| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Cell Biology Baylor College of Medicine Houston, Texas 77030
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
One of the mechanisms by which FSH regulates transcription of the aromatase gene involves stimulation of the cAMP-signaling cascade that activates A-kinases and leads to the phosphorylation of key substrates including specific transcription factors (1, 2). Two regions within the rat aromatase promoter have been identified to mediate cAMP-induced expression in granulosa cells (10, 11, 12). These same two regions are also present in the promoter II of the human aromatase gene (13). The most proximal element (region A) lies between -90 and -66 bp and binds Steroidogenic Factor-1 (SF-1) or adrenal binding protein (Ad4BP), an orphan receptor member of the steroid/thyroid hormone receptor superfamily (14, 15). The hexameric SF-1-binding site [(C/A) AGGTCA] is present within a large number of steroidogenic genes (14, 15) and has been shown to be critical for their transcriptional activation in adrenal and gonadal cells. Targeted deletion of the SF-1 gene results in mice that lack gonads and adrenals and exhibit abnormal gonadotrope development (16), indicating that the expression and activity of SF-1 are critical during early embryonic development and likely involve SF-1-regulated expression of tissue-specific genes in addition to those encoding steroidogenic enzymes (17). In the adult rat ovary, the levels of SF-1 mRNA and protein are relatively constant (10, 11, 18) suggesting that posttranslational modifications as well as transcriptional control may be important for mediating the functional activity of SF-1.
The distal element of the aromatase promoter (region B) lies between
-161 and -138 bp and contains a cAMP-response element (CRE)-like
sequence (TGCACGTCA) (19, 20). This region has been shown to bind
CRE-binding protein (CREB) along with two additional proteins
designated as X and Y (12). Because mutations that prevent binding of
all three proteins result in a decrease in promoter activity (12), we
hypothesized that either CREB alone or that factors X and Y (with
putative functions redundant of CREB) were responsible for the
transactivation of promoter activity at this site. This hypothesis was
based on the known complexity of the CREB/ATF superfamily (21), on the
presence of multiple, alternatively spliced forms of CREB in different
tissues, and on the fertility of the original CREB knockout mice
(21, 22, 23). More recently, Blendy et al. (24) demonstrated
that although the original CREB knockout mice did not express CREB 
-
or 
-isoforms, the levels of CREB ß as well as the CREB/ATF family
member, CRE modulator, were increased. Therefore, factors X and Y that
bind the aromatase CRE region were considered potential members of the
CREB/activating transcription factor (ATF) family.
The functional activity of CREB is dependent upon the phosphorylation of serine 133 (25) that occurs primarily by activation of A-kinase. CREB phosphorylation occurs rapidly (within minutes) after stimulation of cells with hormones or cAMP (25), and the phosphorylation site is antigenic (26). Furthermore, the ability of phosphoCREB to transactivate gene expression involves binding to the coactivator CBP (CREB-binding protein; Refs. 27–29). Although recent reports have also shown that SF-1 can be phosphorylated by A-kinase (30), it is not yet known whether this phosphorylation is obligatory for transcriptional activity. As indicated above, regulation of the aromatase gene in cells is complex. It can be induced in granulosa cells by low levels of FSH/cAMP, rapidly and dramatically down-regulated in these same cells by high levels of LH and cAMP, or expressed at elevated, cAMP-independent levels in an R2C Leydig cell line. These distinct patterns of aromatase expression indicated that several steps and signaling pathways might regulate either the content or activity of factors, such as CREB and SF-1, that bind to regions A and B of the aromatase gene promoter.
Based on the foregoing observations this study was undertaken to delineate whether specific interactions of factors binding to region A and B mediate cAMP-responsive and constitutive activation of the rat aromatase promoter in granulosa cells and R2C Leydig cells, respectively. To accomplish this goal, mutations of region A and region B were generated within the context of an intact promoter. Mutants within the CRE-like sequence were generated to determine the functional role of CREB and whether factors X and Y exerted redundant activities. Mutations within the hexameric SF-1 site were generated to determine whether SF-1 interacted with CREB for transactivation of the aromatase promoter. To determine whether changes in the content and phosphorylation of CREB and SF-1 were related to the activation of the aromatase gene, Western blot analyses, immunocytochemical localization, immunoprecipitation, and electrophorectic mobility shift assays (EMSAs) were performed. Results of these studies indicate that regions A and B interact in an additive manner in granulosa cells and in a synergistic manner in R2C Leydig cells, that levels of phosphoCREB are related to cAMP induction in granulosa cells and constitutive expression in R2C Leydig cells, and that loss of aromatase mRNA is associated with a marked decrease in SF-1 in response to the ovulatory LH surge. Thus, changes in either the phosphorylation state of CREB or the content of SF-1 appear to exert a major impact on transcriptional activation of the endogenous gene, further underscoring the importance of the functional interactions of regions A and B in mediating aromatase gene expression.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
; Ref.12). To elucidate which nucleotides within the
CRE-like region are responsible for the binding of CREB and factors X
and Y, mutant oligonucleotides were synthesized (Fig. 1B) and tested by
EMSAs using nuclear extracts from granulosa cells of preovulatory
follicles (HEF; Fig. 2), in which expression of
aromatase has been shown to be the highest (7). As in previous studies
(12), three protein/DNA complexes corresponding to CREB, X, and Y were
formed when the intact -161 oligonucleotide was used as a labeled
probe (Fig. 2, A-C; lane 2). All three complexes were competed by the
addition of cold competitor -161 oligonucleotide, whereas only the
upper band was competed by an authentic unlabeled CRE oligonucleotide
(Ref. 12; Fig. 2A; lanes 3 and 4). The faster migrating bands are
nonspecific. The binding of all three complexes was also inhibited by
the addition of unlabeled -158/-157 mutant (-158 M)
oligonucleotides (Fig. 2 B; lane 4). The -156/-155 mutant
oligonucleotide (-156M) failed to compete for the binding of CREB but
did compete for the binding of the other two factors, X and Y (Fig. 2B;
lane 5). Mutations of bases -155/-154 or -154/-153 (-155M
and -154M, respectively) rendered these oligonucleotides incapable of
competing for binding of CREB, X, or Y (Fig. 2B; lanes 6 and 7). EMSAs
were also performed using whole cell extracts from R2C Leydig cells and
yielded similar results (data not shown). When increasing
concentrations (50-, 100-, and 200-fold excess) of the competitor
oligonucleotides were added to the reactions, as little as 50-fold
excess of -161, -158M, and -156M competed for the binding of factors
X and Y (Fig. 2C). The binding of CREB was more effectively reduced by
-158M than -161 and was not altered by -156M. Collectively, these
data show that the binding of CREB can be functionally separated from
that of factors X and Y.
|
|
). When granulosa cells were transfected with the -161
arom CAT or -158M arom CAT constructs (which bind all three proteins)
a 6- to 8-fold increase in CAT activity was observed in the presence of
forskolin (Fig. 3A). The -156M arom CAT vector that binds factors X
and Y but not CREB exhibited 65% less forskolin-inducible activity
than the -161 arom CAT (Fig. 3A). Furthermore, the activity of the
-156M arom CAT construct was similar to that of constructs that do not
bind any of the factors, namely, the -154M arom CAT (Fig. 3A) and
-155M arom CAT (data not shown). The results of these transfection
assays indicate that CREB specifically transactivates cAMP inducibility
within region B of the promoter. In the absence of forskolin, all
constructs tested yielded CAT activity levels comparable to the
negative control construct (pCAT Basic), which contains no promoter
sequence.
|
). The
-161 and -158M constructs exhibited high CAT activity (in the absence
of forskolin); whereas, constructs lacking CREB binding (-154M and
-156M arom CAT) had minimal CAT activity (7% of -161 arom CAT).
These results indicate that CREB binding to region B is also required
for high constitutive expression of aromatase in R2C Leydig cells.
Cooperative Interactions Occur between CREB and SF-1 in Both
cAMP-Induced and Constitutive Expression of the Aromatase Promoter
SF-1 has previously been shown to be the factor binding to region
A of the rat aromatase promoter (Refs. 10 and 11; see Fig. 8). To
determine whether SF-1 binding in region A affected either cAMP-induced
or constitutive expression of aromatase, additional aromatase
promoter-CAT constructs were generated. The SF-1-binding site was
mutated either in the context of the intact -161 arom promoter that
contains the functional CRE or in the -95 arom CAT promoter that lacks
the CRE (Fig. 4). EMSAs were performed to verify that
the mutated hexameric motif (AGGTCA to ATTTCA) was unable to bind SF-1
(data not shown).
|
|
). Maximal activity (35% conversion) was
induced by forskolin in cells transfected with the intact -161 arom
CAT construct. Mutation of the SF-1-binding site (-161M arom CAT)
resulted in a 50% reduction in forskolin-induced CAT activity (Fig. 5A). The level of activity was comparable to that of the -156
M and -154M arom CAT constructs, both of which fail to
bind CREB. No induction of transgene activity by forskolin was observed
in vectors with double mutations, -156DM arom CAT and -154DM arom
CAT, which fail to bind CREB and SF-1. The promoter construct
containing the SF-1-binding site alone (-95 arom CAT) did exhibit a
slight but significant 2-fold induction of CAT activity in response to
forskolin. Mutation of the SF-1-binding site (-95M arom CAT) resulted
in complete loss of forskolin-induced CAT activity. These results
indicate that a cooperative or additive interaction between SF-1 and
CREB is required for cAMP activation of aromatase promoter in granulosa
cells.
|
). Whereas the -161 arom CAT exhibited
32% conversion, the -161M arom CAT construct lacking the SF-1-binding
site had only 2% conversion. Thus, mutation of the SF-1-binding site
in the presence of a functional CRE within region B resulted in a
complete loss of CAT activity. Likewise, vectors lacking a functional
CREB-binding site had little activity, and this was not reduced further
in the double mutants (DMs), -156DM arom CAT and -154DM arom CAT. The
-95 arom CAT had low activity that was reduced by mutation of the SF-1
site. These data indicate that both the CREB- and SF-1-binding sites
must be intact and interact in a synergistic manner for transactivation
of the aromatase promoter in R2C cells.
Phosphorylation of CREB and SF-1 in Granulosa Cells and R2C Leydig
Cells
Although CREB homodimers bind DNA at CRE-like sequences,
phosphorylation of CREB at serine 133 is required for it to
transactivate promoter activity (25). Serine 133 resides within a
substrate consensus site for protein kinase A, but other kinases are
also capable of phosphorylating CREB at this same residue (26, 31, 32, 33, 34).
To determine whether CREB was phosphorylated in response to increased
levels of cAMP in granulosa cells and was constitutively phosphorylated
in R2C Leydig cells, Western blot analyses were performed using
specific CREB and anti-phosphoCREB antibodies, the latter of which
recognizes CREB phosphoserine 133 (Ref. 26; Fig. 6). The
amount of CREB was essentially constant in granulosa cells cultured in
the absence of hormones or in the presence of either forskolin or FSH/T
for 0–48 h (Fig. 6A; upper left panel). Similar amounts of
CREB were also detected in R2C cells (Fig. 6A; upper right
panel). When these same samples were analyzed for the content of
phosphoCREB, the anti-phosphoCREB antibody recognized two distinct
immunoreactive proteins (Fig. 6A; lower panel). The 43-kDa
phosphoprotein (larger arrow; lower band) comigrated with
CREB and is considered, therefore, to be authentic CREB protein. The
52-kDa immunoreactive phosphoprotein (smaller arrow; upper
band) migrated more slowly than CREB, was not recognized by the
specific CREB antibody, and therefore is not CREB. Because the
anti-phosphoCREB antibody recognizes a phosphorylation region common to
other CREB family members (26), antibodies to other members of the CREB
family (CRE modulator, ATF-1) as well as other CREB antibodies were
tested, but none recognized the higher molecular weight protein (21, 26). Therefore, this protein is either yet another CREB/ATF family
member or is an unrelated protein that has a phosphorylation domain
similar to that present in CREB.
|
, lower panel; lane 1, large arrow). The addition
of forskolin caused an increase in immunoreactive phosphoCREB at 90 min
that decreased at 6 h but was again increased to a higher amount
at 48 h. Similar results were observed in response to FSH with the
exception that the secondary increase in phosphoCREB occurred as early
as 24 h and was maintained at 48 h. Because alterations in
the amount of phosphoCREB were not due to changes in the concentration
of CREB protein (Fig. 6A; upper panel), these results
indicate that the stimulation of cAMP production in granulosa cells
exerts a biphasic response with respect to CREB phosphorylation and
subsequent activation. Immunocytochemical localization studies showed
that high levels of CREB were present in nuclei of granulosa cells
cultured in the presence or absence of forksolin for 48 h (Fig. 6B; top panel) corresponding to Western analysis data (Fig. 6A; upper panel). However, granulosa cells cultured for
48 h in the presence of forskolin (+) expressed a higher level of
nuclear phosphoCREB as compared with those cultured without forskolin
(-) (Fig. 6B; lower panel). CREB and phosphoCREB were also
detected in cell extracts and nuclei of R2C Leydig cells (Fig. 6).
Interestingly, the highest levels of phosphoCREB, as detected by
Western blotting and indirect immunofluorescent labeling, were observed
in cells expressing the greatest amounts of aromatase mRNA (7, 9).
Because FSH/cAMP are necessary for expression of the endogenous gene in
granulosa cells (7) and for increased transcription transgenes
containing the SF-1 binding site in region A, we sought to determine
whether SF-1 was also phosphorylated in granulosa cells that express
high levels of aromatase mRNA in response to FSH. Accordingly, in
vivo phosphorylation studies were performed using granulosa cells
cultured for 48 h in the presence (+) or absence (-) of FSH (Fig. 7). A phosphoprotein of approximately 60 kDa
corresponding in size to SF-1 (see Fig. 8B) was immunoprecipitated
using an SF-1-specific antibody from cells cultured in the presence of
FSH (Fig. 7, + lane). A similar band corresponding to SF-1 was not
phosphorylated in cells cultured without FSH (- lane). Thus, SF-1 as
well as CREB appear to be an FSH/cAMP-dependent phosphoprotein.
|
). A specific SF-1 complex was formed when
oligonucleotides with the hexameric motif were incubated with preantral
(HE) and preovulatory (HEF) granulosa cell extracts (lanes 2 and 3) and
R2C Leydig cell extracts (lane 6). However, when human CG (hCG) was
given to proestrous rats to mimic the endogenous LH surge, the SF-1/DNA
complex was reduced by approximately 50% (lane 4) and 70% (lane 5) at
2 and 10 h, respectively, after treatment. A similar decline in
SF-1 binding was detected when whole cell extracts were prepared from
granulosa cells cultured for 48 h with FSH (preovulatory) and then
exposed to hCG for 2 or 10 h (data not shown). Second, Western
blot analyses were performed to determine whether loss of SF-1 binding
was due to changes in SF-1 protein content. A single immunoreactive
band corresponding to SF-1 at approximately 60 kDa was observed in
nuclear extracts of preovulatory granulosa cells (Fig. 8B, lane 1).
This immunoreactive SF-1 band was markedly reduced in nuclear extracts
from granulosa cells exposed to hCG for either 2 or 10 h (lanes 2
and 3). SF-1 was also detected in R2C Leydig whole cell extracts (lane
4).
When these same granulosa cell and R2C cell extracts were used in EMSAs
with a labeled aromatase CRE (-161/-138 bp of region A), three
binding complexes were observed with each sample (Fig. 8C).
Furthermore, no changes in amount of CREB binding (uppermost complex;
see Fig. 2A) were observed in preovulatory granulosa cells treated with
hCG (LH) (Fig. 8C). Likewise, the amount of immunoreactive CREB
remained constant during this time period (Ref. 35 and data not shown).
In addition, the phosphorylation state of CREB also did not vary as a
consequence of hCG (data not shown). Taken together, these data
indicate that loss of SF-1 but not CREB is associated with the decrease
in aromatase whereas increased phosphorylation of both factors is
required for induction of the aromatase gene.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
One intriguing aspect of the results obtained in these experiments is that the molecular mechanisms or pathways involved in cAMP activation of the aromatase promoter in granulosa cells differ from those controlling constitutive expression in R2C cells. By using selected mutants of the CREB and SF-1 binding sites within the context of the intact promoter, we show that CREB and SF-1 interact in an additive manner to confer cAMP responsiveness in granulosa cells, whereas they interact synergistically to confer high constitutive activity in R2C cells. Specifically, within granulosa cells, loss of binding of either factor in the context of the intact promoter reduced transgene activity by at least half, whereas complete loss of activity was achieved only when both CREB and SF-1 binding sites were ablated simultaneously. Although the involvement of SF-1 in the induction of a cAMP-responsive gene has been described elsewhere (30), this is the first study to demonstrate that the hexameric SF-1-binding site is required within the context of the intact promoter to provide maximal cAMP inducibility. In R2C Leydig cells that constitutively express high levels of aromatase, the binding of both CREB and SF-1 are critical for elevated, cAMP-independent expression of the transgenes. Aromatase promoter-CAT reporter constructs containing mutations within either CREB- or SF-1-binding sites or both exhibited little or no activity.
Not only are interactions of regions A and B of the aromatase promoter cell specific, but these interactions are associated with specific changes in the phosphorylation and amount of factors that regulate aromatase induction in the ovary by FSH, its down-regulation by LH, and its constitutive expression in R2C Leydig cells. Phosphorylation of CREB was associated with both cAMP induction of aromatase transgenes in granulosa cells and with their constitutive expression in R2C Leydig cells. Although there is abundant evidence that cAMP can activate A-kinase in granulosa cells (38) and transactivate genes containing CRE sites (36, 37), the kinase(s) responsible for the phosphorylation of CREB in the R2C Leydig cells have not yet been identified. Because forskolin (cAMP) does not regulate expression of the endogenous aromatase gene or the transgenes in R2C Leydig cells (9, 10), it is possible that A-kinase is maximally activated by factors present in the serum in which the cells are cultured. Alternatively, other kinases (e.g. C-kinase, calmodulin (CAM) kinase, RSK-2) that are capable of phosphorylating CREB at the serine 133 residue (31, 32, 33, 34), the site obligatory for transactivation (25) and antibody recognition (26), may be activated in R2C cells. Thus, it is possible that cellular signaling pathways other than, or in addition to, A-kinase phosphorylate and activate CREB in granulosa cells and R2C cells.
Phosphorylation of CREB is also dependent on cell differentiation. One
of the novel observations of these studies is that although the amount
of CREB remained constant during granulosa cell differentiation (Fig. 6A; Refs. 12 and 35), the phosphorylation of CREB was markedly elevated
in cells cultured in the presence of FSH or forskolin for 48 h.
Thus, the phosphorylation of CREB is not only rapid and transient (25)
but is subsequently increased and maintained as the cells
differentiate. As a consequence of this biphasic pattern of
phosphorylation in response to FSH, the highest levels of phosphoCREB
in granulosa cells were coincident with the maximal induction of the
endogenous aromatase gene (7) and the acquisition of the preovulatory
phenotype (1). Several factors may account for the greater
phosphorylation of CREB in the differentiated cells. First, although
stimulation of cAMP production by FSH and forskolin is greatest at 60
min in these cultured cells, the nucleotide continues to be synthesized
at elevated levels for 24–48 h (7, 39). Second, total A-kinase
activity may be increased. Previous studies have shown that the
concentrations of the regulatory type IIß (RIIß) subunit of
A-kinase is increased dramatically in preovulatory granulosa cells
in vivo and in vitro (8, 39, 40, 41). Although we
originally thought that the increase in this regulatory subunit might
serve as a sponge to reduce the sensitivity of granulosa cells to high
cAMP and thereby prevent premature luteinization, it is also possible
that these high levels of RIIß reflect a transient increase in total
kinase holoenzyme with the rapid dissociation of the subunits and the
transport of the activated C subunit to the nucleus. Such a hypothesis
would support the observations of McKnight and colleagues (42), who
have shown that overexpression of regulatory subunits leads to more
holoenzyme. Alternatively, the activity of a specific phosphatase may
be decreased as granulosa cells differentiate, thereby leading to
increased phosphorylation of CREB as well as other granulosa cell
proteins (43). These include RIIß (39, 44), SF-1 (Fig. 7), and a
protein that is antigenically distinct from CREB but which is
recognized by the phosphoCREB antibody (Fig. 6A). Whether this protein
is a member of the CREB family or an unrelated protein with a similar
antigenic phosphorylation site remains to be determined.
SF-1 is known to play a key role in ovarian cell function based on targeted disruption of the SF-1 gene in transgenic mice (16), altered function of granulosa cells cultured in the presence of antisense oligonucleotides (45), and the regulation of transcription of specific transgenes containing the hexameric SF-1-binding site (10, 18, 30). However, little is yet know about the phosphorylation of SF-1 and whether phosphorylation of this factor regulates its transcriptional activity. In the adult ovary and developing follicles, SF-1 mRNA and protein levels appear to be relatively constant (10, 11, 18), suggesting that activation of this orphan receptor occurs through a posttranslational modification. We show herein that granulosa cells cultured with FSH for 48 h contain similar levels of SF-1 but greater amounts of phosphorylated SF-1 than those cultured in the absence of FSH. It is not yet known whether this phosphorylation occurs as a consequence of A-kinase or whether it alters the activity of SF-1 in granulosa cells. However, Zhang and Mellon (30) have recently demonstrated that A-kinase can phosphorylate recombinant SF-1 in vitro on serine and threonine residues, thereby indicating that SF-1 is a putative substrate for A-kinase. Although the levels of SF-1 protein do not exhibit major changes during follicular growth or in response to low levels of FSH in culture (10), elevated levels of LH (high cAMP?) acting on differentiated, preovulatory granulosa cells cause a dramatic decrease in SF-1 protein. This loss of SF-1 protein is associated with the rapid loss of aromatase mRNA in granulosa cells of ovulating follicles. Because the amount of CREB and phosphoCREB did not change during this time (data not shown), the down-regulation of aromatase appears to be associated more with the loss of SF-1 than to any change in CREB. These results provide a physiological basis to support the pharmacological actions of antisense oligonucleotides that decrease SF-1 and aromatase in cultured cells (45).
In summary, these studies provide evidence that the additive interactions of regions A and B of the aromatase promoter in granulosa cells and the synergistic interactions of these same regions in R2C cells are dependent on the phosphorylation of CREB and SF-1, the amounts of these factors, as well as the presence (activation) of additional coregulatory molecules (46, 47, 48), one of which is likely to be such CREB binding protein, CBP.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Promoter Constructs
Construction of the -161 aromatase-CAT reporter construct has
been previously described (12). The mutant CRE aromatase promoter-CAT
reporter constructs (-158M, -156M, -155M, -154M) were generated by
chemical synthesis of oligonucleotides (Genosys, The Woodlands, TX)
containing the designated mutations within the -161/-138 region (Fig. 1). Annealed oligonucleotides were ligated into the -138 aromatase CAT
vector (digested with HindIII and XhoI),
previously described (12). Constructs containing the SF-1-binding site
mutation within the context of the intact region (-161M) or within the
CRE mutant constructs [DMs designated -156DM and -154DM] were
generated by chemical synthesis (Genosys) of a mutant oligonucleotide
corresponding to region A (-95/-50 bp) in which the SF-1-binding site
was mutated from AGGTCA to ATTTCA. The annealed mutant SF-1
oligonucleotide was ligated into the -161, -156M, or -154M aromatase
CAT vectors at the DRAII and APOI sites. To generate the -95 and SF-1
mutant (-95M) aromatase CAT vectors, the normal or mutant
oligonucleotide (-95/-50 bp) was ligated to the -50/+13 bp fragment
of the aromatase promoter and inserted into the pCAT basic vector. The
sequence and orientation of each mutant were confirmed by sequence
analyses and purified by a CsCl gradient. The pCAT Control vector
(Promega, Madison, WI), which contains the Simian Virus-40 promoter and
enhancer sequences, served as a positive control for transfection
reproducibility. The pCAT basic (Promega), which lacks eukaryotic
promoter and enhancer sequences, was used as control for basal vector
activity.
Electrophoretic Mobility Shift Assays
To test the ability of factors to bind these mutant
oligonucleotides, EMSAs were performed as previously described (48, 49). Briefly, nuclear extracts (NE) were prepared from granulosa cells
of hypophysectomized (H) rats treated sequentially with estradiol (HE;
preantral), FSH (HEF; preovulatory), and an ovulatory dose of hCG as
previously described (12). Oligonucleotides were labeled with
[P32]deoxy-CTP (3000 Ci/mmol, ICN, La Mesa, CA) and DNA
Polymerase-l (Klenow enyzme; large fragment, Promega, Madison, WI) to a
specific activity of 108. For studies investigating the
protein/DNA interactions at the CRE (region B) site, 5 µg NE protein
from preovulatory (HEF) granulosa cells were incubated with labeled
-161/-138 fragment (200 pg, 13 fmol) in the presence of 5.8 µg
poly(deoxyinosinic-deoxycytidylic)acid for 30 min at room temperature.
Specificity was analyzed by adding increasing concentrations of
unlabeled competitor DNA, either the aromatase CRE or a commercial
consensus CRE (Promega) as previously described (12). To determine SF-1
binding, 1.5 µg NE protein from granulosa cells of preantral (HE),
preovulatory (HEF), and ovulatory (hCG, 2 and 10 h) rats were
incubated with the labeled hexameric (-90/-66) motif (10).
Protein/DNA complexes were resolved on 5% polyacrylamide gels in 0.5x
TBE (0.089 M Tris borate, pH 8.3, and 2 mM
EDTA) at 150 V at 22 C after 30 min prerun. The gels were dried and
exposed to Kodak X-AR film at -80 C. Quantification of SF-1 binding
was determined using a Betascope 603 Blot Analyzer (Betagen Corp.,
Waltham, MA).
Transfection and CAT Assays
Granulosa cells were isolated from immature female rats (day 23
of age) (36, 39). Cells were cultured in 35-mm multiwell plates in 3 ml
DMEM-F-12 media (1:1, GIBCO/BRL, Gaithersburg, MD) supplemented with 20
mM HEPES (Curtain Matheson Scientific, Inc., Houston, TX),
100 IU/ml penicillin, 100 mg/ml streptomycin (Sigma, St. Louis, MO),
and 1% FBS (Hyclone, Logan, UT). The cells were transiently
transfected 18 h later using 15 µg plasmid DNA and the calcium
phosphate precipitation method (50). Four hours later the DNA was
removed, the cells were washed with HBSS
-Mg-Cl (GIBCO/BRL) and cultured in the
presence or absence of 10 µM forskolin (dissolved in
ethanol, Calbiochem, San Diego, CA). After 24 h, the cells were
lysed by freeze-thaw procedure, and the protein concentrations were
determined by the method of Bradford using the Bio-Rad (Richmond, CA)
protein assay. CAT activity was analyzed using 20 µg protein for
18 h according to a standard protocol (51). The amount of
radioactivity in the substrate and acetylated products was quantified
using the Betascope 603 Blot Analyzer.
R2C Leydig cells [American Type Cell Collection (ATCC), Rockville, MD] were grown in Ham’s F-10 (GIBCO/BRL) supplemented with 12.5% equine serum (Hyclone), 2.5% FBS (GIBCO/BRL), and 100 IU/ml penicillin, 100 µg/ml streptomycin. Cells were plated overnight at a density of 500,000 cells per 35-mm multiwell plate and transiently transfected in a manner similar to granulosa cells, with the exception that 4 h after DNA addition, cells were glycerol shocked (20% in HBSS +Mg+Cl, GIBCO/BRL) for 2 min at 37 C, washed with HBSS -Mg-Cl (GIBCO/BRL), and cultured for 48 h in the R2C medium mentioned above. CAT assays were performed using 10 µg protein for 2 h.
Western Blot Analysis
Western blot analyses of CREB and phosphorylated CREB
(phosphoCREB) were performed as described previously (26) using
specific antibodies that recognized CREB and phosphoCREB (serine 133).
Granulosa cells were isolated and cultured for varying amounts of time
(see Fig. 6 legend) in the presence or absence of ovine FSH (50 ng/ml,
NIH oFSH-16; National Hormone and Pituitary Program, Baltimore, MD) or
forskolin (10 µM) together with testosterone (10 ng/ml,
Steraloids, Keene, NH). R2C Leydig cells were cultured as described
above. After culture, cells were washed with PBS and lysed with boiling
SDS sample buffer [100 mM Tris (pH 6.8), 2% SDS, 20%
glycerol, 10% ß-mercaptoethanol, and bromophenol blue, 100 C]. Cell
extracts were then boiled (100 C) for 5 min. Samples were
electrophoresed on a 10% SDS-polyacrylamide gel by standard methods
(52). Proteins were transferred to a nitrocellulose membrane
(Schleicher & Schuell, Keene, NH) in a buffer containing 25
mM Tris, 192 mM glycine, and 20% methanol (53)
at 12 V for 18 h at 4 C. Blots were rinsed in Tris-buffered
saline-0.05% Tween-20 (TBS-T) and blocked with 4% BSA (BSA, Fraction
V, United States Biochemical Corp., Cleveland, OH) for 1 h at 22
C. The blots were incubated with rabbit anti-phosphoCREB IgG (1:5,000;
generously provided by Dr. David Ginty, Johns Hopkins University,
Baltimore, MD) in 4% BSA in TBS-T for 18 h at 4 C, with shaking.
To detect CREB, the blots were first blocked with 5% nonfat milk in
TBS-T for 1 h at 22 C, shaking. Blots were then incubated with
rabbit anti-CREB IgG (1:1000 in TBS-T, Ab 244 generously provided by
Dr. Marc Montminy, Salk Institute, La Jolla, CA) at 4 C for 18 h,
shaking. After several TBS-T washes, the presence of CREB or
phosphoCREB was detected using I125 protein A (1:1000
vol/vol in TBS-T, specific activity 1 mCi/ml, ICN) for 3 h at 22 C
with shaking. The blots were washed with TBS-T and exposed to x-ray
film.
Western blot analyses of SF-1 were performed as described previously (54). Briefly, nuclear extracts (10 µg) from granulosa cells of preovulatory (HEF) and ovulatory (HEF treated for 2 and 10 h with hCG) follicles, as well as 50 µg of whole cell extracts from R2C Leydig cells, were boiled (100 C) in an SDS-PAGE loading buffer [0.05 M cyclohexylaminoethane sulfonic acid, 2% SDS, 10% glycerol, 2% ß-mercaptoethanol, and bromophenol blue, pH 9.5] and electrophoresed on a 10% SDS-polyacrylamide gel by standard methods (52). Proteins were transferred to nitrocellulose membrane (Schleicher & Schuell) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol (53) at 12 V for 18 h at 4 C. The blots were rinsed in 140 mM NaCl, 10 mM KPO4, pH 7.5, 10 mg/ml BSA for 6 h at 22 C with shaking. The blots were then incubated with rabbit anti-Ad4BP (SF-1) IgG (1:10,000; generously provided by Dr. Ken-ichirou Morohashi, Kyushu University, Fukuoka, Japan) in 140 mM NaCl, 10 mM KPO4, pH 7.5, 10 mg/ml BSA, 0.1% Triton X-100, 0.02% SDS for 18 h at 4 C. The blots were washed in 140 mM NaCl, 10 mM KPO4, pH 7.5, 0.1% Triton X-100, 0.02% SDS at 22 C. After the washes, SF-1 was detected using [125I]Protein A (1:1000 in wash buffer; specific activity, 1 mCi/ml) for 3 h at 22 C with shaking. The blots were washed and exposed to x-ray film.
Immunocytochemistry
Granulosa and R2C Leydig cells were cultured as above on glass
coverslips for varying times in the presence or absence of FSH or
forskolin. Cells were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences, Fort Washington, PA) in PBS for 30 min at 22 C,
washed in 10 mM glycine in PBS, and treated with 0.5%
NP-40 in PBS. The cells were blocked with 3% BSA in PBS and incubated
with anti-phosphoCREB IgG (1:1000) or anti-CREB IgG (1:500) in 3% BSA
in PBS at 4 C for 18 h. After several PBS washes, cells were
incubated with fluorescein-labeled goat anti-rabbit IgG (1:20, Pierce,
Rockford, IL) in 3% BSA in PBS for 1 h at 22 C. CREB and
phosphoCREB were visualized on a Zeiss Axiophot microscope.
Phosphorylation and Immunoprecipitation of SF-1
Phosphorylation of granulosa cell proteins was performed as
previously described (39). Briefly, granulosa cells were cultured in
serum free DMEM:F12 in the presence or absence of FSH and testosterone
for 48 h. Cells were then washed and preincubated for 2 h in
phosphate-free DMEM (GIBCO). Fresh medium with FSH and testosterone and
2.5 mCi/ml of [32P]orthophosphate (ICN) were added for
2 h. The cells were then washed with PBS and resuspended in whole
cell extract buffer containing phosphatase inhibitors (55). The cells
were lysed by freeze-thaw procedure, and the protein concentrations
were determined by the method of Bradford using the Bio-Rad protein
assay. Immunoprecipitations were performed as follows. Protein A
Sepharose CL-4B beads (Sigma) were preswollen in PBS, 0.1% SDS, 0.2%
Triton X-100, and incubated with equal volumes of anti-Ad4BP IgG (1:100
in the above buffer) for 7 h at 4 C, with shaking. Unbound
antibody was removed by excess washing of the Sepharose beads. An equal
volume of the 32P-labeled whole cell extract protein (50
µg) was then incubated with the beads for 18 h at 4 C, with
shaking. Beads were washed with excess buffer to remove unbound labeled
proteins. Bound 32P-labeled proteins were eluted from the
beads by addition of an equal volume of SDS-PAGE loading buffer and
boiled at 100 C for 10 min. Samples were electrophoresed on a 10%
SDS-polyacrylamide gel by standard methods (52), dried, and exposed to
x-ray film.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by NIH Grants 16272 (to J.S.R.), HD-07165, and NRSA-HD-07991 (to D.L.C.).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158This article has been cited by other articles:
 |
|
 |
|
  R. Sirianni, A. Chimento, A. De Luca, F. Zolea, A. Carpino, V. Rago, M. Maggiolini, S. Ando, and V. Pezzi Inhibition of Cyclooxygenase-2 Down-regulates Aromatase Activity and Decreases Proliferation of Leydig Tumor Cells J. Biol. Chem., October 16, 2009; 284(42): 28905 - 28916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Hernandez Gifford, M. E. Hunzicker-Dunn, and J. H. Nilson Conditional Deletion of Beta-Catenin Mediated by Amhr2cre in Mice Causes Female Infertility Biol Reprod, June 1, 2009; 80(6): 1282 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Yang, J. H. Heaton, H. Brevig, S. Mukherjee, J. A. Iniguez-Lluhi, and G. D. Hammer SUMOylation Inhibits SF-1 Activity by Reducing CDK7-Mediated Serine 203 Phosphorylation Mol. Cell. Biol., February 1, 2009; 29(3): 613 - 625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Yivgi-Ohana, N. Sher, N. Melamed-Book, S. Eimerl, M. Koler, P. R. Manna, D. M. Stocco, and J. Orly Transcription of Steroidogenic Acute Regulatory Protein in the Rodent Ovary and Placenta: Alternative Modes of Cyclic Adenosine 3', 5'-Monophosphate Dependent and Independent Regulation Endocrinology, February 1, 2009; 150(2): 977 - 989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Lewis, M. Rusten, E. A. Hoivik, E. L. Vikse, M. L. Hansson, A. E. Wallberg, and M. Bakke Phosphorylation of Steroidogenic Factor 1 Is Mediated by Cyclin-Dependent Kinase 7 Mol. Endocrinol., January 1, 2008; 22(1): 91 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Stocco, J. Kwintkiewicz, and Z. Cai Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells J. Mol. Endocrinol., October 1, 2007; 39(4): 211 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sirianni, A. Chimento, R. Malivindi, I. Mazzitelli, S. Ando, and V. Pezzi Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation Cancer Res., September 1, 2007; 67(17): 8368 - 8377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Wayne, H.-Y. Fan, X. Cheng, and J. S. Richards Follicle-Stimulating Hormone Induces Multiple Signaling Cascades: Evidence that Activation of Rous Sarcoma Oncogene, RAS, and the Epidermal Growth Factor Receptor Are Critical for Granulosa Cell Differentiation Mol. Endocrinol., August 1, 2007; 21(8): 1940 - 1957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Priyanka and R. Medhamurthy Characterization of cAMP/PKA/CREB signaling cascade in the bonnet monkey corpus luteum: expressions of inhibin-{alpha} and StAR during different functional status Mol. Hum. Reprod., June 1, 2007; 13(6): 381 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sher, N. Yivgi-Ohana, and J. Orly Transcriptional Regulation of the Cholesterol Side Chain Cleavage Cytochrome P450 Gene (CYP11A1) Revisited: Binding of GATA, Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein and Activating Protein (AP)-1 Proteins to a Distal Novel Cluster of cis-Regulatory Elements Potentiates AP-2 and Steroidogenic Factor-1-Dependent Gene Expression in the Rodent Placenta and Ovary Mol. Endocrinol., April 1, 2007; 21(4): 948 - 962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kwintkiewicz, Z. Cai, and C. Stocco Follicle-Stimulating Hormone-Induced Activation of Gata4 Contributes in the Up-Regulation of Cyp19 Expression in Rat Granulosa Cells Mol. Endocrinol., April 1, 2007; 21(4): 933 - 947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-S. Wang, T. Kobayashi, L.-Y. Zhou, B. Paul-Prasanth, S. Ijiri, F. Sakai, K. Okubo, K.-i. Morohashi, and Y. Nagahama Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1 Mol. Endocrinol., March 1, 2007; 21(3): 712 - 725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O Gubbay, M T Rae, A S McNeilly, F X Donadeu, A J Zeleznik, and S G Hillier cAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival. J. Endocrinol., October 1, 2006; 191(1): 275 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. N. Parakh, J. A. Hernandez, J. C. Grammer, J. Weck, M. Hunzicker-Dunn, A. J. Zeleznik, and J. H. Nilson Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin PNAS, August 15, 2006; 103(33): 12435 - 12440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Luo and M. C. Wiltbank Distinct Regulation by Steroids of Messenger RNAs for FSHR and CYP19A1 in Bovine Granulosa Cells Biol Reprod, August 1, 2006; 75(2): 217 - 225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-Y. Chen, L.-J. Juan, and B.-c. Chung SF-1 (Nuclear Receptor 5A1) Activity Is Activated by Cyclic AMP via p300-Mediated Recruitment to Active Foci, Acetylation, and Increased DNA Binding Mol. Cell. Biol., December 1, 2005; 25(23): 10442 - 10453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Baba, J. Mimura, N. Nakamura, N. Harada, M. Yamamoto, K.-i. Morohashi, and Y. Fujii-Kuriyama Intrinsic Function of the Aryl Hydrocarbon (Dioxin) Receptor as a Key Factor in Female Reproduction Mol. Cell. Biol., November 15, 2005; 25(22): 10040 - 10051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. Bouchard, H. Taniguchi, and R. S. Viger Protein Kinase A-Dependent Synergism between GATA Factors and the Nuclear Receptor, Liver Receptor Homolog-1, Regulates Human Aromatase (CYP19) PII Promoter Activity in Breast Cancer Cells Endocrinology, November 1, 2005; 146(11): 4905 - 4916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Park, E. T. Maizels, Z. J. Feiger, H. Alam, C. A. Peters, T. K. Woodruff, T. G. Unterman, E. J. Lee, J. L. Jameson, and M. Hunzicker-Dunn Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad J. Biol. Chem., March 11, 2005; 280(10): 9135 - 9148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zheng and C. R. Jefcoate Steroidogenic Factor-1 Interacts with cAMP Response Element-Binding Protein to Mediate cAMP Stimulation of CYP1B1 via a Far Upstream Enhancer Mol. Pharmacol., February 1, 2005; 67(2): 499 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wu, S. Ghosh, Y. Nishi, T. Yanase, H. Nawata, and Y. Hu The Orphan Nuclear Receptors NURR1 and NGFI-B Modulate Aromatase Gene Expression in Ovarian Granulosa Cells: A Possible Mechanism for Repression of Aromatase Expression upon Luteinizing Hormone Surge Endocrinology, January 1, 2005; 146(1): 237 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stocco In Vivo and in Vitro Inhibition of cyp19 Gene Expression by Prostaglandin F2{alpha} in Murine Luteal Cells: Implication of GATA-4 Endocrinology, November 1, 2004; 145(11): 4957 - 4966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kuntz, D. Chardard, A. Chesnel, M. Ducatez, M. Callier, and S. Flament Expression of Aromatase and Steroidogenic Factor 1 in the Lung of the Urodele Amphibian Pleurodeles waltl Endocrinology, July 1, 2004; 145(7): 3111 - 3114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hiroi, L. K. Christenson, L. Chang, M. D. Sammel, S. L. Berger, and J. F. Strauss III Temporal and Spatial Changes in Transcription Factor Binding and Histone Modifications at the Steroidogenic Acute Regulatory Protein (StAR) Locus Associated with StAR Transcription Mol. Endocrinol., April 1, 2004; 18(4): 791 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Sriraman and J. S. Richards Cathepsin L Gene Expression and Promoter Activation in Rodent Granulosa Cells Endocrinology, February 1, 2004; 145(2): 582 - 591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Val, C. Aigueperse, B. Ragazzon, G. Veyssiere, A.-M. Lefrancois-Martinez, and A. Martinez Adrenocorticotropin/3',5'-Cyclic AMP-Mediated Transcription of the Scavenger akr1-b7 Gene in Adrenocortical Cells Is Dependent on Three Functionally Distinct Steroidogenic Factor-1-Responsive Elements Endocrinology, February 1, 2004; 145(2): 508 - 518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Fan, T. Yanase, Y. Wu, H. Kawate, M. Saitoh, K. Oba, M. Nomura, T. Okabe, K. Goto, J. Yanagisawa, et al. Protein Kinase A Potentiates Adrenal 4 Binding Protein/Steroidogenic Factor 1 Transactivation by Reintegrating the Subcellular Dynamic Interactions of the Nuclear Receptor with Its Cofactors, General Control Nonderepressed-5/Transformation/ Transcription Domain-Associated Protein, and Suppressor, Dosage-Sensitive Sex Reversal-1: a Laser Confocal Imaging Study in Living KGN Cells Mol. Endocrinol., January 1, 2004; 18(1): 127 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Peng, J. W. Kim, W. E. Rainey, B. R. Carr, and G. R. Attia The Role of the Orphan Nuclear Receptor, Liver Receptor Homologue-1, in the Regulation of Human Corpus Luteum 3{beta}-Hydroxysteroid Dehydrogenase Type II J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6020 - 6028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Falender, R. Lanz, D. Malenfant, L. Belanger, and J. S. Richards Differential Expression of Steroidogenic Factor-1 and FTF/LRH-1 in the Rodent Ovary Endocrinology, August 1, 2003; 144(8): 3598 - 3610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tajima, A. Dantes, Z. Yao, K. Sorokina, F. Kotsuji, R. Seger, and A. Amsterdam Down-Regulation of Steroidogenic Response to Gonadotropins in Human and Rat Preovulatory Granulosa Cells Involves Mitogen-Activated Protein Kinase Activation and Modulation of DAX-1 and Steroidogenic Factor-1 J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2288 - 2299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yazawa, T. Mizutani, K. Yamada, H. Kawata, T. Sekiguchi, M. Yoshino, T. Kajitani, Z. Shou, and K. Miyamoto Involvement of Cyclic Adenosine 5'-Monophosphate Response Element-Binding Protein, Steroidogenic Factor 1, and Dax-1 in the Regulation of Gonadotropin-Inducible Ovarian Transcription Factor 1 Gene Expression by Follicle-Stimulating Hormone in Ovarian Granulosa Cells Endocrinology, May 1, 2003; 144(5): 1920 - 1930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sadie, G. Styger, and J. Hapgood Expression of the Mouse Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Gonadotrope Cells Is Stimulated by Cyclic 3',5'-Adenosine Monophosphate and Protein Kinase A, and Is Modulated by Steroidogenic Factor-1 and Nur77 Endocrinology, May 1, 2003; 144(5): 1958 - 1971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Russell, K. M. H. Doyle, I. Gonzales-Robayna, C. Pipaon, and J. S. Richards Egr-1 Induction in Rat Granulosa Cells by Follicle-Stimulating Hormone and Luteinizing Hormone: Combinatorial Regulation By Transcription Factors Cyclic Adenosine 3',5'-Monophosphate Regulatory Element Binding Protein, Serum Response Factor, Sp1, and Early Growth Response Factor-1 Mol. Endocrinol., April 1, 2003; 17(4): 520 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Phillips, J. Bailey, S. C. Robson, and G. N. Europe-Finner Differential Expression of the Adenylyl Cyclase-Stimulatory Guanosine Triphosphate-Binding Protein Gs{alpha} in the Human Myometrium during Pregnancy and Labor Involves Transcriptional Regulation by Cyclic Adenosine 3',5'-Monophosphate and Binding of Phosphorylated Nuclear Proteins to Multiple GC Boxes within the Promoter J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5675 - 5685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wei, M. Sasaki, H. Huang, V. L. Dawson, and T. M. Dawson The Orphan Nuclear Receptor, Steroidogenic Factor 1, Regulates Neuronal Nitric Oxide Synthase Gene Expression in Pituitary Gonadotropes Mol. Endocrinol., December 1, 2002; 16(12): 2828 - 2839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Laurich, A. M. Trbovich, F. H. O'Neill, C. P. Houk, P. M. Sluss, A. H. Payne, P. K. Donahoe, and J. Teixeira Mullerian Inhibiting Substance Blocks the Protein Kinase A-Induced Expression of Cytochrome P450 17{alpha}-Hydroxylase/C17-20 Lyase mRNA in a Mouse Leydig Cell Line Independent of cAMP Responsive Element Binding Protein Phosphorylation Endocrinology, September 1, 2002; 143(9): 3351 - 3360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Val, A. Martinez, I. Sahut-Barnola, C. Jean, G. Veyssiere, and A.-M. Lefrancois-Martinez A 77-Base Pair LINE-Like Sequence Elicits Androgen-Dependent mvdp/akr1-b7 Expression in Mouse Vas Deferens, But Is Dispensable for Adrenal Expression in Rats Endocrinology, September 1, 2002; 143(9): 3435 - 3448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Eimerl and J. Orly Regulation of Steroidogenic Genes by Insulin-Like Growth Factor-1 and Follicle-Stimulating Hormone: Differential Responses of Cytochrome P450 Side-Chain Cleavage, Steroidogenic Acute Regulatory Protein, and 3{beta}-Hydroxysteroid Dehydrogenase/Isomerase in Rat Granulosa Cells Biol Reprod, September 1, 2002; 67(3): 900 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-J. Yang, M. Shozu, K. Murakami, H. Sumitani, T. Segawa, T. Kasai, and M. Inoue Spatially Heterogenous Expression of Aromatase P450 through Promoter II Is Closely Correlated with the Level of Steroidogenic Factor-1 Transcript in Endometrioma Tissues J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3745 - 3753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ben-Zimra, M. Koler, and J. Orly Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating Protein-2 Assumes the Role of Steroidogenic Factor-1 by Binding to an Overlapping Promoter Element Mol. Endocrinol., August 1, 2002; 16(8): 1864 - 1880. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Clyne, C. J. Speed, J. Zhou, and E. R. Simpson Liver Receptor Homologue-1 (LRH-1) Regulates Expression of Aromatase in Preadipocytes J. Biol. Chem., May 31, 2002; 277(23): 20591 - 20597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Borud, T. Hoang, M. Bakke, A. L. Jacob, J. Lund, and G. Mellgren The Nuclear Receptor Coactivators p300/CBP/Cointegrator-Associated Protein (p/CIP) and Transcription Intermediary Factor 2 (TIF2) Differentially Regulate PKA-Stimulated Transcriptional Activity of Steroidogenic Factor 1 Mol. Endocrinol., April 1, 2002; 16(4): 757 - 773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. R. Manna, M. T. Dyson, D. W. Eubank, B. J. Clark, E. Lalli, P. Sassone-Corsi, A. J. Zeleznik, and D. M. Stocco Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family Mol. Endocrinol., January 1, 2002; 16(1): 184 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. AEsoy, G. Mellgren, K.-I. Morohashi, and J. Lund Activation of cAMP-Dependent Protein Kinase Increases the Protein Level of Steroidogenic Factor-1 Endocrinology, January 1, 2002; 143(1): 295 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Salvador, Y. Park, J. Cottom, E. T. Maizels, J. C. R. Jones, R. V. Schillace, D. W. Carr, P. Cheung, C. D. Allis, J. L. Jameson, et al. Follicle-stimulating Hormone Stimulates Protein Kinase A-mediated Histone H3 Phosphorylation and Acetylation Leading to Select Gene Activation in Ovarian Granulosa Cells J. Biol. Chem., October 19, 2001; 276(43): 40146 - 40155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. O'Donnell, K. M. Robertson, M. E. Jones, and E. R. Simpson Estrogen and Spermatogenesis Endocr. Rev., June 1, 2001; 22(3): 289 - 318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. T. Maizels, A. Mukherjee, G. Sithanandam, C. A. Peters, J. Cottom, K. E. Mayo, and M. Hunzicker-Dunn Developmental Regulation of Mitogen-Activated Protein Kinase-Activated Kinases-2 and -3 (MAPKAPK-2/-3) in Vivo during Corpus Luteum Formation in the Rat Mol. Endocrinol., May 1, 2001; 15(5): 716 - 733. [Abstract] [Full Text]
|
|
|
|
|
|
D. Boerboom, N. Pilon, R. Behdjani, D. W. Silversides, and J. Sirois Expression and Regulation of Transcripts Encoding Two Members of the NR5A Nuclear Receptor Subfamily of Orphan Nuclear Receptors, Steroidogenic Factor-1 and NR5A2, in Equine Ovarian Cells during the Ovulatory Process Endocrinology, December 1, 2000; 141(12): 4647 - 4656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Gonzalez-Robayna, A. E. Falender, S. Ochsner, G. L. Firestone, and J. S. Richards Follicle-Stimulating Hormone (FSH) Stimulates Phosphorylation and Activation of Protein Kinase B (PKB/Akt) and Serum and Glucocorticoid-Induced Kinase (Sgk): Evidence for A Kinase-Independent Signaling by FSH in Granulosa Cells Mol. Endocrinol., August 1, 2000; 14(8): 1283 - 1300. [Abstract] [Full Text]
|
|
|
|
|
|
E. Barnea and Y. Bergman Synergy of SF1 and RAR in Activation of Oct-3/4 Promoter J. Biol. Chem., February 25, 2000; 275(9): 6608 - 6619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ito, Y. Park, J. Weck, K. E. Mayo, and J. L. Jameson Synergistic Activation of the Inhibin {alpha}-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate Mol. Endocrinol., January 1, 2000; 14(1): 66 - 81. [Abstract] [Full Text]
|
|
|
|
|
|
T. N. Alliston, I. J. Gonzalez-Robayna, P. Buse, G. L. Firestone, and J. S. Richards Expression and Localization of Serum/Glucocorticoid-Induced Kinase in the Rat Ovary: Relation to Follicular Growth and Differentiation Endocrinology, January 1, 2000; 141(1): 385 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Boerboom, A. Kerban, and J. Sirois Dual Regulation of Promoter II- and Promoter 1f-Derived Cytochrome P450 Aromatase Transcripts in Equine Granulosa Cells during Human Chorionic Gonadotropin-Induced Ovulation: A Novel Model for the Study of Aromatase Promoter Switching Endocrinology, September 1, 1999; 140(9): 4133 - 4141. [Abstract] [Full Text]
|
|
|
|
|
|
S. C. Sharma, J. W. Clemens, M. D. Pisarska, and J. S. Richards Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin Endocrinology, September 1, 1999; 140(9): 4320 - 4334. [Abstract] [Full Text]
|
|
|
|
|
|
K. Kawabe, T. Shikayama, H. Tsuboi, S. Oka, K. Oba, T. Yanase, H. Nawata, and K.-i. Morohashi Dax-1 as One of the Target Genes of Ad4BP/SF-1 Mol. Endocrinol., August 1, 1999; 13(8): 1267 - 1284. [Abstract] [Full Text]
|
|
|
|
|
|
I. J. Gonzalez-Robayna, T. N. Alliston, P. Buse, G. L. Firestone, and J. S. Richards Functional and Subcellular Changes in the A-Kinase-Signaling Pathway: Relation to Aromatase and Sgk Expression during the Transition of Granulosa Cells to Luteal Cells Mol. Endocrinol., August 1, 1999; 13(8): 1318 - 1337. [Abstract] [Full Text]
|
|
|
|
|
|
J. P. Somers, J. A. DeLoia, and A. J. Zeleznik Adenovirus-Directed Expression of a Nonphosphorylatable Mutant of CREB (cAMP Response Element-Binding Protein) Adversely Affects the Survival, but Not the Differentiation, of Rat Granulosa Cells Mol. Endocrinol., August 1, 1999; 13(8): 1364 - 1372. [Abstract] [Full Text]
|
|
|
|
|
|
D. Lopez, T. W. Sandhoff, and M. P. McLean Steroidogenic Factor-1 Mediates Cyclic 3',5'-Adenosine Monophosphate Regulation of the High Density Lipoprotein Receptor Endocrinology, July 1, 1999; 140(7): 3034 - 3044. [Abstract] [Full Text]
|
|
|
|
|
|
E. Silverman, S. Eimerl, and J. Orly CCAAT Enhancer-binding Protein beta and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells J. Biol. Chem., June 18, 1999; 274(25): 17987 - 17996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. LaVoie, J. C. Garmey, and J. D. Veldhuis Mechanisms of Insulin-Like Growth Factor I Augmentation of Follicle-Stimulating Hormone-Induced Porcine Steroidogenic Acute Regulatory Protein Gene Promoter Activity in Granulosa Cells Endocrinology, January 1, 1999; 140(1): 146 - 153. [Abstract] [Full Text]
|
|
|
|
|
|
M. Young and M. J. McPhaul A Steroidogenic Factor-1-Binding Site and Cyclic Adenosine 3',5'-Monophosphate Response Element-Like Elements Are Required for the Activity of the Rat Aromatase Promoter in Rat Leydig Tumor Cell Lines Endocrinology, December 1, 1998; 139(12): 5082 - 5093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Dajee, G. H. Fey, and J. S. Richards Stat 5b and the Orphan Nuclear Receptors Regulate Expression of the {alpha}2-Macroglobulin ({alpha}2M) Gene in Rat Ovarian Granulosa Cells Mol. Endocrinol., September 1, 1998; 12(9): 1393 - 1409. [Abstract] [Full Text]
|
|
|
|
|
|
J. W. Clemens, R. L. Robker, W. L. Kraus, B. S. Katzenellenbogen, and J. S. Richards Hormone Induction of Progesterone Receptor (PR) Messenger Ribonucleic Acid and Activation of PR Promoter Regions in Ovarian Granulosa Cells: Evidence for a Role of Cyclic Adenosine 3',5'-Monophosphate but Not Estradiol Mol. Endocrinol., August 1, 1998; 12(8): 1201 - 1214. [Abstract] [Full Text]
|
|
|
|
|
|
D. Yeagley, J. M. Agati, and P. G. Quinn A Tripartite Array of Transcription Factor Binding Sites Mediates cAMP Induction of Phosphoenolpyruvate Carboxykinase Gene Transcription and Its Inhibition by Insulin J. Biol. Chem., July 24, 1998; 273(30): 18743 - 18750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Eggers, G. Siemann, R. Blume, and W. Knepel Gene-specific Transcriptional Activity of the Insulin cAMP-responsive Element Is Conferred by NF-Y in Combination with cAMP Response Element-binding Protein J. Biol. Chem., July 17, 1998; 273(29): 18499 - 18508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Robker and J. S. Richards Hormone-Induced Proliferation and Differentiation of Granulosa Cells: A Coordinated Balance of the Cell Cycle Regulators Cyclin D2 and p27Kip1 Mol. Endocrinol., July 1, 1998; 12(7): 924 - 940. [Abstract] [Full Text]
|
|
|
|
|
|
A. Mukherjee, J. Urban, P. Sassone-Corsi, and K. E. Mayo Gonadotropins Regulate Inducible Cyclic Adenosine 3',5'-Monophosphate Early Repressor in the Rat Ovary: Implications for Inhibin {alpha} Subunit Gene Expression Mol. Endocrinol., June 1, 1998; 12(6): 785 - 800. [Abstract] [Full Text]
|
|
|
|
|
|
M. Shozu, Y. Zhao, S. E. Bulun, and E. R. Simpson Multiple Splicing Events Involved in Regulation of Human Aromatase Expression by a Novel Promoter, I.6 Endocrinology, April 1, 1998; 139(4): 1610 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Monte, F. DeWitte, and D. W. Hum Regulation of the Human P450scc Gene by Steroidogenic Factor 1 Is Mediated by CBP/p300 J. Biol. Chem., February 20, 1998; 273(8): 4585 - 4591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ito, R. N. Yu, and J. L. Jameson Steroidogenic Factor-1 Contains a Carboxy-Terminal Transcriptional Activation Domain That Interacts with Steroid Receptor Coactivator-1 Mol. Endocrinol., February 1, 1998; 12(2): 290 - 301. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Sadovsky and P. A. Crawford Developmental and Physiologic Roles of the Nuclear Receptor Steroidogenic Factor-I in the Reproductive System Reproductive Sciences, January 1, 1998; 5(1): 6 - 12. [Abstract] [PDF]
|
|
|
|
|
|
T. N. Alliston, A. C. Maiyar, P. Buse, G. L. Firestone, and J. S. Richards Follicle Stimulating Hormone-Regulated Expression of Serum/Glucocorticoid-Inducible Kinase in Rat Ovarian Granulosa Cells: A Functional Role for the Sp1 Family in Promoter Activity Mol. Endocrinol., December 1, 1997; 11(13): 1934 - 1949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Porter, B. Saville, D. Hoivik, and S. Safe Functional Synergy between the Transcription Factor Sp1 and the Estrogen Receptor Mol. Endocrinol., October 1, 1997; 11(11): 1569 - 1580. [Abstract] [Full Text]
|
|
|
|
|
|
T. Mizutani, K. Yamada, T. Minegishi, and K. Miyamoto Transcriptional Regulation of Rat Scavenger Receptor Class B Type I Gene J. Biol. Chem., July 14, 2000; 275(29): 22512 - 22519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Sharma and J. S. Richards Regulation of AP1 (Jun/Fos) Factor Expression and Activation in Ovarian Granulosa Cells. RELATION OF JunD AND Fra2 TO TERMINAL DIFFERENTIATION J. Biol. Chem., October 20, 2000; 275(43): 33718 - 33728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Pincas, J.-N. Laverriere, and R. Counis Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3',5'-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells J. Biol. Chem., June 22, 2001; 276(26): 23562 - 23571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Jacob, J. Lund, P. Martinez, and L. Hedin Acetylation of Steroidogenic Factor 1 Protein Regulates Its Transcriptional Activity and Recruits the Coactivator GCN5 J. Biol. Chem., September 28, 2001; 276(40): 37659 - 37664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
