Early Growth Response Protein 1 Binds to the Luteinizing Hormone-ß Promoter and Mediates Gonadotropin-Releasing Hormone-Stimulated Gene Expression

Michael W. Wolfe and Gerald B. Call

Department of Molecular and Integrative Physiology University of Kansas Medical Center Kansas City, Kansas 66160-7401


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH ß-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LHß-subunit promoter in the gonadotrope cell line, {alpha}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 {alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
Reproduction is critical for the perpetuation of a species. The pituitary gonadotropic hormones, LH and FSH, are important regulators of reproduction in mammals. LH and FSH are members of the glycoprotein hormone family that also includes TSH and the placental CGs (1). These hormones are heterodimeric in structure, composed of a common {alpha}-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 {alpha}- 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 {alpha}-, 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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
The eLHß Promoter Is Responsive to Egr
Overexpression of Egr1 or other members of the Egr family transactivated the -448/+60 eLHß promoter (Fig. 1AGo). Furthermore, this promoter was responsive to low levels of Egr1 (Fig. 1BGo) 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. 1CGo). The 185-bp promoter contains both of the Egr sites previously identified in the rat promoter (Fig. 2Go). 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. 1CGo). 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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Figure 1. Transactivation of the eLHß Promoter by Egr Proteins

Transient transfections were performed in {alpha}T3–1 cells to evaluate the ability of Egr1 and other Egr family members to regulate activity of the eLHß promoter. A, The -448/+60 eLHß promoter linked to luciferase (eß -448) or a promoterless control (pGL2) was cotransfected with a control expression vector or the indicated Egr expression vectors, and luciferase activity was measured. B, The ability of Egr1 to induce activity of the -448/+60 eLHß promoter in a dose-responsive manner was assessed by cotransfection of 3–100 ng of the Egr1 expression vector. C, The region of the eLHß promoter required for eliciting a response to Egr1 was determined by evaluating Egr1 induction of 5'-deletion mutants of the promoter. The data in panels A–C are expressed as fold induction (mean ± SEM) over that achieved after cotransfection with the control expression vector.

 


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Figure 2. DNA Sequence Alignment of the Proximal Promoter Regions from the Equine, Bovine, Human, Mouse, and Rat LHß Genes

Nucleotide sequences for the bovine (B; 38), human (H; 39), mouse (M; 40), and rat (R; 41) LHß promoters were aligned relative to that of the eLHß promoter (E; 43). The numbering below the sequence is based on the eLHß promoter with +1 representing the start site of transcription. Uppercase letters indicate promoter sequence while lowercase letters represent transcribed sequence. Shaded regions identify the distal (d) and proximal (p) sequences that have homology to SF-1 and Egr response elements.

 
It has previously been shown that, unlike other LHß promoters, the equine promoter is active basally in {alpha}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. 3Go).



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Figure 3. The Egr Response Elements Are Not Required for Basal Expression of the eLHß Promoter in {alpha}T3–1 Cells

Basal luciferase activity (relative light units, RLU; mean ± SEM) of a promoterless control vector as well as the -448/+60 eLHß promoters containing native sequence (eß -448) or mutations in the distal (eßµdEgr), proximal (eßµpEgr), or both (eßµdpEgr) Egr sites.

 
Egr1 and SF-1 Act Synergistically to Transactivate eLHß
A similar scenario occurs with SF-1 regulation of the eLHß promoter in this cell line. SF-1 does not play a major role in regulating basal expression of eLHß in {alpha}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. 4Go). 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. 4Go). 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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Figure 4. Transactivation of Wild-Type and Mutant eLHß Promoters by Egr1 and SF-1

Transient transfections were performed in {alpha}T3–1 cells to assess the ability of Egr1 and SF-1 alone or in combination to regulate activity of the eLHß promoter. The following promoters were evaluated: the -448/+60 eLHß promoter containing native sequence (eß -448), eß -448 with mutations in the distal (eßµdEgr), proximal (eßµpEgr), or both (eßµdpEgr) Egr sites, eß -448 with mutations in the distal (eßµdSF1), proximal (eßµpSF1), or both (eßµdpSF1) SF-1 sites, and the eß -100 promoter (-100/+60), which retains the proximal SF-1 and Egr sites. These constructs were also cotransfected with a control expression vector. The data are expressed as fold induction (mean ± SEM) over that achieved after cotransfection with the control expression vector.

 
The Distal and Proximal Egr Sites in the eLHß Promoter Can Bind Egr1 from Stimulated {alpha}T3–1 Cells
Although the eLHß promoter has two Egr sites, the functional significance of these sites in {alpha}T3–1 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. 5AGo). Each oligo also contained its adjacent SF-1 binding site. The equine Egr sites are highly homologous to a consensus Egr site (Fig. 5AGo) 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 {alpha}T3–1 nuclear extracts. Our initial attempts at detecting Egr binding to the eßpEgr were also unsuccessful (Fig. 5BGo). 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. 5BGo, lanes 5 and 6). Inclusion of antibodies to Sp1, Sp3, SF-1, or Egr1 (Fig. 5BGo, 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 {alpha}T3–1 cells fail to express Egr1.



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Figure 5. Egr1 Is Expressed in {alpha}T3–1 Cells after Stimulation with PMA and Can Bind to Both Egr Sites in the eLHß Promoter

A, Homology of the distal (eßdEgr) and proximal (eßpEgr) Egr sites in the eLHß promoter to that of a consensus Egr site. The nucleotides in bold lowercase letters represent bases that are not homologous with the consensus Egr. Shown below are the Egr oligodeoxynucleotides used in panels B–E including two consensus Egr oligos (cEgr and scEgr). The SF-1 and Egr response elements within these oligos are underlined. B, EMSA was performed using the proximal eLHß Egr as labeled probe and nuclei from {alpha}T3–1 cells cultured under normal conditions. Two protein complexes were observed and are indicated: SF-1 and one that is unknown (does not represent Sp1, Sp3, SF-1, or Egr1). Competitions (50-fold molar excess) were performed using oligos representing the proximal (pEgr), mutated proximal (µpEgr), and distal Egr sites in the eLHß promoter, the two different consensus Egr oligos (cEgr and scEgr), and an oligo representing the distal eLHß SF-1 site (dSF-1). Antibodies to Sp1, Sp3, SF-1, and Egr1 were used to identify proteins within the retarded complexes. The antibody to SF-1 disrupts DNA binding and as such does not result in a supershift. C, EMSAs were performed using the consensus Egr (cEgr), distal eLHß Egr (eßdEgr), and proximal eLHß Egr (eßpEgr) as labeled probes and {alpha}T3–1 nuclei isolated from serum-starved cells with or without simulation with PMA for 1 h. An antibody to Egr1 was used to determine whether any of the complexes contained Egr1. Complexes identified as SF-1, Egr1, and the unidentified protein described in panel B are indicated. Autoradiography was performed for 2 h with the cEgr probe and 6 h with the eßdEgr and eßpEgr probes in panel C. D and E, EMSAs were performed using the cEgr oligo (consensus Egr) as labeled probe and {alpha}T3–1 nuclei isolated from serum-starved cells that had been simulated with PMA (1 µM) for 1 h. Competitions (5- to 500-fold molar excess) were performed using the oligos described above as well as oligos containing a mutation in the eßpEgr site (eßµpEgr) and an oligo representing a consensus Sp1 site.

 
To distinguish between these possibilities, one of the consensus Egr sites (cEgr) was used as probe in an EMSA. This oligo contains a high-affinity consensus site for Egr1–4 (33). The cEgr also failed to interact with a protein that could be identified as Egr1 (Fig. 5CGo, lane 1), but weakly interacted with two other complexes (data not shown). This suggested that {alpha}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 {alpha}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 {alpha}T3–1 cells with PMA resulted in the appearance of a new complex (Fig. 5CGo, lane 2) that could be supershifted by inclusion of an antibody to Egr1 (Fig. 5CGo, lane 3). Furthermore, based on EMSAs using the cEgr probe, Egr1 was the only Egr protein induced by PMA in {alpha}T3–1 cells (Fig. 5CGo, lane 3 and data not shown).

The PMA-stimulated {alpha}T3–1 nuclei were subsequently used to evaluate Egr1 binding to the labeled eßdEgr and eßpEgr oligos (Fig. 5CGo, lanes 4–9). Stimulation of {alpha}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. 5Go, D and E). A 5-fold molar excess of unlabeled cEgr (homologous competitor) reduced binding of Egr1 by 76% (Fig. 5DGo, 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. 5EGo, 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. 5DGo, 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. 5EGo, lanes 6–10). Finally, an Sp1 response element that has some similarity to an Egr site was a fairly ineffective competitor (Fig. 5EGo, 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 {alpha}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. 4Go were reevaluated for their responsiveness to GnRH. Mutation of both Egr sites completely blocked GnRH induction of the eLHß promoter (Fig. 6Go). 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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Figure 6. Mutation of the Egr or SF-1 Sites in the eLHß Promoter Disrupts Induction by GnRH

Transient transfections were performed in {alpha}T3–1 cells with the native and mutant promoters described in Fig. 3Go. Cells were stimulated with GnRH (10 nM) or received media alone for 8 h before harvesting lysates. Data are expressed as the fold induction (mean ± SEM) by GnRH over control.

 
Nab1 Can Repress Egr1 Induction of eLHß
Egr1 contains an internal inhibitory domain consisting of 34 amino acids (29). A single-point mutation within this domain resulted in a 15-fold increase in transcriptional activity of Egr1. This point mutation effectively disrupts an interaction between Egr1 and an additional cellular protein(s). Subsequent studies have identified these proteins as NGFI-A (Egr1) binding proteins, Nab1 and Nab2, which function to repress the activity of Egr1 (29, 30, 31).

Transient transfections were performed to determine the ability of Nab1 to repress Egr transactivation of the eLHß promoter (Fig. 7Go). 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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Figure 7. The Corepressor Nab1 Represses Egr1 Induction of the eLHß Promoter

Luciferase activity (RLU) of the eLHß -448/+60 promoter was evaluated after cotransfection with a control expression vector, Egr1, Egr1 plus Nab1, or Nab1 alone. A constant amount of Egr1 (100 ng) was transfected, whereas the indicated amount (nanograms) of Nab1 was used. The total amount of DNA transfected for each treatment was equalized using the control expression vector.

 
GnRH Induces Expression of Egr1 and Nab1
Nab1 has been shown to be expressed in a number of tissues (29); however, its presence in pituitary gonadotropes has not been reported. Furthermore, it is unknown as to how the expression of Nab1 is regulated. In contrast, expression of Egr1 can be stimulated by activation of the PKC and MAPK pathways (21). GnRH has been shown to activate both of these pathways in gonadotropes (22, 23, 24, 25, 26, 27). The data shown in Fig. 5Go demonstrate that PMA can indeed induce expression of Egr1 in {alpha}T3–1 cells. Therefore, based on these observations and those described above, expression of Egr1, Nab1, and SF-1 was evaluated in {alpha}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. 8Go). 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 {alpha}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 {alpha}T3–1 cells and was induced 2.6-fold by GnRH. This increase was delayed as compared with that observed for Egr1 (Fig. 9AGo). 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. 9BGo). 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. 9EGo. These data indicate that GnRH stimulation of {alpha}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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Figure 8. GnRH Induces a Transient Increase in Expression of Egr1 in {alpha}T3–1 Cells

Total RNA was isolated from {alpha}T3–1 cells maintained in serum containing media (S) or from cells that had been serum starved overnight and subsequently stimulated with GnRH (10 nM) for the indicated periods of time. Northern blot analysis was performed on 10 µg of RNA using a 32P-labeled Egr1 probe. RNA size markers are shown along the left side of the figure.

 


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Figure 9. GnRH Induces Expression of Egr1 and Nab1

Northern blots were performed on total RNA (10 µg) isolated from {alpha}T3–1 cells treated as described in Fig. 8Go. Cells were stimulated with GnRH (10 nM) for the indicated periods of time. Blots were hybridized to 32P-labeled Nab1 (A), SF-1 (B), Egr1 (C), and mouse ribosomal L7 protein (D) probes. The data shown in panels A–D are from the same blot and are representative of data from additional experiments. E, The blots shown in panels A–D were quantitated using a PhosphorImager. The quantitative data obtained from the PhosphorImager were corrected for RNA loading using the data obtained from the L7 blots. Shown is the change in steady-state RNA levels (mean ± SEM) for Nab1, SF-1, and Egr1 relative to levels from serum- treated cells (arbitrarily set at 1) from at least three different experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
Little is known about the molecular events involved in GnRH regulation of transcription of the LHß-subunit gene. Recent reports have examined the signal transduction pathways used by GnRH to induce promoter activity (26, 27). Previous studies (22, 23, 24, 25) have reported that stimulation of {alpha}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 {alpha}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. 2Go). 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 {alpha}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 {alpha}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. 4Go, Egr1 + SF-1 compared with Fig. 6Go). 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 {alpha}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 {alpha}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 {alpha}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. 9EGo. 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
Materials
Restriction enzymes and other enzymes were obtained from the Promega Corp. (Madison, WI), Life Technologies (Gaithersburg, MD), and New England Biolabs, Inc. (Beverly, MA). All oligodeoxynucleotides were obtained from Life Technologies except for the scEgr oligo, which was originally obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and the Sp1 oligo purchased from Promega Corp. The luciferase reporter vector (pGL2 basic) was obtained from Promega Corp. The GnRH agonist (des-Gly10,[D-Ala6]-GnRH ethylamide) and PMA were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies to Egr1, Egr2, Egr3, Sp1, and Sp3 were purchased from Santa Cruz Biotechnology, Inc. The antibody to SF-1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). All radionuclides were purchased from New England Nuclear Life Science Products, Inc. (Boston, MA). DNA sequencing was conducted using Sequenase purchased from United States Biochemical Corp. (Cleveland, OH) or through cycling sequencing using reagents purchased from PE Applied Biosystems (Foster City, CA) and subsequently run on the ABI 310 sequencer (PE Applied Biosystems). PCR amplification of DNA was performed using Taq polymerase (Life Technologies) or Deep Vent DNA polymerase (New England Biolabs, Inc.). All other chemicals and reagents were obtained from Pharmacia Biotech (Piscataway, NJ), Fisher Scientific (Pittsburgh, PA), Sigma Chemical Co., and Life Technologies.

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 {alpha}T3–1 cells (47) were plated in DMEM with 5% FBS, 5% horse serum, and antibiotics. On the day before transfection, {alpha}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 {alpha}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. 5Go) 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
{alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
During review of this manuscript another report was published indicating that GnRH and PMA could induce Egr1 expression in {alpha}T3–1 cells and that this led to increased expression of the rat LHß promoter (49).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Pamela Mellon for the {alpha}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
 
Address requests for reprints to: Michael W. Wolfe, Ph.D., Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7401. E-mail: mwolfe2{at}kumc.edu

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 

  1. Pierce J, Parsons TF 1981 Glycoprotein hormones: structure, function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177–199[Medline]
  3. Jameson JL, Hollenberg AN 1993 Regulation of chorionic gonadotropin gene expression. Endocr Rev 14:203–221[Medline]
  4. Cattanach BM, Iddon C, Charlton HM, Chiappa SA, Fink G 1977 Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269:338[Medline]
  5. Saade F, London DR, Clayton R 1989 The interaction of gonadotropin-releasing hormone and estradiol on luteinizing hormone and prolactin gene expression in female hypogonadal (hpg) mice. Endocrinology 124:1744–1753[Abstract]
  6. Lutz B, Rugarli EI, Eichele G, Ballabio A 1993 X-linked Kallmann syndrome. A neuronal targeting defect in the olfactory system? FEBS Lett 325:128–134[CrossRef][Medline]
  7. Crowley Jr WF, Filicori M, Spratt KL, Santoro NF 1985 The physiology of gonadotropin-releasing hormone (GnRH) in men and women. Recent Prog Horm Res 41:473–531[Medline]
  8. Levine JE, Ramirez VD 1982 Luteinizing hormone-releasing hormone release during the rat estrous cycle and after ovariectomy as estimated with push-pull cannulae. Endocrinology 111:1439–1448[Medline]
  9. Wierman ME, Rivier JE, Wang C 1989 GnRH-dependent regulation of gonadotropin subunit mRNA levels in the rat. Endocrinology 124:272–278[Abstract]
  10. Haisenleder DJ, Dalkin AC, Ortolano GA, Marshall JC, Shupnik MA 1991 A pulsatile gonadotropin-releasing hormone stimulus is required to increase transcription of the gonadotropin subunit genes: evidence for differential regulation of transcription by pulse frequency in vivo. Endocrinology 128:509–517[Abstract]
  11. Shupnik MA 1990 Effects of gonadotropin-releasing hormone on rat gonadotropin gene transcription in vitro: requirement for pulsatile administration for luteinizing hormone-ß gene stimulation. Mol Endocrinol 4:1444–1450[Abstract]
  12. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen W, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312[Abstract]
  13. Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of luteinizing hormone ß gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem 271:6645–6650[Abstract/Free Full Text]
  14. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
  15. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Milbrandt J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273:1219–1221[Abstract]
  16. Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji D, Driancourt M, Rao CV, Charnay P 1998 Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. Mol Endocrinol 12:107–122[Abstract/Free Full Text]
  17. Halvorson LM, Masafumi I, Jameson JL, Chin WW 1998 Steroidogenic factor-1 and early growth response protein 1 act through two composite DNA binding sites to regulate luteinizing hormone ß-subunit gene expression. J Biol Chem 273:14712–14720[Abstract/Free Full Text]
  18. Milbrandt J 1987 A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 238:797–799[Medline]
  19. Christy BA, Lau LF, Nathans D 1988 A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. Proc Natl Acad Sci USA 85:7857–7861[Abstract]
  20. Lemaire P, Revelant O, Bravo R, Charnay P 1988 Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells. Proc Natl Acad Sci USA 85:4691–4695[Abstract]
  21. Gashler A, Sukhatme VP 1995 Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res 50:191–224[Medline]
  22. Mitchell R, Sim PJ, Leslie T, Johnson MS, Thomson FJ 1994 Activation of MAP kinase associated with the priming effect of LHRH. J Endocrinol 140:R15–18
  23. Reiss N, Levi LN, Shacham S, Harris D, Seger R, Naor Z 1997 Mechanism of mitogen-activated protein kinase by gonadotropin-releasing hormone in the pituitary {alpha}T3–1 cell line: differential roles of calcium and protein kinase C. Endocrinology 138:1673–1682[Abstract/Free Full Text]
  24. Roberson MS, Misra-Press A, Laurance ME, Stork PJS, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone {alpha}-subunit promoter. Mol Cell Biol 15:3531–3539[Abstract]
  25. Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C. Endocrinology 137:304–311[Abstract]
  26. Weck J, Fallest PC, Pitt LK, Shupnik MA 1998 Differential gonadotropin-releasing hormone stimulation of rat luteinizing hormone subunit gene transcription by calcium influx and mitogen-activated protein kinase-signaling pathways. Mol Endocrinol 12:451–457[Abstract/Free Full Text]
  27. Saunders BD, Sabbagh E, Chin WW, Kaiser UB 1998 Differential use of signal transduction pathways in the gonadotropin-releasing hormone-mediated regulation of gonadotropin subunit gene expression. Endocrinology 139:1835–1843[Abstract/Free Full Text]
  28. Wolfe MW The equine luteinizing hormone ß promoter contains two functional steroidogenic factor-1 response elements. Mol Endocrinol (submitted)
  29. Russo MW, Sevetson BR, Milbrandt J 1995 Identification of NAB1, a repressor of NGFI-A and Krox20-mediated transcription. Proc Natl Acad Sci USA 92:6873–6877[Abstract]
  30. Svaren J, Sevetson BR, Apel ED, Zimonjic DB, Popescu NC, Milbrandt J 1996 NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol Cell Biol 16:3545–3553[Abstract]
  31. Swirnoff AH, Apel ED, Svaren J, Sevetson BR, Zimonjic DB, Popescu NC, Milbrandt J 1998 Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol Cell Biol 18:512–524[Abstract/Free Full Text]
  32. Cesnjaj M, Zheng L, Catt KJ, Stojilkovic SS 1995 Dependence of stimulus-transcription coupling on phospholipase D in agonist-stimulated pituitary cells. Mol Biol Cell 6:1037–1047[Abstract]
  33. Levi NL, Hanoch T, Benard O, Rozenblat M, Harris D, Reiss N, Naor Z, Seger R 1998 Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary {alpha}T3–1 cell line is mediated by protein kinase C, c-Src, and CDC42. Mol Endocrinol 12:815–824[Abstract/Free Full Text]
  34. Swirnoff AH, Milbrandt J 1995 DNA-binding specificity of NGFI-A and related zinc finger transcription factors. Mol Cell Biol 15:2275–2287[Abstract]
  35. Kaiser UB, Sabbagh E, Saunders BD, Chin WW 1998 Identification of cis-acting deoxyribonucleic acid elements that mediate gonadotropin-releasing hormone stimulation of the rat luteinizing hormone ß-subunit gene. Endocrinology 139:2443–2451[Abstract/Free Full Text]
  36. Kaiser UB, Sabbagh E, Chen MT, Chin WW, Saunders BD 1998 Sp1 binds to the rat luteinizing hormone ß (LHß) gene promoter and mediates gonadotropin-releasing hormone-stimulated expression of the LHß subunit gene. J Biol Chem 273:12943–12951[Abstract/Free Full Text]
  37. Tashjian Jr AH, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352[Medline]
  38. Hinkle PM, Tashjian Jr AH 1973 Receptors for thyrotropin-releasing hormone in prolactin-producing rat pituitary cells in culture. J Biol Chem 248:6180–6186[Abstract/Free Full Text]
  39. Haisenleder DJ, Yasin M, Dalkin AC, Gilrain J, Marshall JC 1996 GnRH regulates steroidogenic factor-1 (SF-1) gene expression in the rat pituitary. Endocrinology 137:5719–5722[Abstract]
  40. Virgin JB, Silver BJ, Thomason AR, Nilson JH 1985 The gene for the ß subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region. J Biol Chem 260:7072–7077[Abstract/Free Full Text]
  41. Jameson JL, Chin WW, Hollenberg AN, Chong AS, Habener JF 1984 The gene encoding the ß-subunit of rat luteinizing hormone. J Biol Chem 259:15474–15480[Abstract/Free Full Text]
  42. Talmadge K, Vamvakopoulos NC, Fiddes JC 1984 Evolution of the genes for the ß subunits of chorionic gonadotropin and luteinizing hormone. Nature 307:37–40[Medline]
  43. Kumar TR, Matzuk MM 1995 Cloning of the mouse gonadotropin ß-subunit-encoding genes, II. Structure of the luteinizing hormone ß-subunit-encoding genes. Gene 166:335–336[CrossRef][Medline]
  44. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract]
  45. Sherman GB, Wolfe MW, Farmerie TA, Clay CM, Threadgill DS, Sharp DC, Nilson JH 1992 A single gene encodes the ß-subunits of equine luteinizing hormone and chorionic gonadotropin. Mol Endocrinol 6:951–959[Abstract]
  46. Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol 6:1249–1258[Abstract]
  47. Windle JJ, Weiner RI, Mellon PL 1990 Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 4:597–603[Abstract]
  48. Hagenbuchle O, Wellaur PK 1992 A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture. Nucleic Acids Res 20:3555–3559[Abstract]
  49. Halvorson LM, Kaiser UB, Chin WW 1999 The protein kinase C system acts through the early growth response protein 1 to increase LHß gene expression in synergy with steroidogenic factor-1. Mol Endocrinol 13:106–116[Abstract/Free Full Text]