pituitary gonadotropins do
not regulate CREB gene expression but rather stimulate CREB
phosphorylation, leading to its activation (41). We also demonstrated
that CREB is a key mediator of FSH-stimulated inhibin -subunit gene
expression in the ovarian granulosa cell (42). Given the increasing
evidence for modulatory effects of CREM isoforms on CREB-mediated gene
transcription, we wanted to test the hypothesis that CREM isoforms play
a role in attenuating the transcriptional response of the inhibin
-subunit gene to gonadotropins and cAMP in ovarian granulosa cells.
In the present study we investigate the expression and regulation of
CREM/ICER mRNA and protein in immature rats treated with exogenous
gonadotropins, in adult rats during the estrous cycle, and in cultured
rat granulosa cells treated with hormones in vitro. We also
test directly the effect of ICER overexpression on inhibin -subunit
gene promoter activity in a granulosa cell line. Our findings are
consistent with an important role for CREM/ICER in mediating the
repression of the inhibin - subunit gene that is observed in the
ovary immediately after the preovulatory LH surge.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 form of CREM, as shown
schematically in Fig. 1
. Figure 2 indicates that CREM mRNA is induced in
the ovary within 4 h of PMSG treatment and returns to basal levels
by 48 h after PMSG treatment. In PMSG-primed animals, CREM mRNA is
strongly induced within 1 h of human CG (hCG) treatment and again
returns to basal levels by 12 h after hCG treatment.
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2 5'-probe corresponding to the kinase-inducible
and glutamine-rich domains was used to detect full-length forms of CREM
(all probes are indicated in Fig. 1
). As shown in Fig. 3, the ICER-specific mRNA expression
pattern in the hormonally treated rat ovary corresponded closely to
that observed previously using the full-length CREM probe. In contrast,
transcripts hybridizing to the 5'-CREM probe were expressed at very low
levels and were not induced by gonadotropins in the immature rat ovary
(Fig. 3). Thus, inducible CREM expression in the ovary appears to
represent a selective activation of the internal P2 promoter leading to
the generation of ICER mRNAs.
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, ICER transcripts of 1700 and 1900
nucleotides were observed only at 1800 and 2000 h on proestrus.
These times correspond well to the preovulatory LH surge, which began
at 1600 h and peaked at 2000 h on proestrus (Fig. 4B).
Treatment of the animals with either pentobarbitol or a GnRH antagonist
(WY-45760), which block the preovulatory LH surge (43, 44, 45), also
abolished ICER mRNA induction at 1800 h proestrus (Fig. 4C). These
data indicate that, in the ovary, ICER is rapidly induced and then
down-regulated in response to the LH surge in animals progressing
through a normal reproductive cycle.
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, ICER
mRNA is localized predominantly to thecal and interstitial cells after
PMSG stimulation. In contrast, after hCG stimulation, ICER mRNA is also
localized to the granulosa cells of most large preovulatory follicles.
Interestingly, in all cases, ICER mRNA was found to colocalize with LH
receptor mRNA (Fig. 5). This suggests that the induction of ICER by
PMSG in thecal and interstitial cells may be mediated by the weak LH
activity of PMSG as opposed to it predominant FSH-like properties
(46).
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. As shown in Fig. 6, ICER mRNA is
induced in the thecal and interstitial cell-enriched fraction by both
PMSG and hCG treatment, whereas ICER mRNA is induced in the granulosa
cell-enriched fraction only after hCG treatment. Figure 6 also
indicates that four predominant ICER transcripts were detected by
RT-PCR. Each of these was cloned and sequenced, and this analysis
indicated that they correspond to the four previously characterized
forms of ICER, designated I, I, II, and II (39). The exon
structures of these four ICER isoforms are schematically shown in Fig. 1.
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, all four of the ICER transcripts
described in the previous section were detected. These transcripts
could be induced by a high dose of recombinant human FSH in the naive
cells, by a lower dose of hCG in the primed cells, or by the
nonspecific agent forskolin in both cell populations. In the
PMSG-primed cells treated with hCG in vitro, ICER expression
was highly elevated at 1 h, decreased substantially at 4 h,
and returned to basal levels at 12 h after hormone addition (Fig. 7).
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demonstrates that both recombinant FSH
and hCG induce ICER mRNAs in these cells, and this induction is
inhibited by greater than 90% by H89. Thus, ICER mRNAs are rapidly and
transiently induced through a PKA-dependent mechanism in GRMO2
granulosa cells.
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-Subunit Gene Repression-subunit gene repression after LH administration was examined using
the immature rat model. RNAs prepared from granulosa cells that had
been isolated from the ovaries of rats treated with PMSG for various
times or treated with PMSG for 48 h followed by hCG for various
times were used to assess ICER and inhibin -subunit mRNA expression.
As shown in Fig. 9, the levels of inhibin
-subunit mRNA increased in response to PMSG stimulation and peaked
at 48 h of hormone treatment. After hCG administration, inhibin
-subunit mRNA was substantially decreased by 4 h and had
returned to basal levels by 12 h of hormone treatment. In these
same RNA samples, ICER mRNAs were not affected by PMSG treatment, were
strongly induced within 1 h of hCG treatment, and thereafter were
down-regulated. Thus, there is a tight temporal correlation between the
onset of ICER expression and the hCG-induced suppression of inhibin
-subunit gene expression.
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, in the thecal-interstitial
fraction three protein species ranging from 17–20 kDa were induced at
low levels within 4 h of PMSG treatment and declined to basal
levels by 48 h after PMSG treatment. Subsequent hCG treatment
rapidly induced ICER proteins in these cells. ICER protein expression
peaked at 4 h after hCG treatment and was down-regulated by
12 h after hCG treatment. In the granulosa cell fraction, shown in
Fig. 10B, ICER proteins were also induced at 4 h after hCG
treatment and then down-regulated (PMSG alone had no effect on
granulosa cell ICER protein expression, consistent with the mRNA
results). These results indicate that ICER protein is maximally
expressed at 4 h after hCG stimulation, corresponding closely to
the time when inhibin -subunit mRNA begins to decrease.
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-subunit promoter,
electrophoretic mobility shift assays were performed. A radiolabeled
double-stranded oligonucleotide probe corresponding to the inhibin
-subunit CRE was incubated with whole cell extracts from GRMO2 cells
that were transiently transfected with an ICER I isoform expression
construct using a vaccinia virus-T7 RNA polymerase-based system (50).
As shown in Fig. 11, this extract
produced a shift of the -inhibin CRE probe that could be competed
with an excess of the unlabeled -inhibin CRE or a consensus CRE, but
not with a nonspecific oligonucleotide. This band was not observed when
extracts from nontransfected GRM02 cells were used. When an antibody
against the CREM protein (which detects ICER) was included in the
incubation, a supershifted complex of lesser mobility was observed,
indicating that ICER is a component of the protein complex formed on
the inhibin -subunit CRE.
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-subunit gene, the same ICER I expression construct
described above was used. This construct, or an antisense control, was
cotransfected into the GRMO2 granulosa cell line along with an inhibin
-subunit promoter-luciferase reporter construct, and basal and
forskolin-induced luciferase activity was measured. Figure 12A shows the structures of the two
constructs used, while Fig. 12B shows the luciferase activities
normalized for total protein. As expected, the inhibin -subunit
promoter was induced by forskolin in these cells, although the
induction is modest at this early time after forskolin stimulation (4
h). The sense ICER construct substantially reduced both basal and
forskolin-induced promoter activity, while the antisense ICER construct
had essentially no effect. These studies indicate that ICER-I can act
as a functional repressor of basal and cAMP-stimulated inhibin
-subunit gene expression in granulosa cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit gene.
Unlike CREB, which is fairly ubiquitously expressed, CREM and its
isoforms are expressed in a tissue- and cell-specific manner (27). In
particular, CREM isoforms are often highly expressed and regulated in
neuroendocrine tissues and cells, with well studied examples being the
pineal gland (40, 61) and the testis (30, 37, 62). We therefore sought
initially to establish whether CREM was expressed in the ovary and
whether its expression was regulated. Using several different animal
models (immature female rats treated with exogenous gonadotropins or
adult female rats progressing through the estrous cycle) we found that
CREM is expressed in the ovary and that CREM mRNA is induced by the
gonadotropins, particularly hCG or LH. Our data also indicate that the
inducible CREM transcripts found in the ovary correspond to mRNAs
encoding the previously reported isoforms of the repressor ICER, and
that ICER mRNAs are expressed in several cell types in the ovary but
are selectively induced in the granulosa cells of preovulatory
follicles in response to hCG or LH. We also found that ICER proteins
are induced in the ovary in response to the gonadotropins and that
maximal protein expression is delayed several hours beyond RNA
expression, as expected. Using primary cultures of rat granulosa cells
or the immortalized mouse granulosa cell line GRMO2, we demonstrate
that ICER gene expression can be induced by a variety of treatments
known to increase intracellular cAMP levels, and that ICER induction
involves the actions of PKA, as assessed by sensitivity to the PKA
inhibitor H89. Finally, there is a very strong temporal association
between ICER mRNA and induction and inhibin -subunit mRNA
down-regulation in response to hCG treatment in immature rats or the
preovulatory LH surge in adult cycling rats.
The above studies indicate that ICER is induced in the appropriate cell
type, the granulosa cell, and at the appropriate time, after the LH
surge, to play a role in mediating the down-regulation of
FSH-stimulated ovarian genes such as the inhibin -subunit gene. To
test directly whether ICER is able to impact the regulation of the
inhibin -subunit gene, we first demonstrated that ICER can interact
with the nonconsensus CRE found in the inhibin -subunit promoter
using gel mobility shift and antibody supershift approaches. We next
overexpressed ICER in a granulosa cell line and assessed the effect of
excess ICER on inhibin -subunit gene expression. Our finding that
ICER can substantially repress both basal and cAMP-stimulated
expression of this gene in vitro is supportive of a direct
role for ICER in the regulation of inhibin -subunit gene expression
in vivo.
Based on our previous studies of gonadotropin-induced CREB
phosphorylation in the ovary (41) and the findings presented here on
gonadotropin-induced ICER expression, we can propose a model for how
FSH and LH might exert their opposing actions on inhibin -subunit
gene expression in ovarian granulosa cells. During the follicular phase
of the estrous cycle, FSH stimulates the proliferation of ovarian
granulosa cells, and this is accompanied by increasing expression of
the inhibin -subunit gene. FSH, by stimulating intracellular cAMP
production and activating PKA, also induces the phosphorylation of CREB
on serine 133, making it competent to transactivate CRE-containing
target genes such as the inhibin -subunit gene (41, 42). Once
ovarian granulosa cells acquire functional LH receptors, the
preovulatory LH and FSH surges trigger granulosa cell differentiation
and subsequent luteinization. The LH surge, through cAMP and
PKA-dependent pathways, also transiently induces the transcription of
the four ICER mRNAs. We propose that one function of ICER is to rapidly
repress the transcription of CRE-containing and CREB-activated target
genes such as the inhibin -subunit gene. ICER itself is then
down-regulated through an autoregulatory mechanism, thus completing a
cycle of cAMP-dependent gene activation and repression (39).
A question of primary importance with respect to this model is why FSH
and LH, both acting predominantly through cAMP-dependent mechanisms,
have such divergent actions? One likely answer is provided by our data
showing that FSH (PMSG) treatment induces ICER in thecal and
interstitial cells, but not in granulosa cells. Whether this represents
the weak LH-like activity of PMSG on luteal and interstitial cell LH
receptors or a direct FSH effect is not known, but in either case ICER
is not induced in granulosa cells in vivo by this hormonal
treatment associated with granulosa cell proliferation and inhibin
-subunit mRNA induction. A second likely answer comes from studies
on ICER activation by cAMP signaling. The CREM intronic P2 promoter
includes four clustered CRE-like elements termed CAREs, or
cAMP-autoregulatory elements (39). These elements mediate cAMP
induction through activating factors such as CREB, and subsequently
bind ICER, resulting in an autorepression of ICER expression and a
resetting of the system. Because several of the CAREs are fairly poor
consensus CRE elements, it is possible that they are occupied by
activators only when the cAMP-PKA-signaling pathway is fully activated.
The magnitude of the intracellular cAMP signal generated in the
granulosa cells of preovulatory follicles by the LH surge is much
larger than that generated by FSH in the granulosa cells of maturing
follicles (19, 20), providing a potential mechanism for the selective
activation of ICER by LH in vivo. Our studies showing that
ICER expression can also be induced in vitro by high doses
of recombinant FSH are consistent with the idea that the magnitude of
the cAMP signal is a critical determinant of the ICER response.
The CREM gene has been disrupted by homologous recombinant in embryonic
stem cells, and the homozygous mutant mice generated from these cells
lack both CREM and ICER expression (63, 64). The male mutant mice
exhibit severe defects in spermatogenesis, consistent with a proposed
role of CREM and ICER in this process (63, 64). Paradoxically, the
female mutant mice are reported to be fertile, although detailed
studies of reproductive phenotypes in the female have not yet been
reported. This finding might suggest that ICER is not critical as a
transcriptional regulator during LH-induced ovulation and luteinization
in the rodent ovary. An alternative explanation is that other factors
are able to compensate for the loss of ICER expression in the ovary.
For example, similar repressor forms of the CREB gene are expressed
through alternative RNA processing mechanisms in the testis (35). While
we did not detect RNAs for these repressor forms of CREB in the ovary
of normal animals (41), they might be induced in the ovary in the
absence of ICER in the mutant animals and serve a similar function. It
is also unlikely that ICER is the only factor involved in the
repression of cAMP-induced gene expression in response to the LH surge.
Indeed, ICER expression in granulosa cells is very transient, whereas
there is an extended down-regulation of target genes such as the
inhibin -subunit gene, which remains repressed in luteinized cells
in the rodent ovary (5, 65). It has recently been shown that LH
treatment leads to a down-regulation of steroidogenic factor-1 (SF-1)
protein in rat granulosa cells, and this appears to be an important
part of the mechanism that underlies the down-regulation of rat
aromatase CYP19 gene expression (66). The inhibin -subunit gene
promoter contains several consensus SF-1 binding sites, and SF-1 is
likely to be involved in its regulation in granulosa cells. An
additional factor that is likely to be important for down-regulation of
cAMP-regulated genes is the bZip protein C/EBPß (67). Recent studies
indicate that female mice homozygous for a disruption of the C/EBPß
gene are infertile and exhibit defects in ovulation (68).
Interestingly, several genes that are normally down-regulated by the LH
surge, including aromatase and prostaglandin endoperoxide synthase-2
(PGS-2), fail to be down-regulated in the ovaries of the C/EBPß
mutant mice (68). C/EBPß mRNA is rapidly induced by hCG in granulosa
cells (66), but peak levels of C/EBPß mRNA in preovulatory follicles
are not observed until 4–7 h after hormone administration (68). Thus,
C/EBPß might be critical for the longer term repression of this class
of genes, whereas ICER might play a more predominant role in the
immediate early responses to the LH surge. Further studies of the roles
of transcription factors such as SF-1 and C/EBPß in inhibin
-subunit gene expression should be informative in this regard.
The dephosphorylation of CREB attenuates its transcriptional activity (69, 70), and this represents another important mechanism for suppressing cAMP-induced transcriptional responses. However, we have previously reported that hCG rapidly stimulates CREB phosphorylation in PMSG-primed rat granulosa cells (41), suggesting that CREB dephosphorylation cannot fully explain the ability of LH to attenuate FSH-induced gene expression in granulosa cells. Furthermore, ICER mRNA induction requires CREB (71), indicating that CREB is likely to be phosphorylated and active at the time of the LH surge. Recent studies in pituitary AtT20 cells have investigated the relative contributions of CREB dephosphorylation and ICER induction in attenuating cAMP-induced transcriptional responses and also addressed the important concept of the dynamics of cAMP-induced transcriptional responses (72). These experiments and others define a refractory period after the initial stimulation of cAMP-dependent genes that is determined in part by the duration of the initial stimulus (73, 74). These findings may have strong parallels in the ovary that would help to explain our data showing that while the primary gonadotropin surges on proestrus evening strongly induce ICER expression, the secondary FSH surge early on the morning of estrus does not. The intense and sustained primary LH surge that induces ICER expression might, in addition to initiating the events that lead to target gene repression, serve to make granulosa cells refractory to further ICER induction in response to the secondary FSH surge, thus allowing FSH to act in a stimulatory fashion to induce the gene expression and cell proliferation events that are critical to the recruitment and maturation of a new cohort of ovarian follicles. It seems reasonable to speculate that cycles of gonadotropin-induced and cAMP-mediated induction and attenuation of transcriptional responses may be a key mechanistic component for maintaining the cycles of ovarian follicular development characteristic of reproduction in many mammals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Granulosa Cell Cultures
Immature 21- to 23-day old female rats with or without PMSG
treatment (10 IU, sc, for 24 h) were used for granulosa cell
preparation utilizing follicular puncture essentially as described (42, 76). Ovaries were collected into serum-free medium (4F), which consists
of 15 mM HEPES, pH 7.4, 50% DMEM, and 50% Ham’s F12 with
transferrin (5 µg/ml), human insulin (2 µg/ml), hydrocortisone (40
ng/ml), and antibiotics. After incubating the ovaries at 37 C in 4F
medium containing 0.5 M sucrose and 10 mM EGTA
for 30 min, the ovaries were washed in fresh 4F medium. Individual
follicles were punctured, and the granulosa cells were extruded using a
23 ga needle under a Reichert dissection microscope (Buffalo, NY).
Cells were plated in 4F medium supplemented with 10% FBS (GIBCO BRL,
Grand Island, NY), and incubated in a humidified atmosphere of 5%
CO2 at 37 C. Cultured cells were either untreated or
treated in vitro with 10 µM forskolin (Sigma),
varying doses of hCG, or recombinant human FSH (line 1, sample 329,
NICHD).
Isolation of CREM and ICER cDNAs
Rat CREM cDNAs were cloned from total testicular RNA by RT-PCR
using oligonucleotide primers 1 and 2 (see Fig. 1) corresponding to
nucleotide positions 15–36 and 1115–1135, respectively, of the mouse
CREM cDNA (27). DNA sequence analysis indicated that cDNAs encoding the
and 2 isoforms of CREM protein were obtained. Total RNA isolated
from the ovary of an adult rat at 1800 h proestrus was used to
clone ICER cDNAs by RT-PCR. Oligonucleotide primer 4 corresponding to
nucleotides 1–21 of ICER cDNA (39) and oligonucleotide primer 2 were
used for PCR amplification. All oligonucleotide primers were purchased
from Cruachem (Sterling, VA). The ICER-I expression construct was made
by cloning the ICER-I RT-PCR product (see Fig. 1) into the expression
vector pcDNA-3 (Invitrogen, San Diego, CA), which includes the human
cytomegalovirus (CMV) promoter and 3'-polyadenylation sequences from
the bovine GH gene.
RNA Isolation and Analysis
RNA from ovaries or granulosa cells was isolated by
homogenization in 4 M guanidine isothiocyanate containing
25 mM sodium citrate, 0.5% sarkosyl, and 7
µM ß-mercaptoethanol and extraction with acid-phenol
(77) or by centrifugation through CsCl gradients. For RT-PCR assays,
total ovarian RNA was reverse transcribed using avian myeloblastosis
virus (AMV) reverse transcriptase in the presence of
deoxynucleosidetriphosphates (1 mM) and random hexameric
oligonucleotides, and aliquots of this cDNA were amplified by PCR with
incorporation of [32P]dCTP into the PCR product as
described previously (78). PCR primers 2 and 4 (see Fig. 1) and primers
specific for the inhibin -subunit (79) were used. Rat ribosomal
protein L-19 (80) was used as an internal control in all experiments.
The PCR products were separated by size using electrophoresis on 5%
polyacrylamide gels, the gels were dried, and PCR products were
visualized by autoradiography on X-OMAT-AR film (Eastman Kodak Company,
Rochester, NY) and quantified using a PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). For Northern RNA blot
analysis, 20 µg total RNA were separated by size using
electrophoresis in a 1.2% agarose gel containing 1x
3-[N-morpholino]propane-sulfonic acid (MOPS) buffer and
6% formaldehyde (81) and visualized by acridine orange staining.
Size-fractionated RNA was transferred to Biotrans Nylon membrane (ICN,
Irvine, CA), immobilized on the membrane by UV cross-linking and baking
at 80 C for 30 min. A 47-nucleotide antisense oligonucleotide, primer 3
(see Fig. 1) corresponding to nucleotide positions -1 to -47 (39)
unique for ICER was end-labeled with [32P]dCTP and
terminal deoxynucleotide transferase and used as the hybridization
probe. The blots were washed at a final stringency of 0.1x saline
sodium citrate (SSC)/0.01% SDS at 65 C. ICER mRNA was visualized by
autoradiography on X-OMAT-AR film (Eastman Kodak). Radioactive signal
from the blot was removed by washing in 10 mM sodium
phosphate (pH 6.5) in 50% formamide at 65 C for 1 h, followed by
2x SSC/0.1% SDS at room temperature for 15 min. The blot was then
hybridized to a ribosomal protein S2 (82) probe, prepared by random
priming, as a control to compare the amounts of RNA loaded and
transferred.
In Situ Hybridization
Sections (20-µm) of frozen ovaries were prepared using a
Reichert 820 cryostat and mounted onto gelatin and poly
L-lysine-coated glass slides for in situ
hybridization as described previously (78). Hybridization probes used
were [35S]UTP or [33P]UTP-labeled
riboprobes derived from the full-length 2 CREM cDNA clone, a
5'-NcoI subclone (see Fig. 1) of rat CREM 2, a rat
LH receptor cDNA subclone, or a [35S]dATP-labeled
47-nucleotide long antisense oligonucleotide that is unique to ICER
isoform (primer 3 in Fig. 1). Hybridization was continued for 12–18 h
at 47 C in a humidified chamber. Sense riboprobes were used as
controls. Subsequently, the slides were washed to a final stringency of
0.1x SSC at 65 C after a 1-h treatment with 20 µg/ml RNAse at 37 C.
Slides were then processed for emulsion autoradiography (NTB-2, Eastman
Kodak). Exposure time on emulsion was 2 weeks. After development of the
slides, they were stained with hematoxylin to visualize nuclei. The
sections were then examined and photographed using a macroscope (Wild
M420, Leica, Heerbrugg, Switzerland) or a Nikon Optiphot microscope
(Nippon Kogaku, Inc., Garden City, NY).
Preparation of Protein Extracts and Western Blot Analysis
Protein lysates were prepared in lysis buffer (50 mM
Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150
mM NaCl, 1 mM EGTA, 1 mM
phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, leupeptin,
aprotinin, 1 mM NaF). For cultured cells, after the cells
were washed with PBS and collected by low-speed centrifugation, they
were resuspended in lysis buffer and incubated on ice for 10 min and
then lysed by two cycles of freezing and thawing. The lysates were
centrifuged to remove nuclear debris, and the supernatant was collected
and frozen at -80 C until further use. Frozen tissue samples were
pulverized in dry ice and homogenized in lysis buffer using six to
eight strokes with a Dounce homogenizer. This homogenate was subjected
to two cycles of freezing and thawing and processed as described above.
Protein concentrations were estimated using a Bradford colorimetric
assay (Bio-Rad, Richmond, CA).
Protein lysates (100 µg) were boiled for 5 min in denaturing sample buffer and size separated on a 10–18% continuous gradient SDS-polyacrylamide gel, and proteins were transferred to nitrocellulose (BA-85, Shleicher & Schuell, Keene NH). The membrane was washed with water and blocked with 3% nonfat dry milk in PBS (blocking buffer) for 30 min at room temperature with shaking. The blot was then incubated with primary antibody (anti-CREM at 1:250) in blocking buffer for 48 h at 4 C with gentle shaking. The blot was washed two times with water and once with PBS followed by a 45-min incubation at room temperature with goat antirabbit antibody conjugated to horseradish peroxidase (Promega, Madison WI) in blocking buffer. The blot was then washed twice with water, once with PBS, and once with PBS containing 0.05% Tween 20. The blot was rinsed with water, and antibody-antigen complexes were visualized using an enhanced chemiluminescence system (ECL kit, Amersham. Little Chalfont, Buckinghamshire, U.K.).
GRMO2 Cell Culture, Transfection, and Luciferase Assays
GRMO2 cells (provided by N.V. Innogenetics, Ghent, Belgium)
were cultured as described (47, 48) in HDTIS (DMEM-F12 1:1, 10 µg/ml
insulin, 5 nM sodium selenite, 5 µg/ml transferrin)
supplemented with 2% FBS and sodium pyruvate (100 mg/liter) in a
humidified incubator at 37 C and 5% CO2. GRMO2 cells were
either untreated or treated in vitro with 10
µM forskolin, varying doses of hCG, or recombinant human
FSH with or without the PKA inhibitor H89 (Sigma). Cationic liposomes,
prepared as described previously (83), were used for transient
transfection of GRMO2 cells with DNA (84). Plasmid DNA (2.5 µg) for
each well of a 12-well culture dish was incubated at room temperature
with lipofection reagent for 20–30 min in OptiMEM and added to cells
washed with PBS. After 6 h, the DNA-lipid mixture was replaced
with fresh HDTIS containing 2% FBS, and the cells were incubated for
12 h. Fresh medium, or medium containing 10 µM
forskolin, was then added to the cells. After 4 h of hormone
treatment, the cells were washed with PBS and lysed by gently agitating
on ice in sample buffer (25 mM HEPES, pH 7.8, 15
mM MgSO4, 1 mM dithiothreitol,
0.1% Triton X-100). Luciferase assays were performed essentially as
described (85). Cell lysates (100 µl) were added to 400 µl of assay
buffer (25 mM HEPES, pH 7.8, 15 mM
MgSO4, 5 mM ATP, 1 µg/ml BSA), and 100 µl
of 1 mM luciferin (sodium salt) (Analytical
Bioluminescence, San Diego, CA) were added using an automatic injector;
emitted luminescence was measured using a 2010 luminometer (Analytical
Bioluminescence) for 10 sec. Cell lysates (20 µl) were used for total
protein determination using the Bio-Rad protein assay reagent. For
Western blot analysis and electromobility shift assays, the vaccinia-T7
RNA polymerase hybrid expression system (50) was used. GRMO2 or HeLaT4
cells were infected with vaccinia virus vTF7.3 expressing the
bacteriophage T7 RNA polymerase (obtained under license from Dr.
Bernard Moss, NIH, Bethesda, MD) at a multiplicity of infection of 10
for 30–40 min in PBS/0.1% BSA. Virus was aspirated, and preincubated
DNA-liposome mixture was added to the cells and incubated for 12
h. Cells were immediately harvested and processed for protein
isolation.
Electrophoretic Mobility Shift Assays
A 20-bp double-stranded oligonucleotide probe that spans the
inhibin - subunit CRE sequence was end labeled using
[32P-]ATP and T4 polynucleotide kinase (Promega,
Madison, WI) and purified using a G-25 spin column (Boehringer Mannheim
GmbH, Mannheim, Germany). Whole-cell lysates (5 µg) from
vaccinia-infected transfected cells were used for each binding
reaction. Whole-cell lysates, prepared as described earlier in this
section, were incubated in binding buffer [50 mM Tris-HCl,
pH 7.9, 12.5 mM MgCl2, 10% glycerol (vol/vol),
0.5 mM dithiothreitol, 50 ng/µl poly
deoxy(inosinic-cytidylic)acid] for 20 min at room temperature with 1
ng of 32P-labeled oligonucleotide probe. Where indicated,
100 ng of double-stranded unlabeled competitor oligonucleotide were
added to the binding reaction 5 min before the addition of probe. For
antibody supershift studies, 1 µl of anti-CREM antibody was added to
the reaction, and the reaction was incubated at room temperature for an
additional 20 min. The protein-DNA complexes were resolved on a native
5% polyacrylamide-Tris-borate-EDTA gel. The gel was dried and
exposed to x-ray film (Kodak XAR).
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH P01 Grant HD-21921 and NIH P30 Center Grant HD-28048 (to K.E.M.).
Received for publication October 28, 1997. Revision received March 5, 1998. Accepted for publication March 11, 1998.
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