Stat 5b and the Orphan Nuclear Receptors Regulate Expression of the
2-Macroglobulin (
2M) Gene in Rat Ovarian Granulosa Cells
Maya Dajee,
Georg H. Fey and
JoAnne S. Richards
Department of Cell Biology (M.D., J.S.R.) Baylor College of
Medicine Houston, Texas 77030
Department of Genetics
(G.H.F.) Institute for Microbiology, Biochemistry and Genetics
Friedrich-Alexander University D91058 Erlanger-Nurenberg,
Germany
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ABSTRACT
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2-Macroglobulin (
2M) is a serine protease
inhibitor and cytokine inactivator associated with inflammation and
tissue remodeling. The gene encoding this protein is selectively
induced in the rat corpus luteum by the luteotropic hormone and
cytokine, PRL. The promoter of the
2M gene contains two regulatory
regions that bind a diverse set of transcription factors and confer
functional activity in ovarian granulosa-luteal cells. The PRL
response element (PRLRE) binds PRL-activated (tyrosine-phosphorylated)
signal transducers and activators of transcription (Stat 5b and Stat
5a). 5'-Deletion of the Stat-binding sites or mutation of either one or
both of these sites within the context of the intact promoter abolished
PRL inducibility of
2M promoter-reporter constructs in
granulosa-luteal cells. Cotransfection with a vector expressing a
dominant negative, truncated form of Stat 5b abolished PRL-induced
activation of
2M transgenes. 5'-Deletion of the Stat-binding sites
abolished all promoter-reporter activity in response to PRL. Internal
deletion of a second functional domain 3' of the PRLRE also abolished
PRL inducibility and markedly reduced basal activity, indicating that
functional interactions between these two regions might occur. The
3'-region was shown to bind orphan members of the nuclear receptor
superfamily, steroidogenic factor 1 (SF-1) and chicken ovalbumin
upstream promoter-transcription factor (COUP-TF) and has been called
the orphan receptor response element (ORRE). When site-specific
mutations were made in either the SF-1-binding site or the two COUP-TF
direct repeat (DR1 and DR2) binding sites in the context of the intact
promoter, specific changes in the functional activity of this novel
region of the
2M promoter were observed. Mutation of the SF-1 site
drastically reduced basal activity of the
2M promoter. Mutation of
the COUP-TF sites caused the basal activity of the
2M promoter to
increase markedly. Neither mutation altered the PRL inducibility of
these constructs. Lastly, differentiation of cultured granulosa cells
was required for functional activity of both the PRLRE and the ORRE.
Collectively, these results document for the first time that Stat 5b,
SF-1, and COUP-TF each exert specific effects on the function of the
2M promoter: basal activity is controlled by the balance of SF-1
(positive) and COUP-TF (negative) activities and PRL inducibility is
mediated by activation of Stat 5b. These results add
2M to the list
of nonsteroidal genes regulated by SF-1 in the gonads and provide the
first evidence that COUP-TF has a specific role in regulating ovarian
gene activity. In addition, the ORRE and PRLRE act independently of,
rather than synergistically with, each other to regulate basal and
PRL-induced expression of
2M in ovarian luteal cells.
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INTRODUCTION
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Tissue-specific expression of genes is dependent on the cellular
set of transcription factors as well as the selective activation of
these factors by hormones, growth factors, and cytokines. One
transcription factor that dictates ovarian (gonadal) development is
steroidogenic factor 1 (SF-1), an orphan member of the nuclear receptor
superfamily (1). SF-1 binds as a monomer to (CA)AGGTCA-like motifs and
interacts with other factors to regulate the expression of most steroid
hydroxylase genes (2, 3, 4, 5). During the development of preovulatory
follicles and in granulosa cells in culture, FSH induces expression of
two steroid hydroxylase genes encoding aromatase (CYP19; Ref. 6) and
P450scc (CYP11A; Ref. 7). SF-1 also regulates the expression of genes
encoding peptides, including Mullerian Inhibiting Substance (8)
and oxytocin (9). Thus, SF-1 appears to play a critical role in
regulating genes expressed in differentiated granulosa cells in
response to FSH (6).
When granulosa cells luteinize, they respond to a different set of
extracellular signals, one of which is the cytokine PRL. In the ovary
(10, 11, 12), as in other target tissues (13, 14, 15), PRL activates the JAK
(Janus kinase)/STAT (signal transducers and activators of
transcription) pathway. One gene that is selectively regulated by PRL
in luteal cells encodes
2-macroglobulin (
2M), a large tetrameric
protein that traps and inactivates proteases and cytokines. The
inducibility of this gene by interleukin-6 (IL-6) in rat liver (16) and
by PRL in rat luteal cells (11) has provided a unique opportunity to
delineate the cellular signaling mechanisms used by each cytokine to
control the tissue-specific expression of this gene. In previous
analyses, we have shown that Stat 5 is activated by PRL and binds to
specific
-activating sequences (GAS) sites present in the promoter
of the
2M gene and originally referred to as the IL-6 response
element (IL6-RE) based on the activation of this gene by IL-6 in rat
liver (16). Because a luciferase reporter construct containing 1.2 kb
of the
2M promoter showed high basal activity when transfected into
differentiated granulosa cells in the absence of PRL, we sought to
identify additional factors involved in regulating the promoter of this
gene and the extent to which the high basal as well as PRL inducibility
were dependent on hormone-induced differentiation of granulosa cells in
culture (10).
As a result of additional analyses of the
2M promoter, we observed
that when a 109-bp region directly 3' of the Stat 5-binding sites was
deleted, the
2M promoter-reporter construct lost both its high basal
activity and PRL-regulated induction. When the sequence of this region
was examined, we noted that it contained putative binding sites for
SF-1 and chicken ovalbumin upstream promoter-transcription factor
(COUP-TF). Like SF-1, COUP-TF is a member of the orphan nuclear
receptor superfamily (17, 18). However, unlike SF-1, it is presumed to
be expressed ubiquitously and has been shown to exhibit complex
interactions by binding, as a heterodimer, other members of the nuclear
receptor superfamily (19). COUP-TF homodimers bind to a wide spectrum
of response elements containing, as a core half-site, the A/GGGTCA
motif, the spacing and orientation of which determine the relative
binding affinity of COUP-TF (19). Recent evidence indicates that Stat
proteins may interact functionally with other transcription factors and
coregulatory molecules, including members of the nuclear receptor
superfamily (20, 21, 22, 23, 24). Based on the close juxtaposition and functional
activities of the PRL response element (PRLRE) and the putative orphan
receptor-binding sites, it seemed reasonable to predict that factors
binding to these regions might interact to control maximal expression
of the
2M gene in granulosa-luteal cells.
Based on these considerations and the tissue- specific expression
of this gene in the rat ovary, the following studies were undertaken to
determine which Stat 5 proteins were present and functional in
differentiated granulosa cells, if either SF-1 or COUP-TF bound to the
intact
2M promoter, and if either SF-1 or COUP-TF interacted
functionally with Stat 5 proteins to confer maximal expression of this
gene in response to PRL. The functional activities of the Stat-binding
sites and the orphan receptor-binding sites were analyzed by generating
site-specific mutations in the context of the intact promoter and
transfecting differentiated granulosa cells with specific intact and
mutant promoter-luciferase reporter constructs. Activities were related
to the binding of specific nuclear factors and the stage of
differentiation of the granulosa cells.
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RESULTS
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Two Regions in the Proximal Promoter of the
2M Gene Are Required
for Functional Activity in Granulosa Cells
To identify and characterize the functional regions in the
2M
promoter conferring both PRL and high basal activity in granulosa
cells, specific deletion constructs were generated to remove sequences
either 5' (-371 bp and -209 bp) or 3' (-209
) of the Stat-binding
sites or which deleted the PRLRE (-159 bp) (Fig. 1
). The 1.2-kb
2M-luciferase
construct, as well as the deletion constructs, were transiently
transfected into differentiated granulosa cells that had been cultured
with FSH and testosterone for 48 h. After 4 h,
granulosa cells were treated with or without PRL for 6 h.
Luciferase activity was measured, normalized to the amount of protein
present in the sample, and then expressed as activity/µg protein.
Constructs with 5'-deletions to -371 and -209 bp retained both PRL
inducibility and high basal activity (Fig. 1
). Deletion of the
Stat-binding sites reduced basal activity and abolished PRL- inducible
activity (Fig. 1
). Deletion of a 109-bp region 3' of the PRLRE
(-209
) reduced basal activity to that seen with the minimal -48-bp
2M promoter and abolished PRL inducibility. These results indicated
that the PRLRE as well as the 109-bp region immediately downstream of
the PRLRE might interact functionally to confer maximal promoter
activity in granulosa cells.

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Figure 1. Two Regions of the 2M Promoter Are Required for
Functional Activity
Each of the indicated vectors was transfected in triplicate in
differentiated granulosa cells that had been cultured in the presence
of FSH (50 ng/ml) and testosterone (10 ng/ml) for 48 h. After
4 h of incubation, PRL(1 µg/ml)was added for 6 h.
Luciferase activity (light units) was measured in 20 µl cell lysate
and normalized to the amount of protein. The relative luciferase
activity is represented as the mean (light units/µg) ±
SEM.
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Stat Binding Sites Are Critical for PRL Induction of the
2M
Promoter
To specifically determine the function of the Stat- binding
sites in the context of the intact 1.2-kb
2M promoter, each of the
Stat-binding sites was mutated or both of the sites were deleted. The
pSV1.2-kb
2M construct and the mutant promoter-luciferase pSV
constructs (16) were transfected into differentiated granulosa cells.
As shown in Fig. 2
, PRL induced a 2- to
3-fold increase in luciferase activity from the intact
promoter-reporter construct. Although the level of luciferase activity
of the promoter in the pSV promoter-reporter constructs was low (as
compared with the pGL3 constructs, Fig. 1
), the inducibility by PRL was
similar. Deletion of both Stat-binding sites or mutation of either site
abolished inducibility by PRL. Basal activity was not markedly altered
except for the reduced activity in the proximal Stat 5 mutant
construct. These results clearly show that both Stat- binding sites are
required for PRL inducibility of promoter activity in granulosa cells
but not for the high basal activity of the constructs.

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Figure 2. Both the Stat Factor-Binding Sites Are Required for
the 2M Promoter Activity
To determine the function of the Stat binding sites in the context of
the endogenous intact promoter, either one of the Stat-binding sites
was mutated or both were deleted. These constructs were then
transfected into differentiated granulosa cells. Luciferase activity
was measured in 20 µl cell lysate, normalized to the amount of
protein.
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PRL-Induced DNA-Binding Activity Contains Stat 5a and Stat 5b
To determine the specific factors binding to the PRLRE, we used
electrophoretic mobility shift assays (EMSAs) in which the labeled
oligonucleotide probe consisted of two copies of the proximal
Stat-binding site of the
2M promoter (
2MGAS2) (16). Because
activated (phosphorylated) Stat proteins bind DNA whereas the
nonphosphorylated Stats do not, the activated factors are readily
detected in EMSAs. Accordingly, whole cell extracts (WCEs) were
prepared from differentiated granulosa cells before or 15 min after PRL
treatment, incubated with labeled
2M GAS2 probe, and resolved by
electrophoresis. One slow migrating protein/DNA complex was formed with
the WCE from PRL-treated cells but was not observed in WCE prepared
from untreated cells (Fig. 3A
, arrow). To identify the Stat factor(s) present in the slow
migrating complex, WCEs were preincubated with antibodies to either
Stat 5a or Stat 5b. The PRL-induced complex was partially supershifted
by the Stat 5a antibody, as shown previously (10). A specific Stat 5b
antibody resulted in retardation of the entire PRL-induced protein/DNA
complex (Fig. 3A
, asterisk) as was observed in extracts
isolated from PRL-stimulated corpora lutea (11). These data indicate
that in differentiated granulosa-luteal cells, as in luteal tissue from
pregnant rats (11, 12), PRL activates Stat 5b as well as Stat 5a and
that Stat 5b is the predominant isoform in these cells.

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Figure 3. Activation and Phosphorylation of Stat5b in
Differentiated Granulosa Cells by PRL
A, PRL-induced DNA-binding activity present in granulosa cells contains
Stat 5a and Stat 5b. WCEs isolated from untreated and PRL-treated (15
min) granulosa cells were incubated with labeled oligonucleotide
2MGAS2 (5'- GATCATCC
TTCTGGGAATTCTGATATCCTTCTGGGAATTCTG- 3') and
analyzed on EMSA. The arrow indicates the specific
PRL-induced DNA protein complex formed. WCEs from PRL-treated granulosa
cells were preincubated with antisera to Stat 5a and Stat 5b antibodies
(30 min/4 C) before the addition of radiolabeled double-stranded
oligonucleotide 2MGAS2. The PRL-induced DNA-protein complex is
super-shifted partially by the Stat 5a antibody and completely by Stat
5b antibody (*). B and C, PRL induces tyrosine
phosphorylation of Stat 5b. For immunoprecipitations, 30 µl
immobilized Protein A on tryacryl beads were incubated with 200 µg
WCEs, for 1 h at 4 C with shaking. After a brief spin, the
supernatant was incubated with 1 µl Stat 5b antibody and 20 µl
beads for 24 h at 4 C with shaking. Each of the samples was then
washed three times with WCE buffer to remove unbound proteins. Bound
proteins were eluted from the beads by addition of equal volume of
SDS-loading buffer and boiled at 100 C for 10 min. Samples were
electrophoresed on a 10% SDS-polyacylamide gel, transferred to an
immobilion membrane, and blotted with antiphosphotyrosine antibody (B).
The same blot was stripped and immunoblotted with anti-Stat 5b antibody
(C).
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PRL Induces Tyrosine Phosphorylation of Stat 5b
To demonstrate that the activated Stat 5b in granulosa cells was
phosphorylated as a consequence of PRL stimulation, immunoprecipitation
experiments were performed. WCEs were prepared from granulosa cells
before and at selected time points after PRL treatment. WCEs were
incubated with a specific Stat 5b antibody. The immunoprecipitate was
resolved by SDS-PAGE, and tyrosine-phosphorylated Stat 5b was
identified by an antiphosphotyrosine antibody (PY20) used in the
Western blotting procedure. As shown in Fig. 3B
, WCEs from untreated
cells do not contain detectable amounts of tyrosine-phosphorylated Stat
5b, whereas WCEs prepared within 15 min of PRL treatment contained
significant levels of phospho-Stat 5b. Phosphorylated Stat 5b remained
present but at lower levels at 6 h and 20 h after PRL
treatment (Fig. 3B).
To ensure that relatively equal amounts of Stat 5b were present in each
immunoprecipitate, the same blot was reprobed with an anti-Stat 5b
antibody. As shown in Fig. 3C
, similar amounts of Stat 5b were
immunoprecipitated from each of the samples. These data show that PRL
rapidly stimulates tyrosine phosphorylation of Stat 5b in
granulosa-luteal cells but that, even in the presence of continuous
exposure to PRL, the amount of phospho-Stat 5b decreases progressively
with time, coincident with reduced DNA-binding activity (data not shown
and Ref. 10).
Stat 5b Is a Key Regulator of
2M Gene Expression in the
Ovary
Having established that Stat 5b, rather than Stat 5a, is the major
Stat 5 protein activated in these granulosa-luteal cells and knowing
that Stat 3 fails to bind the
2M PRLRE (10, 11), we sought to
determine whether disruption of Stat 5b binding to the promoter would
abolish promoter activity. For this we cotransfected into
differentiated granulosa cells vectors expressing either wild-type Stat
5b or a truncated, splice variant form of Stat 5b (Stat 5b-40C) that
has been shown to act in a dominant negative fashion to suppress Stat
5b function in other cells (25, 26). A reporter construct containing
four copies of the proximal Stat-binding site ligated to the minimal
2M promoter-luciferase gene (4XGAS
2M) was chosen as the reporter
construct since in previous studies (10, 16) it has been shown to
exhibit a greater response to PRL treatment than the intact
2M
promoter. When wild-type Stat 5b expression vectors were cotransfected
with the 4XGAS
2M luciferase construct, the PRL-induced response was
similar to cells cotransfected with the empty vector (Fig. 4
), indicating that endogenous amounts of
Stat 5b were already in excess. In contrast, PRL-induced activation of
the promoter-reporter construct was significantly repressed by
cotransfection of the vector expressing the truncated variant of Stat
5b (Fig. 4
). These data indicate that PRL induction of
2M in
differentiated granulosa cells is dependent on activation of Stat 5b
and that the truncated splice-variant of Stat 5b can act as a dominant
negative factor in granulosa cells as it does in other cells (25, 26).

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Figure 4. Truncated Stat 5b Acts as a Dominant Negative
Factor in Differentiated Granulosa Cells
Expression vectors of the long (Stat 5b) and truncated form (Stat
5b-40C) of Stat 5b were cotransfected as above with 4xGAS- 2M
promoter-reporter construct, containing four copies of the proximal
Stat-binding site. Luciferase activity is presented as the mean ±
SEM of activity obtained for each construct done in
triplicate.
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SF-1 and COUP-TF Are Present in Granulosa Cells and Bind the
Proximal Promoter of the
2M Gene
The 109-bp fragment (-153/-43 bp) which, when deleted, totally
abolished functional activity of promoter-reporter constructs (Fig. 1
;
-209
2M) was analyzed for potential transcription factor-
binding sites. Putative binding sites for orphan members of the nuclear
transcription factor family, SF-1, and COUP-TF (Fig. 5A
) were present in the 45-bp region of
this fragment. The sites for COUP-TF included an imperfect direct
repeat (DR) separated by one nucleotide (designated DR1) and another
direct repeat that contains a variant repeat of A/GGGTCA separated by
two nucleotides, designated DR2. On the opposite strand between DR1 and
DR2, a putative SF-1 site was identified that contains one nucleotide
mismatch from the consensus hexameric sequence (CA)AGGTCA (Fig. 5A
).
Based on sequence identity and the binding studies described below, we
have referred to this region as the orphan receptor response element
(ORRE).

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Figure 5. Sequence and Functional Binding Sites for COUP-TF
and SF-1 in the 2M ORRE
A, Sequence comparison of the ORRE to consensus binding sites for
members of the nuclear orphan receptor family. The ORRE contains two
potential COUP-TF binding sites designated direct repeats (DR), DR1 and
DR2. The SF-1 binding site is present on the opposite strand between
DR1 and DR2. Consensus sequences are represented in
bold. Identical nucleotides are indicated with
lines. B, SF-1 and COUP-TF bind the 2M promoter. WCEs
obtained from differentiated granulosa cells were incubated with
labeled oligonuleotide ORRE various cold competitors, as indicated, and
analyzed by EMSA. The sequence of each oligonucleotide is described in
Materials and Methods. Arrows indicate
the specific DNA protein complexes formed. WCEs from differentiated
granulosa cells were preincubated with antisera to COUP-TF or SF-1 (30
min/4 C) before the addition of radiolabeled ORRE. Antisera against
COUP-TF reacts with Complex A and antisera against SF-1 reacts with
complex B. Supershifts are indicated by (*).
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To determine whether these factors were present in the differentiated
granulosa cells and could bind to the ORRE, EMSAs were performed. A 50
mer oligonucleotide spanning the ORRE was used as a labeled probe and
incubated with WCE obtained from untreated and PRL-treated granulosa
cells. Since identical complexes were observed with either WCE (data
not shown), only the results obtained with WCE not exposed to PRL are
presented (Fig. 5B
). As shown, two major protein/DNA complexes were
formed. The slower migrating complex, designated complex A, consists of
two bands (thin arrow); the faster migrating complex is
designated complex B (thick arrow). Formation of both these
complexes was inhibited by the addition of 100-fold molar excess of
unlabeled ORRE oligonucleotide, indicating that each of these complexes
contains a protein(s) capable of binding to the probe. When a 20-bp
oligonucleotide encompassing a consensus COUP-TF-binding site
(Materials and Methods) was added to the reaction, the
formation of complex A but not complex B was abolished (Fig. 5B
). When
oligonucleotides spanning either the putative COUP-TF-binding DR1 or
DR2 regions (Materials and Methods) of the
2M promoter
were added, neither altered the formation of complex A or complex B at
the 100-fold molar excess. However, when oligonucleotides containing
both DR1 and SF-1 (DR1SF-1) or DR2 and SF-1 (DR2SF-1) (Materials
and Methods) were added, each reduced the formation of complex A
as well as complex B.
To confirm the identification of proteins present in granulosa cell
extracts, the WCEs were incubated with specific antisera to either
COUP-TF or SF-1. Antisera to COUP-TF specifically supershifed both the
bands of complex A (*). Previous data show that this antiserum
recognizes both COUP-TFI and COUP-TF-II (27). This COUP-TF antiserum
also diminished the binding of in vitro translated COUP-TF
to ORRE (data not shown). The SF-1- specific antiserum retarded complex
B (*) but not complex A, indicating a specific interaction of the
antibody to this major protein-DNA complex. Collectively, these data
indicate that the SF-1 (complex B) and COUP-TF (complex A) bind the
2M promoter. Furthermore, the functional binding sites appear to be
overlapping rather than distinct.
Mutation of the SF-1 Binding Site Alters COUP-TF Binding to the
ORRE
To determine whether the nucleotides required for the binding of
SF-1 and COUP-TF to the
2M promoter were overlapping, mutations
within the ORRE were generated (Materials and Methods).
Mutations in both DR1 and DR2 (Mutant 1; M1) were made to selectively
eliminate the putative binding sites of COUP-TF; mutations in the
SF-1-binding site were generated to block the binding of SF-1 (M2)
(Materials and Methods). When EMSAs were performed with M1
as the labeled probe (Fig. 6A
, left
panel), one major complex was formed (arrow). This
complex was competed with 50-fold and 100-fold molar excess of
unlabeled M1. The antibody to COUP-TF failed to affect the mobility of
the major complex binding to M1, whereas the antibody to SF-1
supershifted the major complex (*). These results indicate that
mutations within DR1 and DR2 inhibited COUP-TF binding without altering
the binding of SF-1 to the ORRE.

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Figure 6. Binding Characteristics of Mutant SF-1 and
COUP-TF-Binding Regions
A, Analysis of mutant SF-1- and COUP-TF-binding sites. WCEs obtained
from differentiated granulosa cells were incubated with labeled
oligonuleotide containing either mutation in both DR1 and DR2 (M1) or
mutation in the SF-1 site (M2) and analyzed by EMSA. The sequences of
both M1 and M2 are described in Materials and Methods.
Arrow indicates the specific DNA protein complex formed
with M1. WCEs from differentiated granulosa cells were preincubated
with antisera to COUP-TF/SF-1 (30 min/4 C) before the addition of
radiolabeled M1/M2. Supershift of the specific complex with antisera
against SF-1 is indicated by (*). B, Mutation of the SF-1 site alters
the affinity of COUP-TF to ORRE. WCEs obtained from differentiated
granulosa cells were incubated with labeled oligonuleotide ORRE. The
oligonuleotide containing mutation in the SF-1 site (M2) was used as a
cold competitor at various concentrations. The arrow
indicates the specific DNA protein complex formed with ORRE. WCEs from
differentiated granulosa cells were preincubated with antisera to
COUP-TF (30 min/4 C) before the addition of radiolabeled ORRE.
Supershift of the specific complex with antisera against COUP-TF is
indicated by (*).
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When the SF-1 mutant (M2) oligonucleotide was used as the labeled
probe in EMSAs, no specific protein-DNA complexes containing SF-1
or COUP-TF were observed (Fig. 6A
, right panel). The absence
of SF-1 binding was expected. However the absence of COUP-TF was
unexpected because both DR1 and DR2 sites were intact. Thus mutation of
the SF-1 site altered not only the binding of SF-1 but also that of
COUP-TF. When labeled M2 was incubated with in vitro
translated COUP-TF, the binding of COUP-TF was markedly diminished
compared with the intact ORRE (data not shown). Based on these
observations, we concluded that the SF-1 site of the
2M promoter
appeared to enhance, stabilize, or directly bind COUP-TF.
To determine if the mutation of the SF-1 site within the
2M promoter
altered the binding affinity of COUP-TF to the ORRE, additional EMSAs
were run in which the intact 50-mer ORRE was used as the labeled probe.
As previously, unlabeled ORRE reduced the formation of complexes A and
B whereas the COUP-TF antibody supershifted complex A but not complex B
(Fig. 6B
). When the SF-1 mutant M2 oligonucleotide was used at 100-,
500-, and 1000-fold molar excess, no reduction in the binding of SF-1
was observed. However, M2 did compete for the binding of COUP-TF
(thin arrow), albeit less effectively than unlabeled ORRE. A
1000-fold molar excess of M2 was required to achieve the same
inhibition as a 100-fold molar excess of the intact ORRE (Fig. 6B
).
These results indicate that mutation of the SF-1 site reduces the
affinity or stability but does not completely prevent the binding of
COUP-TF to the
2M promoter.
SF-1 and COUP-TF Exert Opposing Effects on the Functional Activity
of the
2M Promoter
SF-1 is known to be a positive regulator of many genes (2, 3, 4, 5, 6, 7, 8, 9),
whereas the effects of COUP-TF are more complex and can be either
positive or negative (17, 28). In addition, both exhibit
tissue-specific effects as indicated by the phenotypes of mutant mice
lacking SF-1 (1) or COUP-TFI/II (Refs. 17, 29 ; and Y. Qu, F. A.
Pereira, S. Y. Tsai, and M.-J. Tsai, personal communication). To
analyze the functional roles of SF-1 and COUP-TF in regulating
activation of the
2M promoter, specific mutations that abolished the
binding of SF-1 or COUP-TF (Fig. 6
) were generated within the
-371
2M promoter-reporter construct using PCR- directed
mutagenesis techniques (Materials and Methods). These
constructs were then transfected into differentiated granulosa cells.
After 4 h, cells were treated with/without PRL for 6 h.
Luciferase activity was measured, normalized to the amount of protein
present in the sample, and then expressed as activity/µg protein. As
expected, PRL induced a 2- to 3-fold increase in luciferase activity
from the intact -371
2M promoter-reporter construct (Fig. 7
). Mutation of the SF-1 site (mSF-1)
that prevents SF-1 binding and reduces the affinity of COUP-TF binding
(Fig. 6A
/B) markedly decreased basal activity of the reporter gene but
did not prevent the 3-fold increase in response to PRL (Fig. 7
). In
contrast, the COUP-TF mutant (mCOUPTF) construct that retains an intact
SF-1 binding site but fails to bind COUP-TF showed a 5-fold increase in
basal promoter activity. Although the fold increase by PRL was somewhat
reduced, an effect of PRL was observed. When both the SF-1 and the
COUP-TF sites (mSF-mCOUPTF) were mutated, basal and PRL-stimulated
luciferase activities were similar to that of the intact
promoter-reporter construct. Collectively, these data document that
within the ORRE of the
2M promoter, SF-1 acts as a positive
regulator whereas COUP-TF acts as a repressor of transcriptional
activation. Combined with the data from the EMSAs, these results
indicate that although mutation of the SF-1 site decreases the affinity
of COUP-TF binding to the
2M promoter, COUP-TF binds this region
(albeit with reduced affinity) and retains potent repressor activity.
Thus, both of these nuclear receptors appear to control the basal
activity of the
2M promoter in granulosa-luteal cells but do not
markedly alter the response to PRL.

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Figure 7. SF-1 and COUP-TF Are Required for 2M Promoter
Activity
Mutations of specific bases within the SF-1- and/or COUP-TF-binding
sites were generated using PCR mutagenesis technique. Each of the
indicated vectors was transfected in triplicate in differentiated
granulosa cells as above. Luciferase activity (lights units/µg) is
presented as the mean ± SEM of activity obtained for
each construct done in triplicate.
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Molecular Mechanisms by which FSH and T Control Expression of
2M
in Granulosa Cells
The identification of SF-1 and COUP-TF as factors binding to and
regulating the functional activity of the ORRE of the
2M promoter
prompted us to determine whether these factors regulated the basal
activity of this promoter at different stages of granulosa cell
differentiation. Specifically, FSH has been shown to regulate the
expression of numerous genes in granulosa cells that contain SF-1
binding sites. The list of genes includes aromatase (6), P450scc (7),
MIS (8), and, more recently, the receptor for PRL (31). This latter
observation indicated that not only the basal activity of
2M
promoter, but also the PRL inducibility of
2M gene might be
dependent on several aspects of FSH-mediated differentiation of
granulosa cells. Therefore, we analyzed and compared both basal and
PRL- induced expression of
2M in immature granulosa cells and in
cells differentiated in the presence of FSH and T. The expression of
the endogenous
2M gene has been compared with the activity of
2M
promoter-luciferase-reported constructs transfected into similar
immature and differentiated cells. Furthermore, the expression of the
endogenous gene and the transgene has been related to the expression of
PRL receptors (PRLRs), activation of Stat 5, and the relative levels of
SF-1 and COUP-TF.
To analyze the expression of the endogenous gene, granulosa cells were
isolated from estradiol-primed immature rats. The cells were either
cultured in serum-free medium without hormones for 24 h or were
cultured with FSH/T for 48 h. PRL was added to the hormone-free
(undifferentiated) and hormone-induced (differentiated) cell cultures
for 24 h. Total RNA was isolated, and
2M transcripts were
analyzed by Northern blots. As shown in Fig. 8A
,
2M mRNA was not present in the
undifferentiated cells in the absence or presence of PRL. In contrast,
PRL did induce
2M mRNA in the differentiated cells. Thus, in the
absence of FSH/T, PRL is unable to stimulate transcription of the
endogenous
2M gene, confirming previous studies in vivo
(11) and in vitro (32, 33).
Similar cultures were then set up and transfected with the -371
2M
promoter-reporter construct to analyze the activity of these chimeric
genes in the absence and presence of PRL. As shown in Fig. 8B
, basal
luciferase activity of the -371
2M promoter-reporter construct was
low in undifferentiated granulosa cells and was not increased by the
addition of PRL. In contrast, basal luciferase activity was 10-fold
higher in the differentiated cells, and PRL induced a further 2- to
3-fold increase in activity. The marked differences in the activity of
the transgene in these two stages of granulosa cell differentiation
were not related to transfection efficiency since an SV40
enhancer-promoter construct gave similar activity (20,000 mean light
units ± SEM per µg protein) when transfected into
each culture. Based on these observations that FSH- and T-induced
differentiation altered both basal and PRL-regulated activities, we
sought to determine what changes in the PRL-response system or in the
ratio of SF-1 and COUP-TF might account for these marked
differences.
To determine whether PRL could stimulate activation of Stat 5b/5a in
undifferentiated granulosa cells, WCEs were isolated from FSH/T-treated
(48 h) or untreated (24 h) granulosa cells before and after PRL
treatment (1530 min). Several protein/DNA complexes were
formed when WCE prepared from immature granulosa cells before and after
their exposure to PRL were added to the labeled
2MGAS2 probe.
However, only extracts prepared within 15 min after PRL addition to
FSH/T-treated cells (Fig. 9A
) formed a
specific slow migrating complex, which is composed primarily of Stat 5b
with lesser amounts of Stat 5a (Figs. 3A
and 9A
). Immunoblot analysis
using WCEs prepared from FSH/T treated (48 h) or untreated (24
h) granulosa cells before and after PRL treatment (15/30 min),
and Stat 5b antibody shows similar levels of protein in both the
immature and the FSH/T-stimulated cells (data not shown). Thus,
although Stat 5b protein is present in undifferentiated granulosa
cells, PRL is unable to activate it in these cells (i.e.
before exposure to FSH/T).

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Figure 9. PRL Receptor Activity and mRNA Content in
Undifferentiated and Differentiated Granulosa Cells
A, PRL fails to activate Stat 5b in undifferentiated granulosa cells.
WCEs were isolated from cultured granulosa cells before and after PRL
treatment (15 min) from undifferentiated (-FSH/T, 24 h) or
differentiated (+FSH/T, 48 h) cells. WCEs were incubated with a
radiolabeled, double-stranded oligonucleotide 2MGAS2 as above. The
protein/DNA complexes were analyzed by EMSA. B, Expression of PRLR
mRNAs in undifferentiated and differentiated granulosa cells. RNA was
isolated from uncultured, undifferentiated (-FSH/T, 24 h) and
differentiated (+FSH/T, 48 h) granulosa cells. Specific primers to
the long and the short form of the PRLRs were used in a RT-PCR assay
using 350 ng RNA. PCR amplification was performed for 20 cycles as
described in Materials and Methods. PCR products were
resolved on a nondenaturing gel and quantified using Storm860
PhosphoImager.
|
|
One explanation for the transactivation of
2M in differentiated
cells, but not in immature cells, is the level of PRLRs in these cells.
Three forms of the PRLRs have been identified in the rat. The short
receptor is an alternatively spliced product of the long form (L-PRLR).
The Nb2 form expressed in T cells is identical to L-PRLR but lacks 198
aa in the cytoplasmic domain. Studies have shown the long form to be
functional in the ovary. Specific primers to the long and the short
form of the PRLRs were used in an RT-PCR assay using RNA isolated from
uncultured cells, undifferentiated granulosa cells (-FSH/T, 24
h), and differentiated cells (+FSH/T, 48 h). The amplified
receptor mRNA products were normalized to the endogenous ribosomal
L19-amplified band (Fig. 9B
). The long form of the PRLR was detected at
significant levels in immature granulosa cells before culture. However,
in the absence of hormone, the levels of PRLR decreased progressively
at 24 and 48 h of culture (Fig. 9B
). In contrast, cells cultured
with FSH/T for 48 h expressed 5-fold more message for the long
PRLR and exhibited detectable amounts of the short form of the receptor
(Fig. 9B
). These data indicate that the ability of PRL to induce
2M
in these differentiated cells is related, in part, to the
induction-maintenance of PRLR in these cells. However, since PRL does
not induce
2M in immature cells that also express the long form of
the PRLR, additional factors regulating either the PRL-signaling
cascade or the A-kinase system must be regulated by FSH/T as the
granulosa cells differentiate.
Based on the opposing actions of SF-1 and COUP-TF in regulating the
basal activity (Fig. 7
), we sought to determine whether changes in the
relative amounts of these factors might be related to increase of
2M
mRNA expression in differentiated cells compared with immature cells.
EMSAs were performed using labeled ORRE and extracts prepared from
either immature/undifferentiated granulosa cells (in which
2M is not
induced) or from FSH/T- treated/differentiated granulosa cell extracts
(Fig. 10A
). Quantification of the
specific complexes was performed to determine the relative amounts of
the SF-1 and COUP-TF binding to the ORRE. As shown (Fig. 10B
), a
relative increase in SF-1 binding (
6-fold) compared with COUP-TF was
detected in the FSH/T-treated granulosa-lutein cells. These results
indicated that a relative increase in the positive regulator SF-1 may
enhance the basal promoter activity.

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Figure 10. Levels of SF-1 and COUP-TF-Binding Activity in
Undifferentiated and Differentiated Granulosa Cells
A, Relative increase in SF-1 and COUP-TF binding to the ORRE in
differentiated granulosa cells. WCEs obtained from immature and
differentiated (FSH/T, 48 h) granulosa cells were incubated with
labeled ORRE oligonuleotide. The protein/DNA complexes were analyzed by
EMSA. B, Quantitative representation of SF-1 and COUP-TF binding to the
ORRE. The SF-1 and COUP-TF protein/DNA complexes were quantified with
Storm860 PhosphoImager. FSH-dependent differentiation of granulosa
cells increases the relative amount of SF-1 binding to the ORRE
compared with COUP-TF. Ratios (relative units) of SF-1 to COUP-TF
binding are indicated in parentheses.
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DISCUSSION
|
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The expression of
2M in the rat ovary is dependent on the
differentiation of granulosa cells to luteal cells (10, 11) as well as
the presence, activation, and binding of a diverse set of transcription
factors to the promoter of the
2M gene. In this study we have
identified and functionally characterized the interactions of two
regions of the
2M promoter. One region contains a PRLRE that is
comprised of two Stat factor-binding sites and binds both Stat 5a and
Stat 5b (Refs. 10, 11 and results herein). The other region contains
an ORRE that binds the orphan nuclear receptors SF-1 (2, 3, 4) and COUP-TF
(17, 18). We show herein that maximal functional activity of the
2M
promoter depends on the independent activation of each region by the
binding and interactions of these specific transcription factors,
hormonal stimulation, and a differentiated granulosa-luteal cell
phenotype.
When the studies described herein were originally initiated, we had
shown that Stat 5a was expressed in rat granulosa cells, activated by
PRL, and bound to the PRLRE of the
2M promoter. We have extended
these observations by showing that both Stat-binding sites are critical
for PRL-induced activity of the
2M promoter. Neither site alone in
the context of the intact promoter exhibits significant functional
activity. These results indicate that the two sites may interact to
stabilize or increase the binding of coregulatory molecules that
interact with the transcriptional machinery. As new information about
the complexity of the Stat 5 family of transcription factors was
reported (34, 35), reagents to these new family members allowed us to
extend our initial observations. Using antibodies specific for Stat 5b,
we show that not only is it present in the rat granulosa cells, it is
expressed at higher levels than Stat 5a, is activated by PRL, and binds
to the PRLRE of the
2M gene. These results obtained in
granulosa-luteal cells in culture support those observed in luteal
cells in vivo (10, 11). Antibodies to Stat 5b have allowed
us to document that in differentiated granulosa cells Stat 5b is
tyrosine phosphorylated and translocated to the nucleus in response to
PRL. In addition, truncated, splice-variant forms of Stat proteins have
been cloned. For the Stats in which truncated forms have been
identified, Stat 1 (36, 37) Stat 3 (38), and Stat 5 (25, 26), the
mutants lacking the transactivation domain have been shown to
exert dominant-negative functions in transcription assays (25, 26, 36, 37, 38). When we coexpressed the truncated Stat 5b variant with the
2M promoter-luciferase reporter construct, PRL-induced activity was
markedly impaired, supporting the hypothesis that Stat 5 is the major
PRL-regulated activator of
2M promoter activity in the
differentiated granulosa cells. Taken together, our results provide
evidence that PRL-mediated signaling events that transactivate the
2M gene in the granulosa-luteal cells occur by mechanisms similar to
those that regulate cytokine signal transduction of target gene
activity in other tissues, such as mammary gland (13, 39, 40) and liver
(35, 41). That Stat 5, especially Stat 5b, plays a major role in
transducing PRL action in ovarian cells has been supported recently by
the generation of mutant mice. Although female mice null for Stat 5a do
not exhibit an overt ovarian phenotype (42), mice null for Stat 5b
alone (43) are infertile and exhibit abnormal ovarian functions. Based
on the luteal cell-specific expression of
2M in vivo as
well as its regulation by PRL and Stat 5b in cultured cells, we predict
that corpora lutea of the infertile mice either fail to express
2M
or express it at a markedly reduced level.
Our results have also provided new evidence to indicate that other
regions, in addition to the PRLRE, regulate the expression of
2M in
granulosa-luteal cells. The region directly 3' of the PRLRE binds both
SF-1 and COUP-TF and has been designated herein as the ORRE of the
2M promoter. When this region of the
2M promoter was deleted,
leaving the PRLRE intact, transgene activity was reduced to baseline
and PRL inducibility was abolished. Based on these initial
observations, we predicted that Stat 5 binding to the PRLRE interacted
synergistically with the orphan receptors binding to the 3'- regulatory
region to confer maximal promoter activity. This initial hypothesis was
supported by observations of others who have shown that Stats
synergistically interact with a variety of other transcription factors
and coregulatory molecules. In mammary epithelial cells, Stat 5
interacts with the glucocorticoid receptor for activation of the
ß-casein gene by PRL (23, 44). In liver cells, Stat 3
synergizes (interacts) with glucocorticoid receptor for activation of
the
2M gene by IL-6 (24). Stat 1 and Sp1 interact to activate ICAM-I
promoter in human tracheobronchial epithelial cells (21). In addition,
Stat 2 has been shown to bind CBP/p300 and enhance gene activation by
interferon-
(20). When site-specific mutations were generated in
either the Stat 5, SF-1, or COUP-TF binding sites within the context of
the intact
2M promoter, a different set of functional interactions
was observed.
Mutation of either one or both Stat 5-binding sites markedly reduced
PRL inducibility. As mentioned above, these results indicate that the
functional activity of the PRLRE is dependent on the binding of Stat 5
to each site and that these two sites interact to confer maximal PRL
inducibility. However, mutation of the Stat sites did not alter the
high basal activity observed with the intact
2M promoter-reporter
constructs compared with the -48
2M minimal promoter construct.
Contrary to our initial hypothesis, these results indicated that the
Stat sites acted independently of the factors binding to the ORRE.
However, when site-specific mutations of the SF-1 site or the two
COUP-TF binding sites were generated, marked changes in the basal
activities of the mutant constructs were observed. The mutant SF-1
construct that failed to bind SF-1 exhibited markedly reduced basal
activity, whereas the COUP-TF mutant construct that failed to bind
COUP-TF, but did bind SF-1, exhibited elevated basal activity. These
results indicated that SF-1 is a transcriptional activator and enhances
2M promoter activity, whereas COUP-TF acted as a corepressor to
antagonize the activity of SF-1 and thereby reduce the functional
activity of the intact
2M promoter. Thus, the
2M gene can be
added to a growing list of genes that are regulated by SF-1 in ovarian
cells and highlights the importance of SF-1 in many diverse functions
within the gonads and adrenals as suggested by the phenotype of mice
null for SF-1 (1). Furthermore, these observations provide the first
evidence that COUP-TF is present and functional in ovarian granulosa
cells and can regulate the expression of an ovarian gene. Although the
actions of COUP-TF are complex and have been shown to be both
stimulatory and inhibitory (17), our data indicate that COUP-TF acts as
a corepressor of SF-1 action in the intact promoter of the
2M gene.
This observation supports results obtained by one other study in which
the effects of SF-1 were blunted by COUP-TF (5).
Although the mechanism by which COUP-TF blocks the actions of SF-1 on
the
2M promoter have not been entirely defined, our data show an
interesting juxtaposition and interaction of SF-1- and COUP-TF-binding
sites. Of note, mutation of the SF-1 site reduced by 10-fold the
binding affinity of COUP-TF for the DR1 and DR2 regions of the
2M
promoter. From this, we conclude that the binding of COUP-TF to DR1 and
DR2 overlaps the SF-1 site or adjacent sites. However, despite the
dramatic reduction in the binding affinity of COUP-TF to the SF-1
mutant region, COUP-TF retained potent repressor activity as revealed
by the markedly reduced basal activity of the SF-1 mutant construct.
These interactions of SF-1 and COUP-TF indicate that any change in the
relative levels or activity of these factors could markedly alter the
basal activity of the
2M gene and thus its expression.
Both PRLRE and ORRE are dependent on the differentiation of granulosa
cells. In the case of the PRLRE, granulosa cell differentiation is
obligatory, in part, because of the increased expression of PRLRs on
the cell surface. Increased PRLR number enhances the activation of Stat
5b, which is constituitively expressed in the same amount in immature
as well as differentiated granulosa cells. In the case of the ORRE, the
molecular basis for the enhanced activity of this region in
differentiated cells, as opposed to immature cells, remains to be
determined but likely involves altered functional activity and complex
interactions of SF-1 or COUP-TF on the ORRE. As shown herein, the
amounts and relative ratios of these two factors binding to the
2M
ORRE are different in immature and differentiated granulosa cells.
There is a relative 6-fold increase in SF-1 binding in differentiated
granulosa cells compared with the immature granulosa cells. Therefore,
increased basal activity of the
2M promoter may depend on the
increased binding of this transcriptional activator. In addition,
functional changes are likely to involve the selective phosphorylation
of either factor or a coregulatory molecule. Phosphorylation of SF-1
would be predicted to increase activity of the ORRE, whereas
phosphorylation of COUP-TF might decrease its repressor activity on the
ORRE. The phosphorylation of either factor might alter the recruitment
of other coregulatory molecules. Phosphorylation of SF-1 has been shown
to occur in differentiated granulosa cells (45) and, therefore, may
be a critical event in increasing both basal activity of
2M as
well as increasing the expression of PRLR. In combination, these two
events promote enhanced transcription of
2M by PRL. As mentioned
above, most cells do not express SF-1 but do express COUP-TF.
Therefore, it is possible that in these cells the repressor activity of
COUP-TF is unopposed by any other transcription factor, thereby
reducing expression of the gene.
In summary, we have shown that expression of the
2M gene in ovarian
granulosa-luteal cells involves not only PRL-induced activation of Stat
5b/a but also the opposing interactions of SF-1 and COUP-TF on an ORRE.
The close juxtaposition of these regions initially indicated that there
might be a functional synergism between the factors binding to the
PRLRE and the ORRE. However, using site-specific mutations, rather that
deletion analyses, we have been able to show that these regions act
independently of each other in the context of the intact
2M
promoter.
 |
MATERIALS AND METHODS
|
---|
Materials
17ß-Estradiol was purchased from Sigma Chemical Co. (St.
Louis, MO); testosterone from Steroids (Keene, NH); ovine LH (NIH
oLH-23), ovine FSH (NIH oFSH-76), and ovine PRL from the National
Hormone and Pituitary Program (Baltimore, MD); forskolin (10
µM dissolved in ethanol) from Calbiochem (San Deigo, CA);
and Biotrans nylon membrane (0.2 µm) and
[
32P]deoxy-CTP from ICN Biochemical (Cleveland, OH).
TNT-coupled reticulocyte lysate system, large fragments of
E.coli polymerase, T4 poly nucleotide kinase, and
restriction enzymes were purchased from Promega Corp. (Madison WI); T4
DNA ligase and ß-Agarase from New England Biolabs (Beverly, MA),
luciferin from BMB (Indianapolis, IN); coenzyme A from Pharmacia
(Piscataway, NJ); Immobilon-P from Millipore Corp. (Bedford, MA);
protein assay reagents and electrophoresis supplies from Bio-Rad
(Richmond, CA); tissue culture reagents from GIBCO (Grand Island NY);
rainbow molecular weight markers and the enhanced chemiluminescence
(ECL) detection system from Amersham (Arlington Heights, IL); and RNA
ladder (0.249.5 kb) from Bethesda Research Laboratories and Life
Technologies Inc. (Gaithersburg, MD). Immobolized protein A on tryacryl
beads was purchased from Pierce (Rockford, IL). A 3.5-kb fragment of
the
2M cDNA was obtained from 4.2 kb full-length clone isolated from
a rat liver cDNA expression library (46). This cDNA recognizes a single
transcript (5.2 kb) for the
2M message by Northern analysis in
tissues examined to date. Kodak film XAR-5 was from Eastman Kodak Co.
(Rochester, NY). COUP-TFI cDNA was kindly provided by Dr. Ming-Jer
Tsai, Baylor College of Medicine (Houston, TX). cDNA of Stat 5b and its
splice variant, Stat 5b
40C, were generous gifts from Dr. Georg H.
Fey, Department of Genetics, University of Erlangen-Nurenberg
(Erlangen, Germany). A rabbit antiserum IgG fraction generated against
the C-terminal peptide of Stat 5a was generously provided by Dr.
Jeffrey Rosen, Baylor College of Medicine (Houston, TX). Antibodies to
phosphotyrosine (PY20) were obtained from Transduction Laboratory
(Lexington, KY). Antibodies were obtained from several sources.
Antibodies to Stat 5b were generous gifts from Dr. Lothar
Henninghausen, Laboratory of Biochemistry and Metabolism, National
Institute of Diabetes, Digestive and Kidney Diseases, NIH (Bethesda,
MD); antibodies to SF-1 (Ad4BP) from Dr. Ken-ichirou Morohashi, Kyushu
University (Fukuoka, Japan); and antibodies to COUP-TF from Dr.
Constantin Flytzanis and Dr. Ming-Jer Tsai, Baylor College of Medicine
(Houston, TX).
Animals
Intact immature Sprague Dawley (23 days old) female rats were
obtained from Harlan (Indianapolis, IN) and maintained under a 16-h
light, 8-h dark schedule, with food and water ad libitum.
Animals were treated in accordance with the NIH Guide for the Care and
Use of Laboratory Animals. All protocols were approved by the
Institutional Committee on Animal Care and Use, Baylor College Of
Medicine (Houston, TX). Rats were delivered on day 23 of age and
weighed 5055 g. Rats were injected sc on days 2426 with 1.5 mg
17ß-estradiol in 0.2 ml propylene glycol.
Granulosa Cell Culture
Ovaries isolated from immature rats primed with 17ß-estradiol
were punctured with a 22-gauge needle to isolate granulosa cells. The
granulosa cells were pooled and treated with trypsin (20 µg/ml) for 1
min, followed by the addition of soybean trypsin inhibitor (300
µg/ml) and DNAse (100 µg/ml) to remove necrotic cells (47). After
washing the cells twice, they were cultured for 48 h in
DMEM-Hams F12 (DMEM:F12; 1:1) supplemented with a low dose of oFSH
(50 ng/ml) and T (10 ng/ml) at 37 C in 95% air and 5%
CO2. Granulosa cells were cultured for 24 h in the
absence of FSH and T or for 48 h in the presence of FSH and T (47, 48). At each time period, cells were treated with PRL (1 µg/ml) or
forskolin (10 µM). At indicated time points, total RNA
was isolated for Northern analyses, WCEs were prepared for EMSAs, and
immunoblots or the cells were used for transient transfections of
various expression vectors.
RNA Isolation and Northern Analysis
Extraction buffer containing 1% Nonidet P-40 was used for
isolation of cytoplasmic RNA from granulosa cells (49). RNA samples
were extracted with phenol-chloroform, ethanol precipitated,
resuspended in water containing 0.1% dimethyl pyrocarbonate, and
quantitated by measuring the absorbance at 260 nm. For Northern blot
analyses, 20 µg RNA were denatured at 55 C for 15 min in 45%
formamide-5.4% formaldehyde and resolved by electrophoresis at room
temperature in a formaldehyde-agarose gel. To confirm equal loading of
RNA samples and to determine the migration of the standards in the RNA
ladder, the gel was stained with acridine orange. After transfer of the
RNA to nylon membranes, the blots were baked, prehybridized, and
hybridized with P32-labeled
2M cDNA probe. Blots were
washed four times, 5 min/wash at room temperature in 2x saline sodium
citrate, 0.1% SDS, followed by two washes, 15 min each, in
0.1% saline sodium citrate and 0.1% SDS at 55 C and exposed to film
at -80 C.
Immunoprecipitations and Immunoblot Analyses
Protein A on tryacryl beads (30 µl) was incubated with 200
µg WCEs for 1 h at 4 C with shaking. After a brief spin, the
supernatant was incubated with 1 µl Stat 5b antibody and 20 µl
diluted beads for 24 h at 4 C with shaking. Each of the samples
was then washed three times with WCE buffer to remove unbound protein.
Bound proteins were eluted from the beads by addition of an equal
volume of SDS-loading buffer and boiled at 100 C for 10 min. Samples
were resolved by 10% one-dimensional SDS-PAGE at 20 mA for 67 h.
Electrophoretic transfer to Immobilon polyvinylidene difluoride
(PVDF) membrane was performed at 12 V overnight at 4 C. After
blocking with 5% milk for 2 h, filters were incubated with PY20
antiphosphotyrosine antibody (1:250). The filters were then washed with
Tris-buffered saline, 0.05% Tween (20 mM Tris, 150
mM NaCl, pH 7.5, 0.05% Tween) (TBST). The blots were
incubated with 1:10,000 of antirabbit-horseradish peroxidase or
antimouse-horseradish peroxidase. Filters were then washed six times
for 5 min each with TBST. ECL reagents were used to detect specific
proteins as described by Amersham. The same blot was stripped
(2% SDS, 50 mM ß-mercaptoethanol, 50 mM
Tris, pH 6.8) for 2 h at 55 C. After extensive washes with TBST,
the blot was reprobed with anti-Stat 5b antibody (1:10,000).
Plasmid Constructs
The luciferase constructs pSVoA1.2 kb
2M (from -1152 to +54
bp) and the Stat mutants have been described previously (16). All the
luciferase constructs in Fig. 1
were generated by initially isolating
the 1.2-kb HindIII fragment from pSVoA1.2 kb
2M and
ligating it to pGL3 basic luciferase vector from Promega to obtain 1.2
kb
2M. -371
2M was generated by ligating the 425-bp fragment
(StuI:HindIII digest of the 1.2-kb fragment) to
pGL3 Basic SmaI:HindIII site. -209
2M was
generated by ligating the 298-bp fragment
(RsaI:HindIII digest of the 1.2-kb fragment) to
pGL3 Basic SmaI:HindIII site. -159
2M was
generated by digesting -371
2M with EcoRI and
SacI, which deletes the PRLRE. -209
2M was digested with
EcoRI:PvuII to generate
-209
2M in which
the ORRE is deleted. -48
2M, the minimal
2M promoter, was created
by a PvuII digest of -371
2M. 4XGAS
2M, containing four
copies of the proximal Stat-binding site, has been described previously
(16, 24). Stat 5b and a splice variant of Stat 5b (Stat 5b-40C) were
cloned to the expression vector pSVsport GIBCO BRL (Grandland,
NY) and have been described previously (32).
The mutations (indicated in lower case) within the ORRE
(Fig. 5a
) of the
2M promoter were created by PCR using the -317
2M as a template. The primers used include mutant antisense
oligonucleotides to either the COUP-TF (M1) site or the SF-1 site (M2)
and the external sense and antisense primers that would hybridize at
each end of the promoter region. The double mutant was generated with
the same external primers, mutant oligonucleotide, and the COUP-TF
mutant construct as the template. The sequence of the oligonucleotides
is as follows:
mCOUP-TF:
5'-ATtGttTAATTAttGCCAAGGTTAATTCCT-aAttCGTTAGtCAG-3'
mSF-1:
5'-ATGGCCTAATTACCGCCAAttTTAATTCCTGA-CCCGTTAGTCCAG-3'
double mutant: 5'-ATTGCCAAttTTAATTCCTAA-3'
sense: 5'-CGGGGTACCCCGCCTCCTTGCCAACTATT-CC-3'
antisense: 5'-GTGCAAACAGCTGCTCCCACCC-3'
Figure 11
is a schematic representation of the
PCR-directed mutagenesis. The cloning procedure was as described in PCR
protocols (50). The PCR products were separated and purified by 1.5%
low-melt agarose gel using an overnight ß-agarase digestion (50).
After the second round of PCR, the purified products were isolated,
digested with KpnI and PvuII, and ligated using
sites present in the multiple cloning cassette of -371
2M
construct. All plasmids were sequenced in both directions by Sangers
dideoxy method (50).
Transient Transfection Assay
Plasmid DNA was purified from bacteria by alkaline lysis and
centrifugation on CsCl gradients (51). Differentiated granulosa cells
in culture were transfected with different plasmid constructs using a
calcium phosphate precipitation technique (52). After 30 min incubation
at room temperature, 300 µl of the precipitate were added to the
cultures (3 ml) and incubated for 4 h at 37 C. Each construct was
transfected in triplicate for each treatment. SV40 luciferase construct
containing the SV40 promoter enhancer elements was used as a positive
control in each of the transfection experiments. Cells were then washed
twice with Ca++- and Mg++-free HBSS before
feeding with fresh DMEM/F12 media containing FSH (50 ng/ml) and T (10
ng/ml) with or without PRL (1 µg/ml) or forskolin (10
µM) for 6 h. Cells were then washed with 1x PBS and
lysed with lysis buffer (0.2 M Tris, pH 8, 0.2% Triton
X-100). The soluble protein obtained after centrifugation was assayed
for luciferase activity using a Monolight 2010 Luminometer (Analytical
Luminesence Laboratories, San Diego, CA). The luciferase activity in 20
µl of each sample was normalized to the amount of protein, as
measured by the Bradford assay (Bio-Rad), and expressed as relative
light units/µg protein.
Oligonucleotides and Nomenclature
The functionally active PRLRE of the
2M promoter contains two
Stat- binding sites, AACTGGAAA and TTCTGGGAA (GAS) sequences, as
described previously (24, 10). The sequence of
2MGAS2
oligonucleotide used in EMSA is
5'-GATCATCCTTCTGGGAATTCTGATATCCTTCTGGGAATTC-TG3'.
The ORRE sequence shown in Fig. 5A
was used to generate mutant
oligonucleotides:
M1:
5'-CTGaCTAACGaaTtAGGAATTAACCTTGGCAATAA-TTAaaCaAT-3'
M2: 5'-ATGGCCTAATTACCGCCAAttTTAATTCCTGACCC-GTTAGCCAG-3'
Sequences of other oligonucleotides used in EMSAs are as follows:
DR1: 5'-CTGGCTAACGGGTCAGGAA-3'
DR2: 5'-TGGCGGTAATTAGGCCAT-3'
DR1SF-1: 5'-CTGGCTAACGGGTCAGGAATTAACCTTGG-3'
DR2SF-1: 5'-GGAATTAACCTTGGCGGTAATTAGGCCAT-3'
Consensus COUP-TF: 5'-TGTCTTAGAGGTCAAAGGTCAAAT-3' (28)
Hexameric SF-1: 5'-GAGTCTCCCAAGGTCATCCTTGT-TT-3' (6)
Oligonucleotides were synthesized, purified, and provided by Genosys,
Inc. (Houston, TX).
Whole Cell Extracts and EMSA
Granulosa cells were cultured with FSH and T for 24 h or
48 h and then treated with or without PRL (1 µg/ml) for 15 min,
6 h, and 20 h. The cells were harvested by scraping and lysed
by three cycles of freeze thawing to prepare soluble extracts (53).
Protein samples were measured (Bradford; Bio-Rad). All samples were
stored at -70 C until EMSA and immunoblot analyses were done. EMSAs
were performed as described previously (54). Briefly, WCE (1015 µg
per lane) were incubated for 30 min at room temperature with
50,00060,000 cpm of end-labeled double-stranded oligonucleotide with
or without the competitor DNA and 5 µg
poly(deoxyinosinic-deoxycytidylic)acid in a final volume of 20
µl in a buffer containing 100 mM KCl, 15 mM
Tris-HCl (pH 7.5), 5 mM dithiothreitol, 1 mM
EDTA, 5 mM MgCl2, and 12% glycerol. When
antibodies were used, WCEs were incubated with 1 µl of the antiserum
for 30 min on ice before addition of the labeled probe. Binding
reactions were resolved by 5% acrylamide, 0.5x TBE (0.5 M
Tris-0.5 M boric acid-0.01 M EDTA) buffer. Gels
were dried under vacuum and heat and exposed to Kodak XAR film at -80
C.
RT-PCR
For each RT reaction, 350 ng RNA were reverse transcribed as
previously described (55, 56). PCR was performed as previously
described (55). The sequence of primers used for the PRLRs is as
follows:
Forward primer: 5'-ATACTGGAGTAGATGGAGCCAGGAGAGTTC-3' and specific
Reverse LPRLR: 5'-CTTCCGTGACCAGAGTCACTGTCG-GGATCT-3'
Reverse SPRLR: 5'-TCCTATTTGAGTCTGCAGCTTCAGTAGTCA-3'
Primers for L19 were used as an internal control. PCR was performed for
20 cycles (Perkin-Elmer/Cetus, Norwalk, CT) with a denaturing
temperature of 92 C (1 min), an annealing temperature of 60 C (2 min),
and an extension temperature of 72 C (3 min). Reaction (20 µl) was
electrophoresed on a 5% polyacrylamide gel in 0.5x TBE buffer. The
gels were dried and the bands quantified with Storm860 PhosphoImager
(Molecular Dynamics, Sunnyvale, CA). Specific PCR products
amplified for the LPRLR-422 bp and the SPRLR-332 bp were normalized to
the L19 band from the same sample. Gels were also exposed to HyperFilm
(Amersham) for 624 h at room temperature.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Jeffrey Rosen and Dr. Ming-Jer
Tsai, Department of Cell Biology, Baylor College of Medicine, for their
helpful suggestions and reagents that were provided during the course
of these experiments. The authors also thank Dr. Lothar Henninghausen,
Laboratory of Biochemistry and Metabolism, National Institute of
Diabetes, Digestive and Kidney Diseases, NIH (Bethesda, MD) for the
antibodies to Stat 5b, and Dr. Ken-ichirou Morohashi, Kyushu University
(Fukuoka 812, Japan) for the SF-1 (Ad4BP) antibodies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. e-mail: joanner@ bcm.tmc.edu.
Received for publication February 20, 1998.
Revision received April 22, 1998.
Accepted for publication May 12, 1998.
 |
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