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Department of Molecular and Integrative Physiology University of Kansas Medical Center Kansas City, Kansas 66160-7401
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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T3–1, and the importance of these factors
and cis-acting elements in regulation of the promoter by
GnRH. All four members of the Egr family were found to induce activity
of the equine promoter. The region responsible for induction by Egr was
localized to the proximal 185 bp of the promoter, which contained two
Egr response elements. Coexpression of Egr1 and SF-1 led to a
synergistic activation of the equine (e)LHß promoter. Mutation of any
of the Egr or SF-1 response elements attenuated this synergism.
Endogenous expression of Egr1 in T3–1 cells was not detectable
under basal conditions, but was rapidly induced after GnRH stimulation.
Reexamination of the promoter constructs harboring mutant Egr or SF-1
sites indicated that these sites were required for GnRH induction. In
fact, mutation of both Egr sites within the eLHß promoter completely
attenuated its induction by GnRH. Thus, GnRH induces expression of
Egr1, which subsequently activates the eLHß promoter. Finally, GnRH
not only induced expression of Egr1, but also its corepressor, NGFI-A
(Egr1) binding protein (Nab1), which can repress Egr1- induced
transcription of the eLHß promoter. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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-subunit noncovalently linked to a unique ß-subunit that
confers biological activity (2, 3).
It has been known for more than two decades that the hypothalamic
factor, GnRH, stimulates the synthesis and secretion of gonadotropins
from the anterior pituitary gland. Mice that are homozygous for a
mutation in the gene encoding GnRH (hpg) are sexually
immature and have arrested germ cell development (4). These mice lack
detectable levels of GnRH, leading to low pituitary levels of LH and
FSH. Administration of exogenous GnRH to hpg mice restored
pituitary levels of - and LHß-subunit mRNAs as well as LH content
and secretion (5). A similar phenomenon exists in humans (Kallmann’s
syndrome) where GnRH neurons fail to migrate from the olfactory placode
into the hypothalamus (6). These individuals lack hypothalamic
secretion of GnRH and as a consequence are infertile due to
hypogonadotropic hypogonadism (lack of synthesis and secretion of LH
and FSH).
Not only is GnRH critical for synthesis and secretion of gonadotropins, the pattern in which it is secreted has a profound impact on gonadotrope function. GnRH is released in a pulsatile fashion from the hypothalamus. The frequency and amplitude of the GnRH pulses vary with sex of the individual, age, and physiological state (7, 8). Alterations in the GnRH pulse profile can change the secretory profile for LH and FSH as well as the biosynthesis of the gonadotropin subunits (9, 10, 11).
Resolution of the molecular mechanisms responsible for
pituitary-specific expression and GnRH regulation of the LHß-subunit
gene has lagged behind studies of LH secretion. Homozygous disruption
of the Ftz-F1 gene, which encodes the orphan nuclear
receptor steroidogenic factor-1 (SF-1), resulted in the loss of -,
LHß, and FSHß expression in mice (12). The bovine and rat LHß
promoters have been shown to be regulated by SF-1 (13, 14). Mutation of
the single SF-1 site in the bovine promoter severely attenuated
activity of this construct and disrupted its regulation by GnRH in
transgenic mice (14). Thus, SF-1 plays a role in regulating activity of
the LHß-subunit gene.
Targeted disruption of the immediate-early response gene, early
growth response protein 1 (Egr1), has been reported to result in the
selective loss of LH synthesis and secretion (15, 16). Expression of
the LHß subunit gene was severely, if not completely, diminished
while little or no change was observed in steady-state mRNA levels for
the -subunit. A subsequent study has identified two Egr1 DNA
response elements in the rat LHß promoter, and they appear to be
conserved within the LHß promoters of other species (17). The
response elements within the rat promoter bind Egr1, enabling Egr1
to functionally interact with SF-1 (binding to adjacent sites)
to transactivate the LHß promoter (15, 17). Based on its homology
with other LHß promoters, the bovine promoter contains two putative
Egr sites and a single SF-1 site. Mutation of this SF-1 site disrupted
GnRH regulation of the transgene in vivo. The importance of
the Egr sites in GnRH induction of the bovine promoter has not been
determined.
Expression of Egr1, also called NGFIA (18), Zif268 (19), and Krox24 (20), can be induced through activation of the protein kinase C (PKC) and mitogen- activated protein kinase (MAPK) pathways (21). These are also pathways activated by GnRH in gonadotropes (22, 23, 24, 25, 26, 27). In the present study we investigated the role played by Egr1 in regulating activity of the equine (e)LHß promoter and determined the involvement of Egr1 in GnRH induction of eLHß. Mutation of the Egr sites within the equine promoter reduced or completely attenuated the ability of Egr1 to transactivate the promoter. Data are also presented indicating that Egr1 and SF-1 act in a synergistic manner to regulate LHß promoter activity. Furthermore, both Egr and SF-1 sites are required for full activation of the equine LHß promoter by GnRH. Finally, evidence is presented suggesting that GnRH can induce Egr1 expression as well as that of the corepressor, Nab1, which represses transcription induced by Egr1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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). Furthermore, this promoter was
responsive to low levels of Egr1 (Fig. 1B) and was maximally responsive
to 100 ng of Egr1 (data not shown). As little as 3 ng of the Egr1
expression vector elicited an increase in activity of the eLHß
promoter. Egr1 was equally effective in transactivating eLHß
constructs containing 2200, 448, 387, and 185 bp of 5'-flanking
sequence (Fig. 1C). The 185-bp promoter contains both of the Egr sites
previously identified in the rat promoter (Fig. 2). Furthermore, these LHß Egr sites
are highly homologous across species. Truncation of the eLHß
promoter from -185 to -100 severely attenuated the ability of Egr1 to
transactivate the promoter (Fig. 1C). Interestingly, the -100
construct retained the proximal Egr site; nonetheless, it was
unresponsive to Egr1. These data tend to suggest that the proximal Egr
site is not functional in the eLHß promoter.
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T3–1 cells (28). The role
played by the Egr response elements in maintaining basal activity of
the eLHß promoter was assessed either individually or in combination
after mutation of these sites. Surprisingly, none of these
mutations adversely affected basal promoter activity (Fig. 3).
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T3–1 cells, but can induce
promoter activity when overexpressed (28). Previous studies have
indicated that Egr1 and SF-1 can interact in solution and that they
work in a synergistic manner to transactivate the rat LHß promoter
(15, 17). Based on these data, the functional importance of the SF-1
and Egr sites for achieving full transactivation of the eLHß promoter
by Egr1 was evaluated. Mutation of either Egr site severely attenuated
the ability of Egr1 to transactivate the eLHß promoter (Fig. 4). Mutation of both Egr sites completely
blocked responsiveness to Egr1. Similarly, mutation of either SF-1 site
attenuated the ability of Egr1 to induce eLHß promoter activity (Fig. 4). The double Egr or SF-1 mutations completely eliminated the
synergism observed between these two factors in transactivating the
eLHß promoter. Furthermore, it appeared that the synergism was also
lost when only a single SF-1 site was disrupted. These data suggest
that both Egr sites and both SF-1 sites are required to achieve full
transactivation of the eLHß promoter by Egr1.
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T3–1 CellsT3–1 cells was only revealed in an
overexpression model. The ability of Egr1 to specifically bind to these
Egr sites was assessed by performing electrophoretic mobility shift
assays (EMSAs) using oligodeoxynucleotides representing the distal and
proximal Egr sites (eßdEgr and eßpEgr, respectively; Fig. 5A). Each oligo also contained its
adjacent SF-1 binding site. The equine Egr sites are highly homologous
to a consensus Egr site (Fig. 5A) and to the rat LHß Egr sites, which
were previously shown to bind in vitro translated Egr1 (17).
Surprisingly, these investigators were unable to detect Egr1 binding to
these sites using T3–1 nuclear extracts. Our initial attempts at
detecting Egr binding to the eßpEgr were also unsuccessful (Fig. 5B).
The proximal Egr probe did interact with SF-1 and a slower migrating,
unidentified protein complex. Binding of this additional complex could
not be competed for by inclusion of a 50-fold molar excess of two
different consensus Egr sites (Fig. 5B, lanes 5 and 6). Inclusion of
antibodies to Sp1, Sp3, SF-1, or Egr1 (Fig. 5B, lanes 8–10) had little
effect on the complex. Binding was also unaffected by antibodies
against Egr2 or 3 (data not shown). The proximal Egr site in the rat
LHß promoter differs from the homologous equine site by a single
nucleotide (CACCCCCAC vs. CtCCCCCAC for rat and equine,
respectively) and has been shown to bind in vitro translated
Egr1 (17) and respond to Egr1 when overexpressed (15, 17). Therefore,
either this single nucleotide change completely disrupted Egr1 binding
or T3–1 cells fail to express Egr1.
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, lane
1), but weakly interacted with two other complexes (data not shown).
This suggested that T3–1 cells do not express Egr1, which supports
the data of others (17). In other cell lines it has been shown that
expression of Egr1 is low or absent under basal conditions but can be
induced after an acute stimulation with the PKC activator, phorbol
12-myristate 13-acetate (PMA) (21). Therefore, nuclei were
isolated from T3–1 cells that had been stimulated with PMA (1
µM) for 1 h and subsequently evaluated for the
presence of Egr proteins by EMSA. Treatment of T3–1 cells with PMA
resulted in the appearance of a new complex (Fig. 5C, lane 2) that
could be supershifted by inclusion of an antibody to Egr1 (Fig. 5C, lane 3). Furthermore, based on EMSAs using the cEgr probe, Egr1 was the
only Egr protein induced by PMA in T3–1 cells (Fig. 5C, lane 3 and
data not shown).
The PMA-stimulated T3–1 nuclei were subsequently used to evaluate
Egr1 binding to the labeled eßdEgr and eßpEgr oligos (Fig. 5C, lanes 4–9). Stimulation of T3–1 cells with PMA resulted in the
appearance of a new protein complex on the distal (lane 5) and proximal
(lane 8) eLHß Egr oligos that could be supershifted by the Egr1
antibody (lanes 6 and 9, respectively). The supershifted complex was
evident after a longer exposure of the gel (data not shown). Binding of
this complex to the cEgr, eßdEgr, and eßpEgr was unaffected by
inclusion of an antibody to Sp1 (data not shown). In support of
previous findings (17), it is interesting to note that an SF-1/Egr1/DNA
ternary complex was not observed in these experiments. Thus, both Egr
sites in the eLHß promoter can bind Egr1, but this appears to be a
weak interaction as compared with that observed with the cEgr
probe.
A series of competitions were conducted to determine the relative
affinities of the LHß Egr sites as compared with that of the
consensus Egr (Fig. 5, D and E). A 5-fold molar excess of unlabeled
cEgr (homologous competitor) reduced binding of Egr1 by 76% (Fig. 5D, lane 1 vs. 2). In contrast, a 50-fold molar excess of the
Santa Cruz Consensus Egr (scEgr) competitor was required to
reduce binding by 65% (Fig. 5E, lanes 1 vs. 5), suggesting
that the affinities of these consensus sites differed by at least
10-fold. Addition of increasing amounts of either the distal or
proximal eLHß Egr site competed for Egr1 binding to the cEgr probe
(Fig. 5D, lanes 6–15); however, a larger molar excess was required,
indicating a weaker interaction (lower affinity) as compared with the
cEgr and scEgr sites. A 250-fold molar excess of the distal or proximal
site was required to reduce Egr1 binding to the cEgr probe by 46% and
33%, respectively. Mutation of the Egr site within the eßpEgr
sequence disrupted its ability to compete for Egr1 binding (Fig. 5E, lanes 6–10). Finally, an Sp1 response element that has some similarity
to an Egr site was a fairly ineffective competitor (Fig. 5E, lanes
11–15).
Mutation of the Egr and SF-1 Sites within the eLHß Promoter
Disrupts Induction by GnRH
We have shown that activation of PKC via PMA can induce expression
of Egr1 in T3–1 cells; however, the importance of the Egr1 and SF-1
response elements for GnRH induction of the eLHß promoter has not
been determined. The promoter constructs shown in Fig. 4 were
reevaluated for their responsiveness to GnRH. Mutation of both Egr
sites completely blocked GnRH induction of the eLHß promoter (Fig. 6). This appeared to be predominantly due
to mutation of the distal Egr site. Disruption of SF-1 binding through
mutagenesis of both SF-1 sites also reduced GnRH responsiveness of
eLHß from an induction of 11-fold to 3-fold. Mutation of the distal
SF-1 site had no detrimental effect alone, while mutation of the
proximal site reduced responsiveness to GnRH. Interestingly, the -100
eLHß promoter retained 50% of the responsiveness to GnRH as compared
with the -448 construct.
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Transient transfections were performed to determine the ability of Nab1
to repress Egr transactivation of the eLHß promoter (Fig. 7). Overexpression of Nab1 repressed Egr1
transactivation of the eLHß promoter in a dose-dependent manner, but
had no effect on basal activity of the promoter. These data indicate
that Egr1 can transactivate the eLHß promoter and that the
corepressor Nab1 can disrupt transactivation by Egr1.
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demonstrate that PMA can indeed induce expression of
Egr1 in T3–1 cells. Therefore, based on these observations and
those described above, expression of Egr1, Nab1, and SF-1 was evaluated
in T3–1 cells at various time points after stimulation with GnRH.
Initial experiments revealed that GnRH induced a rapid, yet transient,
increase in RNA transcripts for Egr1, characteristic of an
immediate-early response gene (Fig. 8).
Increased expression of Egr1 was evident by 10 min, peaked at 50 min,
and had returned to unstimulated levels by approximately 180 min after
stimulation with GnRH. The presence of Nab1 in T3–1 cells and the
ability of GnRH to induce its expression, as well as that of SF-1, were
subsequently evaluated. Basal expression of Nab1 was observed in
T3–1 cells and was induced 2.6-fold by GnRH. This increase was
delayed as compared with that observed for Egr1 (Fig. 9A). GnRH induction of Nab1 was first
evident at 90 min and continued to increase through 180 min. In
contrast, GnRH did not appear to induce expression of SF-1 (Fig. 9B).
In fact, the data suggest that levels of the SF-1 mRNA tended to
decrease over time. Data from several experiments were combined and are
summarized in Fig. 9E. These data indicate that GnRH stimulation of
T3–1 cells results in a transient burst in expression of Egr1,
little or no change in SF-1 expression, and a somewhat delayed
induction of the Egr1 corepressor, Nab1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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T3–1 cells with GnRH results in activation of
the PKC and MAPK pathways and can lead to induction of expression of
immediate-early gene products such as c-jun and
c-fos (32, 33). In the present study we have examined the
ability of the immediate-early gene product, Egr1, and the orphan
nuclear receptor, SF-1, to regulate transcriptional activity of the
equine LHß promoter. Similar to the rat LHß promoter (17), the
eLHß promoter contains two Egr and two SF-1 sites. These sites are
located within the proximal 185 bp of the promoter. This segment of the
promoter is the shortest tested to date that still maintains full
responsiveness to Egr1 and SF-1 in an overexpression model.
Interestingly, the -185/+60 portion of the eLHß promoter also
retains full responsiveness to GnRH (our unpublished data).
The initial indication that Egr1 played a role in gonadotrope function
was discovered after targeted mutagenesis of the Egr1 gene (15).
Disruption of Egr1 expression led to fertility problems in these mice,
which was later determined to be due to a diminished or complete lack
of expression of LHß. These findings were subsequently confirmed by
another laboratory (16). It is interesting to note that the loss of
Egr1 expression in pituitary gonadotropes was not compensated for by an
increase in expression of the other Egr family members, or, if this did
occur, they could not functionally replace Egr1 in regulating
expression of LHß. Our data would indirectly indicate that Egr2,
Egr3, and Egr4 are not expressed in pituitary gonadotropes. All four
Egrs can transactivate the eLHß promoter and presumably the mouse
LHß promoter. One would predict that if present in gonadotropes, they
could functionally replace Egr1 after disruption of the Egr1 gene.
Furthermore, Egr1 was the only Egr protein that we could detect in
T3–1 cells. These findings and those from the gene disruption
studies strongly support the contention that Egr1 is the only family
member expressed in pituitary gonadotropes.
The two Egr sites within the eLHß promoter appear to be of low
affinity. Egr1 binding to the distal and proximal sites was extremely
weak as compared with that of the consensus Egr site. We estimate these
sites to have affinities that are 10-fold weaker than the scEgr site
and approximately 100-fold weaker than the cEgr. This occurred even
though there was a considerable amount of homology between these sites.
The guanosine located at position 4 in the equine sites is a
conservative change that should not have a dramatic effect on Egr1
binding (34). The remaining deviations from the consensus site, and in
particular the cytosine at position 1 in the distal LHß Egr site,
must be more deleterious to Egr1 binding. Nonetheless, the Egr sites in
the eLHß promoter are functional. It has been previously reported
that the affinity of the proximal rat LHß Egr site to Egr1 is 10-fold
greater than that of the distal site (17). However, the affinity of
Egr1 to the rat LHß sites was not compared with that of a consensus
Egr site. We would predict that the Egr sites on the LHß promoters
from other species would have similar characteristics. The distal
eLHß Egr site is completely homologous to the distal Egr sites in the
bovine and human LHß promoters. The proximal Egr in the eLHß
promoter differs by a single nucleotide from that in the bovine and
human, which also differ at the same nucleotide position from the
proximal Egr in the rat and mouse LHß promoters (Fig. 2). None of
these sites are completely homologous to the high-affinity Egr response
element, GCG(T/G)GGGCG, identified previously (34).
Our data indicate that the two Egr sites identified in eLHß are the
only ones present within the proximal 448 bp and potentially 2200 bp of
the promoter. Furthermore, these data as well as those from Halvorson
et al. (17) indicate that the presence of two Egr sites
makes the promoter more responsive to Egr and that this is more than an
additive effect. In the present work, this can be partially explained
by the fact that the T3–1 cell line was used, which
endogenously expresses SF-1, and that SF-1 and Egr1 act synergistically
to increase promoter activity. Synergism between Egr1 and SF-1 is
supported by the fact that coexpression of Egr1 and SF-1 led to a
greater than additive induction of eLHß promoter activity and was
lost upon mutation of either the Egr or SF-1 binding sites. These
findings are in agreement with those of others (15, 17). Furthermore,
it has been reported that Egr1 and SF-1 can interact in solution in the
absence of DNA; however, we and others (17) have been unable to detect
similar protein-protein interactions in an EMSA. The previous study
used in vitro translated proteins and, therefore, the lack
of formation of a ternary complex in an EMSA should not have been due
to limiting amounts of protein. It is unknown as to whether other
proteins are required to stabilize this complex and, hence, visualize
it in an EMSA. If this is the case, they must be limiting in our EMSA
conditions. Thus, the molecular events responsible for eliciting the
Egr1-SF-1 synergism are currently obscure.
Data from the present study demonstrate that the eLHß promoter is
responsive to GnRH in T3–1 cells. Responsiveness to GnRH was
dependent upon the presence of the Egr and SF-1 sites in the eLHß
promoter. Mutation of both Egr or SF-1 sites severely attenuated the
ability of GnRH to transactivate the eLHß promoter. Disruption of
either Egr site individually had a slight negative impact on GnRH
induction, suggesting that only one site was required. In contrast,
mutation of the proximal SF-1 site had a more dramatic effect on GnRH
induction than did the distal site. Furthermore, retention of the
proximal Egr and SF-1 sites resulted in a response to GnRH that was
approximately 50% of that of a construct containing all four sites
plus additional 5'-flanking sequence. Thus, a minimum of one Egr and
SF-1 site is required for GnRH induction of LHß promoter
activity.
Two groups have recently identified regions of the rat LHß promoter that are required for induction of the promoter by GnRH and can convey GnRH responsiveness to a heterologous promoter (26, 35). A region upstream of -245 was identified by both groups, while a more proximal segment (-207/-82) was determined to augment responsiveness of the upstream region (35) or not to be required at all (26). It was concluded that a GnRH response element(s) was located within the distal portion (-491/-352) of the rat promoter (35). Interestingly, GnRH was able to induce the rat promoter in the absence of SF-1, and the addition of SF-1 increased basal activity, but not fold induction by GnRH. Data from a subsequent study indicated that the -491/-352 region of the rat LHß promoter contained multiple Sp1 sites (36). Sp1 was shown to interact with these sites, and mutation of two of the Sp1 sites reduced GnRH induction of the promoter by 50%. It should be pointed out that these later studies (27, 35, 36) were performed in GH3 cells. This is a rat pituitary somatolactotropic cell line (37, 38) that does not have GnRH receptors (these studies used an overexpression model to get the GnRH receptor expressed) and as such may not truly represent responses in gonadotropes. Furthermore, GH3 cells do not express SF-1 (35). Thus, an important transcription factor that is known to be expressed in gonadotropes and involved in expression of the LHß subunit gene is absent in GH3 cells.
Data presented from the present study are some of the first to identify
specific DNA response elements that are responsible for GnRH induction
of an LHß-subunit promoter in a gonadotrope cell line. Furthermore,
they reinforce the previously shown interactions between Egr1 and SF-1.
However, they differ somewhat from that obtained through use of the
overexpression model (Fig. 4, Egr1 + SF-1 compared with Fig. 6). We
suspect that this is due to differences in the manner and extent of
expression of Egr1 and SF-1 (48 h vs. a transient increase
or no increase at all) and potentially due to GnRH activation of
additional intracellular events.
The PKC and MAPK pathways are known activators of Egr1 expression (21).
We demonstrate that GnRH induces a transient burst in expression of
Egr1, characteristic of an immediate-early gene product. GnRH did not
induce expression of SF-1 in T3–1 cells. In fact, our data suggest
that steady-state levels of SF-1 mRNA declined after stimulation with
GnRH. These findings are in contrast to data indicating that GnRH can
increase SF-1 mRNA levels by approximately 2-fold (39). These data were
obtained using an in vivo model and pulsatile administration
of GnRH, which could account for the discrepancies with our data.
Nonetheless, our data indicate that GnRH induction of eLHß in
T3–1 cells can be partially accounted for by an increase in
expression of Egr1 with little change in basal expression of SF-1. It
should be pointed out that SF-1 is expressed in T3–1 cells in the
absence of stimulation, whereas Egr1 is not. Therefore, the limiting
component for achieving the synergism between SF-1 and Egr1 that leads
to the induction of LHß is an increase in expression of Egr1. If Egr1
and SF-1 are both induced by GnRH in vivo, this would result
in an even stronger response of the LHß promoter to GnRH.
GnRH not only induced expression of Egr1, it also induced expression of the Egr1 corepressor, Nab1. Furthermore, both of these nuclear factors can regulate activity of the eLHß promoter. Nab1 was initially identified due to its ability to interact with a 34-amino acid domain of Egr1 and to repress transcription mediated by Egr1 (29). A subsequent study determined that Nab1 functioned as a corepressor by binding to Egr1 and actively repressing gene transcription (31). Nab1 is expressed at low levels in most tissues of the adult mouse (29) and has been suggested to be expressed in a constitutive manner (30). Therefore, we were surprised by the fact that GnRH induced its expression. The time course for GnRH induction of Nab1 was delayed as compared with Egr1 and was reminiscent of serum stimulation of Nab2 expression in NIH 3T3 cells (30). Furthermore, although continuous exposure to GnRH was used in the current study, it is tempting to speculate that the frequency of GnRH stimulation (pulses) will determine the pattern of expression of Egr1 and Nab1, and this may dictate whether transcription of Egr-responsive genes is induced or repressed.
These data provide some important insights into the mechanisms
responsible for GnRH induction of the LHß-subunit promoter. Our data,
as well as that of others, highlight the importance of both Egr1 and
SF-1 in regulating the activity of the LHß-subunit promoter. The
exquisite regulation of the LHß gene by GnRH in vivo may,
in fact, be linked to these two transcription factors. This is
supported by data from the targeted disruption of the SF-1 and Egr1
genes. We believe that the temporal pattern of expression for these
factors after stimulation by GnRH dictates how different GnRH pulse
profiles ultimately influence expression of the LHß-subunit gene.
Thus, the GnRH pulse profile that is most effective in inducing LHß
expression would maximize the expression or activity of activating
factors while minimizing the expression or activity of repressing
factors. This can be illustrated using the data shown in Fig. 9E.
Maximal induction of the LHß promoter by GnRH would require that the
pulses occur at a frequency such that Egr1 is present when Nab1 is low
or that Egr1 is induced to a greater extent than Nab1. Alternate
patterns of GnRH pulses may result in inadequate levels of Egr1 and a
lack of LHß induction or elevated levels of Egr1 occurring when Nab1
levels are also elevated, resulting in repression of the LHß
promoter. Finally, it is important to point out that this is an
oversimplification of GnRH induction of the LHß-subunit gene. Other
transcription factors may be involved, and it is equally likely that
GnRH induces posttranslational modifications in some of these
proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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Plasmid Constructs
The pGL2 basic plasmid was used as a reporter vector for all of
the promoter constructs used in this study. The eLHß promoter
constructs containing various lengths of 5'-flanking sequence and those
containing mutant SF-1 sites have been described previously (28).
The eLHß promoter constructs that contained Egr mutations were made in the context of the -448/+60 promoter in a manner similar to the SF-1 mutant constructs (28). Mutation of the distal Egr site was accomplished by PCR using an upstream oligodeoxynucleotide located in the pGL2 vector (GL1, Promega Corp.), a downstream mutant oligodeoxynucleotide encompassing bases -113 to -73 and eß -448/+60 Luc as template. The dEgr site was mutated from CGCCCCCGG to CGCtgCaGG. This should disrupt binding of the first and second zinc fingers of Egr1 to this response element (34). The PCR product was digested with SstI and BstEII and subsequently gel isolated (-343 to -78). The eß -448/+60 Luc vector was digested with SstI and BglII, and both fragments were isolated. The smaller fragment (-343 to the BglII site 5' to the luciferase gene) was digested with BstEII, and the BstEII/BglII fragment was isolated. The SstI/BglII-digested eß -448/+60 Luc vector, the BstEII/BglII fragment, and the SstI/BstEII PCR fragment were subsequently ligated to generate eßµdEgr Luc.
The proximal Egr site was mutated by a similar PCR strategy. Two PCR reactions were performed using eß -448/+60 Luc as template. The first used GL2 (downstream oligodeoxynucleotide located in luciferase) and an oligodeoxynucleotide encompassing the proximal Egr (bases -64 to -27, sense strand), while the second reaction used GL1 and an antisense oligodeoxynucleotide encompassing the same region of the promoter (-64/-27). The oligodeoxynucleotides encompassing -64 to -27 mutated the pEgr from CTCCCCCAC to tatttCtag. The GL1 to µpEgr PCR product was digested with SstI and XbaI (cuts within the Egr mutation), while the µpEgr to GL2 PCR product was digested with XbaI (cuts within the Egr mutation) and BglII. These fragments were subsequently ligated into the eß -448/+60 Luc vector previously digested with SstI and BglII. Positive clones were evaluated for the correct orientation. Both Egr mutant clones were sequenced to confirm that the appropriate mutations had been made and to ensure that random point mutations had not been generated in the native sequence during PCR (44, 45).
The double-Egr mutant promoter was generated as follows. The eßµdEgr Luc and eßµpEgr Luc plasmids were digested with BstEII to excise the portion of DNA between -78 in the promoter and the BstEII site within the 5'- end of luciferase. The BstEII fragment from eßµpEgr Luc was isolated and ligated into the homologous region of the eßµdEgr Luc plasmid. This essentially replaced the segment of DNA containing the wild-type pEgr with the mutant pEgr.
Overexpression experiments involving SF-1 used a mouse cDNA (46) that had been subcloned into an expression vector that contained the Rous sarcoma virus (RSV) long-terminal repeat to drive expression (28). The Egr and Nab expression vectors were obtained from the laboratory of Dr. Jeffrey Milbrandt and were driven by cytomegalovirus (CMV) promoter sequences (29, 30). In addition, a 1.8-kb fragment from the CMV-NGFIA (Egr1) vector was isolated and subcloned into the previously mentioned RSV expression vector. A similar construct containing a globin cDNA or a CMV expression vector was used as a control in the overexpression experiments.
Cell Culture and Transient Transfections
Cultures of T3–1 cells (47) were plated in DMEM with 5%
FBS, 5% horse serum, and antibiotics. On the day before transfection,
T3–1 cells were plated at a density of 1.8 x 105
cells per well in six-well plates. Cells were transfected with up to
1.5 µg of plasmid DNA (luciferase reporter with or without expression
vectors), 400 ng of RSVßGal (internal control of transfection
efficiency), and 7 µl of LipofectAmine (Life Technologies) according to the manufacturer’s recommendations
(28). Cell lysates were harvested 2 days post transfection. For
experiments involving GnRH induction, DNA constructs were transfected
as described above with the following additions. On the afternoon
before harvesting, the culture medium was replaced with serum-free
DMEM, and the cells were maintained in this medium overnight.
Approximately 16–18 h after the initiation of serum-free conditions,
the medium was replaced with serum-free DMEM containing 10
nM GnRH (des-Gly10,[D-Ala6]-GnRH
ethylamide, Sigma Chemical Co.). Cell lysates were
harvested 8 h later. Plasmid constructs were evaluated in
triplicate within each transfection, and transfections were performed a
minimum of three times.
Reporter Assays
Luciferase assays were performed following the protocol for the
Promega luciferase assay system (Promega Corp.). Briefly,
cells were washed twice in PBS and harvested in 150 µl of reporter
lysis buffer (Promega Corp.). After the addition of
luciferase assay buffer (100 µl), relative luciferase activity (20
µl of lysate) was measured for 10 sec in a Berthold Lumat LB 9501
luminometer (Wallac, Inc., Gaithersburg, MD). The
Galacto-Light ß-galactosidase reporter gene assay system (Tropix,
Bedford, MA) was used to analyze ß-galactosidase activity. Reaction
buffer (100 µl) was added to cell lysates (20 µl) and incubated at
room temperature for 1 h. Light emission accelerator (150 µl)
was added, and light emission was measured for 5 sec in a luminometer.
Luciferase activity (relative light units, RLU) was normalized to the
activity of ß-galactosidase (RLU).
Nuclear Preparation and EMSAs
Nuclei were prepared from T3–1 cells according to the
methods of Hagenbuchle and Wellaur (48). Cells were 70–80%
confluent when harvested and had been maintained in normal media,
serum-free media for 16–18 h, or serum starved for 16–18 h followed
by a 60 min stimulation with 1 µM PMA. Nuclei were
diluted to a concentration of 2–4 x 105 nuclei/µl
and stored at -80 C. Oligodeoxynucleotides (shown in Fig. 5) were
end-labeled with [32P]ATP using T4 kinase. These labeled
DNAs were used as probes in EMSAs. Nuclei (1–2 µl) were incubated
for 15 min at room temperature in binding buffer (10 mM
HEPES, pH 7.9, 100 mM KCl, 5 mM
MgCl2, 10 µM ZnCl2, 1
mM EDTA, 10% glycerol) containing 0.5 µg poly (dA-dT),
0.25 µg poly (dI-dC), and 0.1 µg salmon sperm DNA. Labeled probe
(25–50 fmol) and competitor were then added and incubated for an
additional 15 min at room temperature (total reaction volume of 20
µl). The DNA-protein complexes were resolved on a 4% native
polyacrylamide gel (pre-run for 
30 min) in 0.5x Tris-borate-EDTA
buffer. The gel was subsequently transferred to blotting paper
(Schleicher & Schuell, Inc., Keene, NH) and dried, and
autoradiography was performed using reflection autoradiography film
(New England Nuclear, Boston, MA). In experiments in which antibodies
were used, sera (1–2 µl) were added to the reaction 30 min before
addition of labeled probe. The reaction was allowed to incubate for an
additional 15 min after inclusion of labeled probe. For competition
experiments, Egr1 binding to the cEgr probe was quantitated using a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale,
CA).
RNA Isolation and Northern Blots
T3–1 cells were grown until 70–90% confluent, serum
starved for 16–18 h, and subsequently stimulated with 10
nM GnRH for 10 min to 4 h. At selected time points,
cells were quickly washed with cold PBS and harvested in 5 ml of Trizol
reagent (Life Technologies). Total RNA was isolated
following the manufacturers protocol. Total RNA (10 µg) was
fractionated on a 1% agarose/formaldehyde gel. The RNA was blotted,
hybridized, washed, and stripped using standard protocols (45).
Membranes were hybridized with random oligodeoxynucleotide
primer-generated 32P-labeled probes for Egr1, Nab1, SF-1,
and mouse ribosomal L7 protein to control for RNA loading. Membranes
were autoradiographed and subsequently quantitated using a
PhosphorImager. Blots shown are representative of at least three
different experiments unless otherwise noted.
|
Note Added in Proof |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNote Added in ProofREFERENCES |
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T3–1
cells and that this led to increased expression of the rat LHß
promoter (49).
|
ACKNOWLEDGMENTS |
|---|
T3–1 cell
line, Dr. Keith Parker for the SF-1 cDNA, Dr. Ulf Rapp for the RSV
expression vector, Dr. Leslie Heckert for the mouse L7 cDNA, and Dr.
Jeffery Millbrandt for the Egr and Nab expression vectors. |
FOOTNOTES |
|---|
This work was supported by NIH Grant R29-DK50668 (M.W.W.) and was performed with the assistance of the Imaging/Photography and Cell Culture Cores of the NIH-supported Center of Reproductive Sciences (P30-HD-33994).
Received for publication September 24, 1998. Revision received January 26, 1999. Accepted for publication February 3, 1999.
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M. W. Wolfe The Equine Luteinizing Hormone {beta}-Subunit Promoter Contains Two Functional Steroidogenic Factor-1 Response Elements Mol. Endocrinol., September 1, 1999; 13(9): 1497 - 1510. [Abstract] [Full Text]
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