©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Luteinizing Hormone Gene Promoter Activity by the Orphan Nuclear Receptor, Steroidogenic Factor-1 (*)

(Received for publication, September 6, 1995; and in revised form, December 13, 1995)

Lisa M. Halvorson (1) (2)(§)(¶) Ursula B. Kaiser (1)(§) William W. Chin (1)

From the  (1)Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115 and the (2)Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, alphaT3-1, where it is believed to enhance expression of the common gonadotropin alpha-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone beta-subunit (LHbeta) gene promoter revealed the presence of a consensus GSE at -127 to -119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both alphaT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHbeta-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHbeta promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHbeta-luciferase reporter construct. When cotransfected with -209/+5 of the LHbeta promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHbeta-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHbeta promoter. These data suggest that SF-1 may participate in the expression of the LHbeta gene by the gonadotrope.


INTRODUCTION

The pituitary gonadotropins, luteinizing hormone and follicle-stimulating hormone, are critical modulators of gamete maturation and gonadal steroidogenesis. These hormones are composed of a common alpha-subunit linked noncovalently to unique beta-subunits which specify physiologic actions(1) .

Several DNA regulatory elements have been defined for the alpha-subunit gene promoter. GnRH(^1)-stimulated expression is believed to be mediated through a regulatory element located between positions -346 and -244 in the human alpha-subunit gene promoter, a region separate from those involved in basal and cAMP-stimulated expression(2) . Activation of a cAMP response element (CRE) appears to be important for expression in both pituitary and placental cell types, while placental-specific expression occurs through the activation of a trophoblast-specific element acting in concert with the CRE(3) . Pituitary-specific expression of the alpha-subunit gene has been attributed to the presence of both a pituitary glycoprotein basal element and a gonadotrope-specific element (GSE)(4, 5) .

The consensus GSE sequence (TGACCTTGT), defined in the common alpha-subunit by Mellon and colleagues, resembles a nuclear receptor binding half-site(4, 6) . Variations of this sequence, alternatively called the Ad4 response element, are also present in the promoter regions of multiple genes which play a role in steroidogenesis, sexual differentiation, and adult reproductive function(7) . The GSE/Ad4 element has been shown to interact with the transcription factor, steroidogenic factor-1 (SF-1), in a number of genes, including the steroidogenic P450, the aromatase, and the Müllerian inhibiting substance genes(8, 9, 10) . SF-1 is an orphan member of the nuclear hormone receptor superfamily. Best known for its selective expression in adrenal and gonadal cells, it has more recently been identified in the pituitary gland with localization to the gonadotrope(6, 7) .

In studies of the human alpha-subunit gene promoter, SF-1 has been shown to bind to the GSE region by electrophoretic gel mobility shift assay. Furthermore, reporter constructs which contain the alpha-subunit GSE site are expressed at higher levels in cell lines which contain endogenous SF-1 than in those cells which lack SF-1, consistent with a role for SF-1 in tissue-specific transcriptional activation of the alpha-subunit gene(4, 6) .

In contrast with the alpha-subunit, the cis-acting elements responsible for expression of either the LHbeta- or FSHbeta-subunit mRNAs are poorly understood. Interestingly, transgenic mice null for the gene which encodes SF-1 not only express the alpha-subunit in low levels, but also fail to express the beta-subunits, suggesting a functional role for SF-1 in LHbeta gene expression(7) . As previous studies of the bovine LHbeta gene promoter have shown that the proximal 776 base pairs are sufficient to direct pituitary-specific expression in transgenic mice (11) , we analyzed the corresponding region of the rat LHbeta promoter for the presence of an SF-1-binding site, or GSE.

The rat LHbeta gene promoter contains a consensus GSE at position -127 to -119 relative to the transcriptional start site (Fig. 1). Interestingly, this sequence is highly conserved across species among the LHbeta genes, suggesting physiologic significance(11, 14) . Inasmuch as the consensus GSE sequence is present in the LHbeta gene promoter, we wished to determine whether this putative GSE region has functional significance. We, therefore, investigated the ability of SF-1 to bind to and transactivate the rat LHbeta gene promoter.


Figure 1: A consensus SF-1-binding site is present in the rat LHbeta gene promoter. The core GSE sequence as defined in the human glycoprotein alpha-subunit (4) is aligned with the putative GSE sites of the LHbeta gene in rat(12) , cow(13) , pig(14) , and human (15) . Also shown are regions of homology with the SF-1-binding sites in the 21-hydroxylase(8) , aromatase(9) , and Mullerian inhibiting substance (10) gene promoters. Variant nucleotides are underlined.




MATERIALS AND METHODS

Oligonucleotides Used in Electrophoretic Mobility Shift Assay (EMSA)

The nucleotide sequence of the rat LHbeta gene promoter was based on Fig. 3of Jameson et al.(12) with position -1 assigned to the nucleotide immediately 5` to the transcriptional start site. The LHSF oligonucleotide used in EMSA corresponds to bases -134 to -113 of the rat LHbeta gene (sense strand: 5`-TCCTTTCTGACCTTGTCTGTCT-3`). The LHSFM oligonucleotide sequence is identical to LHSF except for the conversion of the CC nucleotide pair at positions -124 and -123 to an AA pair (sense strand: 5`-TCCTTTCTGAAATTGTCTGTCT-3`). The Pit-1 oligonucleotide used in competition studies corresponds to -137 to -65 of the rat growth hormone promoter and contains two Pit-1/growth hormone factor-1 binding sites (sense strand: 5`-GGGAGGAGCTTCTAAATTATCCATCAGCACAAGCTGTCAGTGGCTCCAGCCATGAATAAATGTATAGGGAAA-3`) (16) . Except for the Pit-1 oligonucleotide, all oligonucleotides used for EMSA contained 5`-BamHI and 3`-BglII restriction sites in addition to the sequences listed above.


Figure 3: Mutation of the putative LHbeta-GSE sequence defines nucleotides essential for binding by alphaT3-1 nuclear extract. Binding reactions included alphaT3-1 nuclear extracts and either the P-labeled wild-type LHbeta gene oligonucleotide (LHSF, lanes 1-4) or the P-labeled mutated oligonucleotide (LHSFM, lanes 5-8). Competition with 500-fold molar excess of unlabeled LHSF, LHSFM, or Pit-1 oligonucleotide was performed as indicated. The specific and nonspecific binding complexes are indicated by an arrowhead and asterisk, respectively.



Sense and antisense oligonucleotides were annealed and end-labeled with [-P]ATP by T4 polynucleotide kinase and purified over a NICK column (Pharmacia Biotech Inc.).

Nuclear Extract and in Vitro Translated Proteins

The method of Andrews and Faller (17) was used to prepare crude nuclear extracts from a mouse gonadotrope-derived cell line (alphaT3-1), a monkey kidney fibroblast cell line (CV-1), and a rat somatolactotrope cell line (GH(3)). In vitro translated SF-1 protein was generated from a plasmid containing 2.1 kilobase pairs of the mouse SF-1 cDNA using the TNT coupled reticulocyte lysate system (Promega, Madison, WI)(18) . The resultant product was determined to be of appropriate size by comparison with [S]methionine-labeled protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Electrophoretic Mobility Shift Assays

Nuclear extract (5 µg) or in vitro translated protein (1, 3, or 5 µl) was incubated with 50,000 cpm of oligonucleotide probe in DNA-binding buffer (20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl(2), 10 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, 1 mg/ml bovine serum albumin, and 5% (v/v) glycerol) for 30 min on ice. For competition studies, excess unlabeled oligonucleotide was added 5 min prior to the addition of probe. Where indicated, antiserum (1 µl) was added 30 min following the addition of probe, and incubation was continued for 2 h. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5 times Tris borate-EDTA buffer and subjected to autoradiography.

Plasmids Used in Transfection Studies

The largest LHbeta reporter construct used for these studies contained 794 base pairs of the 5`-flanking sequence of the rat LHbeta gene and the first 5 base pairs of the 5`-untranslated region fused to a luciferase reporter gene, pXP2(19) . Deletions in this construct were created by subcloning polymerase chain reaction products containing the LHbeta promoter sequences into the pXP2 vector using BamHI/HindIII sites which were introduced by the primers. The -209LHbeta-MUT plasmid was created by introducing a two base pair mutation into the -209LHbeta construct using the transformer site-directed mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, California). The selection primer was located in the pXP2 polylinker and the 3`-end of the LHbeta flanking sequence and converted a unique HindIII restriction site to a unique MluI site (sense strand: 5`-GGTAGGGAAGGTATCACGCGTGTCGACCCGGGTACC-3`). The mutagenic primer spanned region -147 to -104 of the LHbeta promoter and eliminated a TthIII1 restriction site in addition to introducing the desired mutation (sense strand: 5`-GCTGGTCCCTGGCTTTTCTGAAATTGTCTGTCTCGCCCCCAAAG-3`). To create GSE2-GH50, an oligonucleotide was designed which contained two copies of the putative GSE region as a tandem repeat flanked by BamHI/BglII restriction sites (sense strand: 5`-GATCCTTTTCTGACCTTGTCTGTCTCGCCTCTGACCTTGTCTGTA-3`). This oligonucleotide was inserted upstream of the minimal growth hormone promoter, GH50, in the pXP1 luciferase reporter plasmid(19, 20) . All reporter constructs were confirmed by dideoxysequencing.

The SF-1 expression vector contained 2.1 kilobase pairs of the mouse SF-1 cDNA driven by cytomegalovirus promoter sequences(18) . The Pit-1 expression vector was created by placing 915 base pairs of the rat Pit-1/growth hormone factor-1 cDNA sequence from pBluescript SK(-) (Stratagene, La Jolla, CA) into the pcDNAI vector (Invitrogen, San Diego, CA) using HindIII/NotI restriction enzyme sites(21) .

Cell Culture and Assays

Monkey kidney fibroblast (CV-1) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells growing in 3.5-cm tissue culture wells (Flow Laboratories, McLean, VA) were transfected with expression (0.1 µg/well) and reporter (1.65 µg/well) plasmids using the calcium phosphate precipitation method(22) . Control wells received the appropriate ``empty'' expression vector (0.1 µg/well). Cotransfection with an RSV-beta-galactosidase plasmid (1 µg/well) allowed correction for differences in transfection efficiency between wells. The cells were harvested 48 h following transfection and the cell extracts analyzed for both luciferase (23) and beta-galactosidase (24) activities. Luciferase activity was first normalized to the level of beta-galactosidase activity. Results were then calculated as fold change relative to expression in the presence of the control empty expression vector. Data are shown as the mean ± S.E. and represent a minimum of three independent experiments with each point run in triplicate in each experiment.

Sources of SF-1 and Pit-1 Antibodies and Plasmids

The rabbit SF-1 antiserum as well as the vectors containing the SF-1 cDNA were kindly provided by Dr. K. L. Parker (Duke University). The SF-1 antibody was generated against a glutathione S-transferase-SF-1 fusion protein(25) . The Pit-1 antiserum, directed against amino acids 136-150 of rat Pit-1/growth hormone factor-1, was provided by C. Bancroft (Mt. Sinai School of Medicine) (26) . The Pit-1 cDNA in pBluescript SK(-) was provided by Dr. L. E. Theill (University of California, San Diego).


RESULTS

alphaT3-1 Nuclear Extract Binds to the Putative GSE Region of the LHbeta Gene Promoter

The alphaT3-1 cell line has previously been shown to express both SF-1 mRNA and protein(6, 7) . Nuclear extracts from this cell line, presumed to contain SF-1, have been shown to bind to the alpha-subunit promoter GSE(4, 6) . We therefore utilized EMSA to investigate whether these extracts were able to interact with the region of the rat LHbeta gene promoter that contains the putative GSE sequence (oligonucleotide LHSF). As shown in Fig. 2, the interaction of alphaT3-1 nuclear extracts with P-labeled LHSF produced a specific protein-DNA complex as demonstrated by successful competition with unlabeled LHSF (lanes 1 and 2).


Figure 2: alphaT3-1 nuclear extracts and in vitro translated SF-1 bind the putative LHbeta-GSE region with similar specificity. Binding reactions included P-labeled LHSF as a probe and, as indicated, either alphaT3-1 nuclear extract (lanes 1-4) or 1, 3, or 5 µl of in vitro translated SF-1 (lanes 5-11). Competition with 500-fold molar excess of unlabeled LHSF is shown for both the nuclear extract and in vitro translated SF-1 (lanes 2 and 8). Incubation with antiserum specific to SF-1 (lanes 3 and 9) or Pit-1 (lanes 4 and 10) was also performed using both protein preparations. Note that 3 µl of in vitro translated SF-1 were used in the cold competition and antibody studies (lanes 8-10) and therefore band intensity should be compared against lane 6. Lane 11 contains a probe and the unprogrammed reticulocyte lysate used for in vitro translation. The arrowhead indicates the specific binding complex. A nonspecific band, indicated by the asterisk, is present in unprogrammed reticulocyte lysate.



In order to confirm that the complex identified in Fig. 2contained SF-1, we investigated the effect of a SF-1-specific antibody on the formation of the alphaT3-1 nuclear extract-LHSF complex. This antibody has previously been shown to block the ability of SF-1 to bind to the promoter element of a number of genes, including the glycoprotein hormone alpha-subunit, aromatase, and 21-hydroxylase genes(6, 9, 25) . Treatment with this SF-1-specific antiserum substantially decreased the intensity of the protein-DNA complex while the addition of an anti-Pit-1 antiserum, used as a negative control, had no effect (Fig. 2, lanes 3 and 4). This result confirms that the GSE of the LHbeta gene promoter is bound by SF-1, or an immunologically related protein, present in alphaT3-1 nuclear extracts.

Parallel EMSA was performed using the oligonucleotide LHSF as a probe in the presence of nuclear extracts from cell lines that do not contain SF-1. No specific protein-DNA interactions were detected with the use of nuclear extracts from either monkey kidney fibroblast cells (CV-1) or rat somatolactotrope cells (GH(3)) (data not shown).

In Vitro Translated SF-1 Binds to the LHbeta Gene Promoter

Further confirmation that SF-1 binds to the LHbeta-GSE promoter region was obtained by the use of in vitro translated SF-1 in EMSA. A binding reaction containing the labeled LHSF oligonucleotide and in vitro translated SF-1 resulted in a protein-DNA complex mobility similar to that obtained with the alphaT3-1 nuclear extracts (Fig. 2, lanes 5-10). Formation of this complex diminished in the presence of either excess unlabeled LHSF oligonucleotide or blocking antiserum directed against SF-1, but was unaffected by the Pit-1 antibody. Taken as a whole, these data clearly demonstrate that the putative GSE region of the LHbeta gene is recognized by SF-1, as either an endogenous (alphaT3-1 nuclear extract) or an in vitro translated product.

Mutation of the Putative LHbeta-GSE Site Eliminates Binding

In order to localize further the SF-1 recognition site, a 2-base pair mutation was introduced in the wild-type LHbeta oligonucleotide sequence (LHSF) to form LHSFM. The choice of this mutation was based on the loss of DNA binding which resulted from analogous mutations in the Müllerian inhibiting substance and glycoprotein alpha-subunit promoters(6, 10) . EMSA was performed using alphaT3-1 nuclear extracts and either the wild-type LHSF (Fig. 3, lanes 1-4) or the mutant LHSFM (Fig. 3, lanes 5-8) as a probe. The intensity of the complex obtained with the LHSF probe was blunted by unlabeled wild-type LHSF, but not by the mutated sequence or by an unrelated oligonucleotide containing two binding sites for the pituitary transcription factor, Pit-1 (Fig. 3, lanes 2-4). As seen in lanes 5-8, the alphaT3-1 nuclear extract was not able to bind to LHSFM when used as a probe. These results establish that an intact LHbeta-GSE sequence is required for binding by alphaT3-1 nuclear extract.

SF-1 Specifically Increases LHbeta Promoter Activity

We next sought to determine the functional significance of the interaction between SF-1 and the LHbeta gene promoter sequences. In initial investigations utilizing the gonadotrope-derived alphaT3-1 cell line, LHbeta promoter-driven luciferase activity exceeded luciferase activity in the absence of cell extract (background activity) by less than 2-fold. At this level of expression, we were unable to evaluate reliably whether the presence or absence of the putative GSE sequence altered LHbeta promoter activity in response to the endogenous SF-1 present in this cell line. Furthermore, attempts to increase LHbeta gene expression through cotransfection with an SF-1 expression vector were unsuccessful.

These studies were therefore performed in the monkey kidney fibroblast cell line, CV-1, a cell line which has previously been shown to support SF-1-induced transactivation of the bovine P-450 CYP11B promoter. By Northern blot analysis, this cell line lacks the mRNA which encodes the SF-1 homolog, Ad4BP(27) . As stated previously, we have also demonstrated that CV-1 nuclear extract fails to bind the LHbeta-GSE region by EMSA, consistent with the absence of endogenous SF-1 (data not shown). Utilizing the CV-1 cell line, basal LHbeta gene promoter activity exceeded expression of the promoterless reporter plasmid (pXP2) by an average of 15-fold.

In Fig. 4A, CV-1 cells were cotransfected with region -209 to +5 of the LHbeta gene promoter and cytomegalovirus-driven expression vectors containing either the SF-1 or Pit-1 cDNA. The presence of SF-1 markedly increased LHbeta promoter activity (56 ± 5-fold). In contrast, the pituitary transcription factor Pit-1 did not alter luciferase levels, indicating the specificity of the SF-1 response.


Figure 4: An intact putative LHbeta-GSE region confers SF-1 responsiveness to both the LHbeta promoter and a heterologous minimal promoter. CV-1 cells were transiently transfected with luciferase reporter constructs which contained various regions of the rat LHbeta gene promoter. Cells were cotransfected with plasmids encoding either SF-1 or Pit-1 and with an RSV-beta-galactosidase expression vector. Luciferase activity was normalized to beta-galactosidase activity. Promoter activity was then calculated as fold change over expression in the presence of the appropriate control expression vector. Results are shown as the mean ± S.E. of at least nine samples in three independent experiments. A, comparison of LHbeta promoter activity in response to SF-1 versus Pit-1. B, SF-1 stimulation of LHbeta promoter activity with loss of the intact GSE sequence by sequential 5`-deletion or mutagenesis. C, SF-1 responsiveness of the growth hormone minimal promoter (GH50) (20) or GH50 preceded by two copies of the putative LHbeta-GSE region (GSE2-GH50).



Of importance, we have recently confirmed the ability of SF-1 to increase LHbeta promoter activity in the rat pituitary-derived somatolactotrope cell line, GH(3). Utilizing transiently transfected GH(3) cells and conditions similar to those in CV-1 cells, SF-1 increased LHbeta promoter activity in the -209/+5 construct by 15 ± 1.5-fold.

SF-1 Stimulation of Rat LHbeta Gene Promoter Activity Is Dependent on the Presence of an Intact GSE

In order to delineate the region in the LHbeta gene promoter responsible for providing SF-1 responsiveness, reporter constructs were generated which incorporated various deletions in the rat LHbeta gene promoter. Of note, no systematic changes in basal expression (i.e. in the absence of SF-1) were observed in these deletion constructs.

The evaluation of sequential 5`-deletion constructs revealed persistent SF-1 stimulation of LHbeta promoter activity with deletion to position -134, followed by an abrupt loss of the SF-1 response with deletion to position -82 (Fig. 4B). Based on these data, loss of LHbeta promoter sequences across the putative GSE region (positions -127 to -119) correlates with the loss of SF-1-stimulated promoter activity.

Further definition of the SF-1-responsive cis-acting element was obtained by the introduction of a two base pair mutation into the putative GSE site of the -209LHbeta luciferase reporter construct to form -209LHbeta-MUT. This small change, analogous to the mutation which eliminated DNA-binding by nuclear extract (Fig. 3), substantially decreased the ability of SF-1 to increase promoter activity (6 ± 1-fold versus 56 ± 5-fold) (Fig. 4B). Thus, transactivation of the LHbeta promoter by SF-1 appears to be critically dependent on the presence of an intact GSE sequence.

LHbeta Promoter Sequences Confer SF-1 Responsiveness to a Heterologous Promoter

Having demonstrated that the putative GSE site is necessary for SF-1 responsiveness in the context of the LHbeta gene promoter, we next asked whether this sequence was sufficient to confer SF-1 responsiveness to a heterologous promoter. Two copies of the LHbeta-GSE sequence were inserted upstream of the growth hormone minimal promoter, GH50. As seen in Fig. 4C, these sequences conferred a marked SF-1 response to this normally nonresponsive promoter (57 ± 10-fold versus 1.3 ± 0.2-fold).

Other investigators have shown previously that the human alpha-subunit promoter GSE (identical to the putative rat LHbeta-GSE, see Fig. 1) increases thymidine kinase minimal promoter activity in SF-1 containing cell lines, but not in cell lines which lack SF-1(6) . However, interpretation of this study was limited by the possibility that additional cell-specific factors were contributing to the observed differences in transcriptional activity. As our results were obtained in a single cell line, stimulation of promoter activity in the presence of the GSE can be attributed solely to SF-1-induced effects.


DISCUSSION

Our results clearly demonstrate that SF-1 binds specifically to the putative GSE region of the rat LHbeta gene promoter and that, through this interaction, SF-1 substantially increases LHbeta gene promoter activity. Furthermore, we have shown that the introduction of a two base pair mutation within the LHbeta-GSE sequence markedly blunts the ability of SF-1 to either bind to or transactivate this promoter.

It is of interest to note that the SF-1 response was not fully lost in the mutated construct, suggesting a role for additional nucleotides within the GSE in effecting SF-1-induced transactivation. While parallel functional assays have not been performed, it has been clearly demonstrated that DNA binding by SF-1 is severely blunted by mutation of nucleotide pairs at other positions within the GSE/Ad4 element(8) . Alternatively, additional SF-1 binding sites may contribute to the regulation of LHbeta promoter activity. Sequence analysis of the rat LHbeta promoter identifies a number of regions which resemble the consensus GSE. It will be of interest to investigate the possible functional significance of these regions in future studies.

SF-1 has been shown to act at multiple levels of the reproductive axis, including the hypothalamus, pituitary, and gonad(7) . Within the pituitary, Mellon and colleagues have demonstrated SF-1-stimulated expression of the glycoprotein alpha-subunit gene(6) . In conjunction with their results, our data regarding the regulation of LHbeta gene expression suggests that SF-1 may play a critical role in the coordinated expression of both subunit genes required for luteinizing hormone biosynthesis in the gonadotrope.

In the functional studies reported here, we utilized a heterologous system in which both an SF-1 expression vector and a reporter construct containing LHbeta promoter sequences were transiently transfected into a fibroblast cell line, CV-1. In preliminary studies, we have also observed SF-1-mediated increases in LHbeta promoter activity using a pituitary-derived cell line, the rat somatolactotrope cell line, GH(3). Although these studies would ideally have been performed in a gonadotrope-derived cell line, currently available cell lines fail to express either endogenous or exogenous gonadotropin beta-subunits at appreciable levels(28) .

As gonadotrope-derived alphaT3-1 cells are known to contain SF-1(7) , the lack of LHbeta gene expression may seem inconsistent with a role for SF-1 in activation of the LHbeta promoter. However, a number of potential explanations are possible. For example, this cell line may be arrested at a stage of development in which both alpha-subunit (E12.5) and pituitary SF-1 (E13.5) expression have been established, but LHbeta-subunit expression is absent (E16.5)(4, 7, 28) . The alphaT3-1 cells may therefore be appropriately expressing an inhibitory factor(s) which is responsible for suppression of LHbeta promoter activity at this developmental stage. Alternatively, alphaT3-1 cells may lack the SF-1 ligand (currently postulated but as yet unidentified) or an additional cofactor required for LHbeta promoter activation. This explanation seems less likely, however, in view of the magnitude of the observed response in CV-1 cells, a cell line that does not express endogenous SF-1 nor any of the identified SF-1-regulated genes and would therefore be predicted to be less likely to contain the necessary cofactors.

The results presented here clearly demonstrate that the presence of the GSE element is sufficient to direct SF-1 responsiveness in the context of both the LHbeta gene promoter and the heterologous minimal growth hormone promoter, GH50. The question remains, however, as to whether SF-1 is required for LHbeta gene expression. Interestingly, in transgenic mice null for the Ftz-F1 gene which encodes SF-1, GnRH replacement was able to restore gonadotropin expression in four out of five animals(7, 29) . These results suggest that cells from the gonadotrope lineage are present in these animals and are capable of expressing the LHbeta gene despite the lack of SF-1. However, it is important to note that quantitatively normal levels of LH in the absence of SF-1 have yet to be shown. Furthermore, as is true for all gene ``knockout'' paradigms, this transgenic model system does not exclude possible redundancy in the pool of potential transactivating factors for this critical reproductive gene. Thus, while SF-1 may not be absolutely required for LHbeta gene expression, it may in fact be required for normal levels of expression in the intact animal. The magnitude of the SF-1-directed increase in promoter activity observed in both the CV-1 and GH(3) cell lines strongly implies physiological significance.

Our results do not directly address whether SF-1, a member of the nuclear hormone receptor superfamily, functions as a monomer or whether it has the ability, and/or requirement, to undergo dimerization(18) . Members of this family are best known for binding to pairs of recognition half-sites arranged as tandem or inverted repeats. More recently, an alternative mechanism for DNA interaction, monomer binding to a single 5`-extended half-site, has been described for both SF-1 and another orphan nuclear receptor, NGFI-B(30) . The results presented here are at least consistent with the ability of SF-1 to bind to a single GSE element in the LHbeta gene promoter. By sequence analysis, the rat LHbeta-GSE at positions -127 to -119 is present as a single site. Furthermore, as shown by EMSA, SF-1 is able to bind to an oligonucleotide probe in which the LHbeta-GSE site is flanked by fewer than 8 additional base pairs on each side. SF-1 has also been shown to bind to similarly short GSE-containing promoter sequences from a variety of other genes, including the glycoprotein hormone alpha-subunit, aromatase, and Mullerian inhibiting substance genes(6, 9, 10) .

The lack of a second DNA-response element in the region of the LHbeta-GSE does not exclude potential SF-1 dimerization with a non-DNA-binding partner such has been shown to occur between the orphan nuclear receptor NurrI/NGFI-B and the 9-cis-retinoic acid receptor (31) . Intriguingly, mutation of the DAX-1 gene in humans results in adrenal and gonadal hypoplasia similar to that observed in SF-1 deficient mice, implicating DAX-1 as a potential SF-1 dimerization partner(7, 32) .

The current studies clearly define a role for SF-1 in the regulation of basal expression of the LHbeta gene. Within the pituitary gland, SF-1 expression is restricted to the gonadotrope subpopulation and may therefore be an important modulator of cell-specific expression(7) . SF-1 cannot, however, be the sole determinant of gonadotrope-specific expression as it is also expressed in non-LH-producing tissues such as the ventromedial hypothalamus, gonad, and adrenal gland(7, 29) . It will also be of interest to determine whether SF-1-induced increases in LHbeta promoter activity interact with GnRH-stimulated responses. The potential role of SF-1 in both tissue-specific and hormonally-mediated expression of the LHbeta gene awaits further exploration.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HD000849 and the American Gynecological and Obstetrical Society/American Association of Obstetricians and Gynecologists Foundation through the Reproductive Scientist Development Program (to L. M. H.), the Medical Research Council of Canada Clinician-Scientist Award (to U. B. K.), and by National Institutes of Health Grant HD19938 (to W. W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work and both should be considered first authors.

To whom correspondence should be addressed: G. W. Thorn Research Building, Rm. 913, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115. Tel.: 617-732-5856; Fax: 617-732-5123.

(^1)
The abbreviations used are: GnRH, gonadotropin-releasing hormone; CRE, cAMP response element; GSE, gonadotrope-specific element; SF-1, steroidogenic factor-1; LHbeta, luteinizing hormone beta-subunit; FSHbeta, follicle-stimulating hormone beta-subunit; EMSA, electrophoretic mobility shift assay; RSV, Rous sarcoma virus.


ACKNOWLEDGEMENTS

We thank Elena Sabbagh for her technical assistance.


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