The Promoter of Murine Follicle-Stimulating Hormone Receptor: Functional Characterization and Regulation by Transcription Factor Steroidogenic Factor 1

Jérôme Levallet, Pasi Koskimies, Nafis Rahman and Ilpo Huhtaniemi

Department of Physiology University of Turku 20520 Turku, Finland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The promoter of the FSH receptor (R) gene has been cloned from several species. Although some of its regulatory elements have been identified, its function still remains poorly characterized. Using transient transfections of luciferase reporter constructs, driven by various fragments of the murine (m) FSHR promoter, we identified a cell-specific promoter region. This domain is located in the distal part of the mFSHR promoter, -1,110 to -1,548 bp upstream of the translation initiation site, and it contains two steroidogenic factor 1 (SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and protein closely correlated in various steroidogenic cell lines with activity of the transfected mFSHR promoter/luciferase reporter construct carrying the distal activator domain. A dose-dependent increase in FSHR promoter activity was shown in nonsteroidogenic HEK 293 cells transiently transfected with SF-1 cDNA. SF-1 was found to bind to a nonconsensus 5'-CAAGGACT-3' SLBS-3 motif in the distal part of the promoter; formation of the SF-1/SLBS-3 complex could be reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a significant loss of SF-1-mediated mFSHR promoter activity. The second SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In conclusion, we have identified a regulatory domain in the mFSHR promoter participating in the cell-specific regulation of FSHR expression. We demonstrated for the first time that the mFSHR promoter possesses functional SF-1 binding sites and thus belongs to the group of SF-1-regulated genes. These findings provide further evidence for the key role of SF-1 in the regulation of genes involved in gonadal differentiation and endocrine functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadal function is critically dependent on regulatory impulses from the pituitary-gonadal axis. The two gonadotropins, LH and FSH, play a key role in this regulation. FSH acts via a specific G protein-coupled cell surface receptor consisting of a long extracellular domain, the characteristic seven transmembrane-spanning domains, and a short carboxy-terminal intracellular part (1). FSH stimulation results in activation of the Gs protein and subsequent cAMP production. In the male, it supports indirectly spermatogenesis through action on Sertoli cells, together with the Leydig cell product testosterone. In mice with targeted disruption of the FSH ß-subunit or FSH receptor (FSHR) genes, males are fertile despite decreased testis size and epididymal sperm count; however, all female homozygotes are sterile (2, 3, 4). In the female, FSH is therefore indispensable for recruitment and maturation of Graafian follicles through its growth-promoting action and stimulation of granulosa cell estrogen production.

Expression of the FSHR is strictly limited to Sertoli cells in the testis and granulosa cells in the ovary, which may implicate the presence of cell-specific cis-regulatory elements in the FSHR promoter. The FSHR gene has been cloned from various species, including the human, rat, mouse, bovine, ovine, and chicken (5, 6, 7, 8, 9, 10). Various regulatory elements have been identified in the 5'-flanking region of the FSHR gene, including an E box upstream-activating sequence (CANNTG) and an initiator region conserved in the rat, human, and mouse (11). In a murine Sertoli cell line, MSC-1, Heckert et al. (12) identified two upstream stimulatory factors, 1 and 2, as primary components of the complexes binding the E box. In the rat, an activator protein 1 (AP-1) binding site in the FSHR (6) and a cAMP-responsive element (CRE)-like sequence have also been described (13). The FSHR expression is up- regulated by FSH-induced cAMP production in cultured rat granulosa cells and inhibited by some growth factors [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF)] (14). FSHR expression is posttranscriptionally down-regulated by a cAMP-dependent mechanism in rat Sertoli cells (15), and a biphasic effect of gonadotropins on FSHR regulation has also been demonstrated in vivo (16). However, the mechanisms of cell specificity of FSHR expression have not yet been studied, partly because of the lack of permanent cell lines of gonadal somatic cells. The expression of FSHR is extremely sensitive to in vitro conditions and is rapidly lost in tissue culture conditions and upon cell transformation. Immortalized lines of steroidogenic granulosa (KK-1) (17) and Sertoli (MSC-1) (18) cells have been generated by genetically targeted tumorigenesis in transgenic mice. However, these cell lines do not respond to FSH stimulation: the former lost its response to FSH after prolonged culture, and the latter only expressed the functional catalytic subunit of adenylate cyclase.

Recent studies have shown that an orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays an important role in the regulation of gene expression of steroidogenic cells. SF-1 was first recognized to regulate steroidogenic enzymes in the adrenal gland and gonads (19). SF-1 shares common structural organization with other members of the steroid receptor superfamily, but, in contrast to the others, it recognizes specific hexameric response elements in target genes and binds as a monomer (20). SF-1 expression has been detected in steroid hormone-producing organs, including testicular Leydig and Sertoli cells, ovarian theca and granulosa cells, and adrenocortical cells (21, 22) as well as in the ventral medial nucleus of the hypothalamus and in pituitary gonadotrope cells (23, 24). Targeted disruption of the SF-1 gene resulted in pleiotropic impairment of function of the hypothalamic-pituitary gonadal axis and adrenal cortex, supporting the role of SF-1 as a key regulator of endocrine development and function (25).

Herein, we have identified a cell-specific regulatory region of the murine (m) FSHR promoter containing functional noncanonical recognition sites for SF-1. Our aim was to characterize the SF-1 binding sites and to study the regulation of FSHR promoter activity by SF-1 in steroidogenic and nonsteroidogenic cell lines. Two steroidogenic cell lines, derived from FSHR-expressing cells, were selected despite their inability to express the endogenous FSHR in culture, i.e. immortalized murine granulosa cells (KK-1) (17) and Sertoli cells (MSC-1) (18). Although lacking FSHR, these cells nevertheless express other markers specific to FSH- responsive cells. Additionally, a Leydig cell-derived steroidogenic cell line lacking FSHR (mLTC-1) (26), as well as a nonsteroidogenic embryonic kidney cell line (HEK 293) were used.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Structure of the 5'-Flanking Sequence of the mFSHR Gene
Analysis of the 1,548-bp mFSHR 5'-flanking sequence revealed a number of putative binding elements for various transcription factors (Fig. 1Go). These include two CRE-like sequences at positions -227 and -655 bp (in relation to translation initiation codon) and two putative estrogen response element (ERE) half-sites at position -394 bp and -679 bp. The presence of the CREs is consistent with the known transcriptional regulation of FSHR expression by cAMP (14, 15). Additionally, several putative PEA 3 binding sites at positions -521, -758, and -819 bp, reported to function as phorbol ester response elements (27) and AP-1 involved in the protein kinase C pathway (28), were identified. These are in agreement with findings of involvement of additional signaling pathways, in addition to cAMP, in target cell responses to FSH stimulation (29, 30). A consensus Sp 1 binding site (-1,313 bp) was also identified. In addition to its general role in the transcription of housekeeping genes, Sp 1 has regulatory functions in the mouse differentiation process (31). The E box and the initiator region (In R), described previously in the rat FSHR promoter (11), were located at positions -118 bp, and -81 to -107 bp, respectively. The mouse E box shares total homology with those of the rat and ovine, while a 1-bp change was noticed in the human E box. The marked 94% homology between rat and mouse promoters (32) was also evident in the initiator region. Putative GATA (-534 bp) and transforming growth factor-ß (TGFß) (-734 bp) binding sites were also observed. Further analysis of the mFSHR promoter revealed additional potential binding elements. These were three SF-1-like binding sites (SLBS), containing the core sequence CAAGG, but differing at their 3'-ends compared with the consensus sequence. One of these SLBS was located downstream of the main transcription initiation site between nucleotides -289 to -281 bp (5'-TCAAGGAAT-3', SLBS-1), and two were located in the distal region of the promoter at positions -1,171 to -1,163 bp (5'-AACCCTTGG-3' reverse orientation, SLBS-2) and -1,369 to -1,361 (5'-CCAAGGACT-3', SLBS-3) (Fig. 1Go).



View larger version (43K):
[in this window]
[in a new window]
 
Figure 1. DNA Sequence of the 5'-Flanking Region of Mouse FSHR Gene

The nucleotides are numbered assigning position -1 for the first nucleotide 5' of the translation initiation codon. A deduced sequence for the first amino acids translated is shown below the DNA sequence. The main transcription start site of the FSHR gene (7 ) at -534 is indicated by an arrow. Potential binding sites as well as the putative E box and In R regions are underlined. The restriction endonuclease recognition sites used for generation of deletion mutants are boxed.

 
Characterization of a Steroidogenic Cell-Specific Domain in the Distal Region of the mFSHR Promoter
We prepared chimeric constructs of the mouse FSHR promoter, in which the immediate -1,548-bp sequence of the 5'-flanking region, and various deletion mutants thereof, were used to drive expression of the luciferase reporter gene. The promoter activity was investigated in murine granulosa tumor (KK-1) and Sertoli tumor (MSC-1) cells that had lost their endogenous FSHR expression (17, 18), as well as in a Leydig cell line (mLTC-1) that did not express this receptor in vivo. Additionally, a nonsteroidogenic cell line (HEK 293) was used as a control. As shown in Fig. 2Go, the promoter fragments of differential lengths displayed highly variable transcriptional activities in the mLTC-1 and KK-1 cells. Surprisingly, the overall luciferase/ß-galactosidase activity was highest in Leydig mLTC-1 cells (P < 0.05), displaying a maximum of 105-fold increase over the promoterless control plasmid (pBL-0Luc) with the longest promoter sequence (pBL-1548Luc), as compared with a respective 55-fold stimulation in KK-1 granulosa cells. Deletion of region -1,548/-1,110 bp caused a dramatic 85% reduction of promoter activity in mLTC-1 cells and 69% in KK-1 cells. Using the same construct, a significantly less pronounced decrease of luciferase activity was found in HEK 293 (48%) and MSC-1 (31%). A further deletion of the promoter sequence down to -867 bp partially restored the transcriptional activity in mLTC-1 and KK-1 cells, and totally in MSC-1 and HEK 293 cells, whereas removal of the sequence between -867 and -555 bp did not significantly affect the promoter activity in any of the cell types studied.



View larger version (19K):
[in this window]
[in a new window]
 
Figure 2. Deletion Analysis of the Murine FSHR Promoter Function

Different promoter/luciferase constructs were transiently transfected into immortalized murine granulosa (KK-1), Leydig (mLTC-1), and Sertoli (MSC-1) cell lines, and in human embryonic kidney cell line (HEK 293). Luciferase/ß-galactosidase activity is presented as fold-increase over a promoterless luciferase construct (pBL-0Luc), used as negative control. The left side of the figure shows schematically the different deletion mutants and the restriction sites used for their generation. The approximate position of the main transcription initiation site according to Huhtaniemi et al. (7 ) is indicated by arrows. The results shown on the right represent the mean ± SEM of luciferase/ß-galactosidase activities (fold over promoterless construct pBL-0Luc) of three independent experiments measured in triplicate.

 
The minimum promoter length capable of driving luciferase gene expression in the steroidogenic and nonsteroidogenic cells resided between nucleotides -555 to -99. The orientation of this regulatory element was critical for transcriptional activity of the FSHR promoter, since in reverse orientation it was nearly totally devoid of activity. The inability of region -1,548/-555 to promote luciferase activity in the absence of the -555/-99 region was confirmed using the pBL-1548{Delta}-555,-99Luc construct. This construct induced only marginal increases of luciferase activity in all cell types tested. The activity was comparable to that evoked by pBL-99Luc, and it was negligible in comparison to that measured with constructs carrying the minimal promoter sequence. Conspicuously, the residual low promoter activity observed with the short promoter fragments was roughly similar in each cell line, indicating that the steroidogenic cell-specific cis elements reside in the distal region of the FSHR promoter. In the mFSHR promoter, a major transcription initiation site was found at position -534 bp (7). The pBL-555Luc construct displays high promoter activity in KK-1 and mLTC-1 cells, although it contains only 21 bp upstream of the main transcription initiation site. In this study, the positions of transcription initiation sites of the different constructs were not assessed. Multiple transcription start sites have been demonstrated for the rat (6) and human (5) FSHR genes. In this regard, the presence of alternative, quantitatively less marked transcription start sites is also possible with the mFSHR promoter as has been previously suggested (7).

The Cell-Specific mFSHR Promoter Activity Is Correlated with the SF-1 mRNA and Protein Contents
As SF-1 is a likely candidate for transactivation of the FSHR gene in steroidogenic cells, the endogenous SF-1 mRNA expression and SF-1 protein contents were investigated (Fig. 3Go). The endogenous 2.7-kb transcript of SF-1 mRNA (panel A, upper part) and a 53-kDa protein (panel B, upper) were demonstrated in KK-1, MSC-1, and at the highest level in mLTC-1 cells. The size of the protein (53 kDa) is consistent with the reported molecular mass of SF-1 (19, 21). Mouse testicular and ovarian tissues, used as positive controls, also showed SF-1 mRNA expression, at higher levels in the ovary. The nonsteroidogenic embryonic kidney cells (HEK 293) did not express endogenous SF-1 mRNA or protein; however, an abundant 53-kDa SF-1 protein band was detected in these cells after transient transfection of the SF-1 expression plasmid. The endogenous SF-1 mRNA expression was 1:3 and 1:10 in KK-1 and MSC-1 cells, respectively, as compared with mLTC-1 cells (Fig. 3AGo, lower panel), and similar differences were observed at the level of SF-1 protein (Fig. 3BGo, lower panel).



View larger version (39K):
[in this window]
[in a new window]
 
Figure 3. Northern Blot and Immunoblot Analysis of Endogenous SF-1 mRNA and Protein Expression

A, Twenty micrograms of total RNA were prepared from KK-1, mLTC-1, MSC-1, and HEK 293 cells, as well as from normal mouse testicular and ovarian tissues (upper panel). Hybridization was performed with a [{alpha}-32P]dCTP-labeled EcoRI/PstI fragment of the SF-1 cDNA probe generated from pCMV119+-SF-1 plasmid (see Materials and Methods). The ethidium bromide staining of 28S rRNA is shown below as proof of equal RNA loading. B, Ten micrograms of nuclear extract were resolved by SDS-PAGE and blotted onto nitrocellulose filter. The membrane was probed with a polyclonal anti-SF-1 antibody raised in rabbit (1:5,000), and protein bands were visualized by chemiluminescence. Lower panels, The arbitrary densitometric unit (ADU) values of the 2.7-kb SF-1 mRNA transcript (panel A) and of the immunoreactive 53-kDa protein (panel B) as quantified by densitometric scanning. Values represent the mean ± SEM of three separate experiments. Bars bearing asterisks are statistically significantly different (P < 0.05) compared with the level of KK-1 cell SF-1 expression.

 
To obtain evidence for a cell-specific region in the mFSHR promoter, luciferase activity of constructs pBL-1,548, -1,110, and -867Luc (see Fig. 2Go) was compared with activity of the construct carrying the minimum mFSHR promoter (pBL-555Luc) (Fig. 4AGo). The activator domain between -1,548/-1,110 bp increased the promoter activity especially in the steroidogenic cell lines, but with significant effect only noticed in mLTC-1 cells, where a 2-fold increase occurred as compared with pBL-1548Luc activity of HEK 293 cells. However, the repressor domain that encompasses region -1,110/-867 bp provoked a decrease of promoter activity that was similar in steroidogenic and nonsteroidogenic cells. It is of interest that the cell-specific increase of promoter activity with constructs containing the -1,548/-1,110 bp region correlated significantly (P < 0.05) with the levels of endogenous SF-1 mRNA and protein (Fig. 4BGo). In accordance, this distal promoter region possesses two nonconsensus SF-1 recognition sites at positions -1,163 bp (SLBS-2) and -1,361 bp (SLBS-3) (Fig. 1Go). Taken together, these data are in favor of SF-1- dependent regulation of the mFSHR promoter activity through the distal -1,548/-1,110 bp activator domain. To study further the SF-1-dependent mFSHR promoter activity, we concentrated on investigating the mLTC-1 cell line, which has the dual advantage of expressing high levels of endogenous SF-1 and no FSHR. HEK 293 cells were used as a negative control free of endogenous SF-1 expression.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 4. Cell-Specific mFSHR Promoter Activity in Relation to Endogenous SF-1 Expression

Panel A represents the luciferase/ß-galactosidase activities (mean ± SEM) of pBL-1548Luc, pBL-1,110Luc, and pBL-867Luc promoter constructs compared with activity of the construct carrying the minimal promoter (pBL-555Luc) for each cell line, assigned a value of 100%. The bar bearing an asterisk is statistically significantly different (P < 0.05) from the data obtained on HEK 293 cells. Panel B presents the correlation between pBL-1548Luc activity and the level of endogenous SF-1 mRNA (circle) or protein (triangle) level in the four cell lines studied.

 
A Functional SF-1 Binding Site Is Present in the mFSHR Promoter Sequence
To reveal putative interactions between SF-1 protein and the SF-1-like binding sites (SLBS-1, -2, or -3), electrophoretic mobility shift assays (EMSAs) were carried out. Purified nuclear extracts of KK-1, MSC-1, mLTC-1, and HEK 293 cells, transfected or untransfected with the SF-1 plasmid, were incubated with specific radiolabeled oligonucleotides containing the different SLBS sites. In the experimental conditions used, we were unable to detect specific SF-1-shifted complex using oligonucleotides SLBS-1 and SLBS-2, either with nuclear extracts from mLTC-1 or SF-1-transfected HEK 293 cells (data not shown). With oligonucleotide carrying the SLBS-3 site, purified nuclear extract from mLTC-1 cells gave rise to two slowly migrating DNA-protein complexes (Fig. 5Go, lane 3). The intensity of the lower shifted complex was markedly decreased using KK-1, and absent using MSC-1 nuclear extract. However, the intensity of the higher molecular mass complex was not dependent on the cell line (lanes 2–5). With HEK 293 cell nuclear extract, three shifted complexes were found (lane 5), whereas with HEK 293-SF-1 cell nuclear extract, a new shifted complex appeared to the detriment of the other bands (lane 6). Interestingly, this SF-1-specific complex comigrated with the lower binding complex detected with the mLTC-1 and KK-1 cell extracts. To verify the identity of the protein(s), gel-shift assays were performed in the presence of a rabbit polyclonal antibody directed against the full-length bovine SF-1 protein (33) (Fig. 6Go). The addition of a 1:100 dilution of the SF-1 antiserum reduced the intensity whereas a 1:10 dilution totally abolished the SF-1/SLBS-3 complex generated by the HEK 293-SF-1 and mLTC-1 nuclear extracts (panel A and B, respectively). The same dilutions of nonimmunized rabbit serum were not able to reverse the SF-1/SLBS-3 complexes in either cell lines (data not shown). This type of competition, termed gel-shift abrogation, has already been described and relies on ability of the anti-SF-1 antibody to effectively block accessibility of the DNA to the SF-1 DNA-binding domain (34, 35). Collectively, these data support the notion that SF-1 is capable of binding to the SLBS-3 sequence of mFSHR promoter.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 5. Intensity of a Shifted Complex in EMSA Correlates with Cell-Specific SF-1 Protein Content

Oligonucleotides SLBS-3S and SLBS-3AS (see Table 1Go) were annealed, end-labeled, and incubated without (lane 1) or with 10 µg of nuclear extract of KK-1, mLTC-1, MSC-1, and HEK 293 cells (lanes 2–5). The nuclear extract used in lane 6 was from HEK 293 cells transiently transfected with 1 µg of the pCMV119+-SF-1 expression plasmid. The DNA-protein complexes were resolved on a 4% polyacrylamide gel after 1 h incubation at 4 C.

 


View larger version (30K):
[in this window]
[in a new window]
 
Figure 6. Inhibition of SF-1 Binding to SLBS-3 by Rabbit SF-1 Antisera

Nuclear cell extracts (10 µg) from HEK 293 cells (panel A, lane 2) and HEK 293 transfected with SF-1 expression plasmid (panel A, lanes 3–5) or mLTC-1 (panel B, lanes 2–4) were incubated with a rabbit polyclonal SF-1 antibody (1:10 and 1:100) (33 ) for 45 min at 4 C before the addition of 32P-radiolabeled double-stranded SLBS-3 oligonucleotide. Protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide gel.

 
The direct effect of SF-1 on mFSHR promoter activity was investigated in HEK 293 cells devoid of endogenous SF-1 expression (Fig. 7Go). Coexpression of SF-1 cDNA increased, in a dose-dependent manner, the FSHR promoter-driven luciferase activity, reaching 2.3-fold elevation with 1 µg of the pCMV119+-SF-1 sense plasmid. Cotransfection of pCMV119--SF-1 (reverse orientation) failed to stimulate promoter activity. Hence, it can be concluded that SF-1 is able physically to interact with the SLBS-3 domain present in the distal region of the mFSHR promoter. Moreover, the binding of SF-1 to the mFSHR promoter sequence enhances its effect on reporter gene expression.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 7. Dose-Dependent Increases of mFSHR Promoter Activity after Transient Transfection of SF-1 Expression Plasmid into Cultured HEK 293 Cells

The cells were transiently transfected with an FSHR promoter/luciferase reporter gene construct (pBL-1548Luc) and increasing amounts (0.1 to 1 µg) of pCMV119+-SF-1 sense (lanes 2–5) or 1 µg pCMV119--SF-1 antisense (lane 6) expression plasmids. The pCMV-ß-galactosidase plasmid was cotransfected to control for transfection efficiency. The results shown are luciferase/ß-galactosidase ratios in relation to the pBL-1548Luc activity (mean 100%). The molar amount of transfected DNA was equalized using empty pMT2 vector. Each bar represents the mean ± SEM of three separate experiments, each run in triplicate. Different letters above the bars indicate that the difference between them is statistically significant (P < 0.05).

 
Integrity of the SF-1/SLBS-3 Sequence Is Needed for Full Binding Capacity and Transcriptional Activity
To further confirm the specificity of the SF-1/SLBS-3 binding complex, mutations were introduced into SLBS-3. As shown in Fig. 8AGo (lanes 2 and 3), using mLTC-1 cell nuclear extract, the shifted lower molecular mass DNA-protein complex could be competed for by increasing the amount of unlabeled SLBS-3. The same molar excess of competitor, mutated in the SF-1 core sequence, failed to displace SF-1 binding (lanes 4 and 5). The lower molecular mass SF-1 binding complex also disappeared totally after addition of 50-fold molar excess of competitor containing the SF-1 consensus sequence (comparing lanes 2 and 6). An identical experiment was performed with HEK 293-SF-1 extract, showing the same pattern of competition (Fig. 8BGo, lanes 1–4). In lanes 5 and 6, SLSB-2 and SLBS-1 oligonucleotides were used in high excess (x400) as competitors, and decreases in SF-1/SLBS-3 complex intensity were observed in both cases, SLBS-2 behaving as a stronger competitor. The latter finding suggests that SLBS-2 and, to a lesser extent, SLBS-1 could also bind SF-1 protein, although with lesser avidity. A nonspecific AP-1 competitor used at 200- and 400-fold excess failed to affect the SF-1/SLBS-3 binding complexes either using HEK 293-SF-1 or mLTC-1 nuclear extract (data not shown).



View larger version (53K):
[in this window]
[in a new window]
 
Figure 8. Characterization of the Distal FSHR SF-1 Binding Site by Competition Studies

A, EMSA performed using nuclear extract from mLTC-1 cells preincubated in the presence of 50- or 200-fold molar excess of unlabeled double-strand SLBS-3 (lanes 2 and 3), SLBS-3-mut (lanes 4 and 5), and SLBS-3-Con (lanes 6 and 7). A 32P-radiolabeled SLBS-3 probe was added for a further 1-h incubation. The DNA protein complexes were resolved on a polyacrylamide gel. B, EMSA performed as described above using SF-1-transfected HEK 293 nuclear extract. Competitors were use at 200-fold excess in lanes 2–4, and at 400-fold excess in lanes 5 (SLBS-2) and 6 (SLBS-1). The SLBS-3-Mut was prepared using oligonucleotides MUT-3S and MUT-3AS, to change the SF-1 core sequence from CAAGG to CAATT. For SLBS-3-Con, CON-3S and CON-3AS oligonucleotides were used to create a consensus SF-1 site CAAGGTCA. All the oligonucleotides used as double-stranded competitors are listed in Table 1Go.

 
The mutation that abolished SF-1 binding to SLBS-3 (CAAGG changed to CAATT) was introduced to the SLBS-3, SLBS-2, and SLBS-1 sequences (Fig. 9Go). The effects were investigated in HEK 293 cells transfected or untransfected with SF-1 expression plasmid, as well as in mLTC-1 cells. No significant changes in promoter activity were observed when using HEK 293 cells, regardless of the site mutated. However, destruction of the SF-1 binding site of SLBS-3 resulted in a 38–40% decrease (P < 0.05) of promoter activity in both HEK 293-SF-1 and mLTC-1 cells as compared with nonmutated pBL-1548Luc. Additionally, a mutation in SLBS-2 slightly but significantly suppressed the luciferase activity (20–25%) while no effect was shown with mutated SLBS-1. Reduction of FSHR promoter activity in mLTC-1 cells reached 50% when both SLBS-3 and SLBS-2 were mutated (data not shown). The mutation introduced in the SLBS sites created an E box-like element (CANNTG). To eliminate this potential experimental artifact, a second set of mutations (CAAGG to CCGGG) in SLBS-3 and SLBS-2 were assayed. The promoter activity of constructs carrying the newly mutated SLBS-3 site was similarly decreased to 50% and 37% in HEK 293-SF-1 and mLTC-1 cells, respectively. With the second mutated SLBS-2 construct, a reduced promoter activity was also observed in both cell lines (data not shown). The two sets of SLBS-3-mutated primers were also used as oligoprobe in EMSA assay, and no SF-1-specific shifted bands could be detected with either HEK 293-SF-1 or mLTC-1 nuclear extract (data not shown).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 9. Mutation in SF-1 Sites Affects the FSHR Promoter Activity in mLTC-1 and HEK 293-SF-1 Cells

PCR-based mutagenesis was used to generate mutations in the putative SF-1-like site of the FSHR promoter. pBL-1548Luc was used as the template and site-directed mutagenesis was carried out as described in Materials and Methods. The mutated constructs were transiently transfected into mLTC-1, HEK 293, and SF-1-transfected HEK 293 cells, 24 h before luciferase and ß-galactosidase measurements. The results shown are luciferase/ß-galactosidase activity ratios in relation to the nonmutated pBL-1548Luc construct (mean 100%). Each bar represents the mean ± SEM of three separated experiments, each done in triplicate. Bars bearing asterisks are statistically significantly different (P < 0.05) from nonmutated constructs in the same cell line. The left panel depicts schematically the positions of the mutated SF-1-like sites.

 
The transformation of the SLBS-3 sequence to a consensus SF-1 site (CAAGGTCA) had a positive effect (52% increase, P < 0.05) on SF-1-induced promoter expression in mLTC-1 cells, and a 22% increase was observed in HEK 293-SF-1-transfected cells (Fig. 9Go). These results confirm the role of SLBS-3 and, to a lesser extent, of SLBS-2 in the SF-1-regulated mFSHR promoter activity. Furthermore, the binding capacity and transcriptional ability are closely related and dependent on the SF-1 DNA recognition sequence.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The FSHR promoter belongs to the promoter types that lack the conventional TATA and CCAAT box elements, a characteristic shared by ubiquitously and constitutively expressed genes. However, in contrast to housekeeping genes, that of FSHR is tightly regulated during development and expressed in a cell-specific manner. This subclass of TATA-less promoters usually contains an initiator region with one or several transcription start sites close to the ATG codon. The positive regulatory elements needed for full activity of the rat and human FSHR promoters have been exclusively sought and located in the proximal region of the 5'-untranslated region, close to their respective transcription start sites (5, 11, 13). The majority of the positive regulatory elements described in rat and human minimal promoters were also found in the mouse and sheep proximal promoter region (Fig. 10Go). However, these putative cis-acting domains were located downstream from the main transcription initiation site in the mouse (-534 bp). The present study demonstrated the active participation of the sequence between nucleotides -99 to -555 bp in transactivation of the promoter. Thus, the cis-acting elements located downstream of the transcriptional start site are apparently functional in the mFSHR promoter, as has been reported for several other genes (36, 37). Several regions 3' of the transcription start sites were also important for promoter function in the rat FSHR (12). The molecular mechanisms by which these elements enhance transcription are largely unknown, but they may act in a manner similar to 5'-promoter elements, to help recruitment of components of the general transcription machinery, or they may be important for start site selection or elongation. Further studies are needed to define the proximal sequences of the promoter regions that interact with cellular transcription factors to modulate the expression of gonadotropin receptors and augment in selection of transcription initiation start sites.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 10. Alignment of Putative DNA-Responsive Elements of the FSHR Proximal Promoter Regions

The murine (7 ), rat (6 ), human (5 ), and chicken (9 ) nucleotide sequences are numbered assigning -1 to the first nucleotide above the translation initiation codon (ATG). Capital letters represent identical nucleotides.

 
In this study, we demonstrated that additional regions far upstream of the transcription initiation sites are also involved in the cell-specific mFSHR expression. Deletion of a distal region between bp -1,548 to -1,110, containing two putative SF-1 binding sites, showed a decrease in promoter activity in highly SF-1-expressing mLTC-1 cells. We demonstrated herein that overexpression of SF-1 induced a dose-dependent increase of the mFSHR promoter activity in transiently transfected HEK 293 cells. The SF-1 action was mostly mediated by a nonconsensus SF-1 site at position -1,369 bp (5'-CCAAGGACT-3'). SF-1 was able to specifically bind to this SLBS-3 sequence and transactivate FSHR promoter expression. A mutation of the functional SLBS-3 motif, which abolished its binding ability, significantly, although not totally, suppressed the promoter activity. The luciferase activity and the intensity of the SF-1/SLBS-3 binding complex were closely related to the endogenous SF-1 mRNA expression and SF-1 protein levels in steroidogenic cell lines.

SF-1, alternatively known as adrenal 4-binding protein, is an orphan nuclear receptor that is a key regulator of steroidogenic enzymes (19, 21). In mammals, SF-1 plays a key role in the development and differentiated function of the adrenal glands and gonads. In addition to its role during development, and consistent with its distribution, SF-1 functions as a potent transcription factor for many genes involved in hypothalamic-pituitary-steroidogenic function. In addition to steroidogenic enzymes, SF-1 regulates in mice the expression of ACTH and GnRH receptors, the LH ß-subunit, steroidogenic acute regulatory protein (StAR), anti-Müllerian hormone, and DAX-1 (for references, see Ref. 38). SF-1 possesses multiple putative functional domains, including a characteristic zinc finger DNA-binding domain at the N-terminal region, a hinge region, a dimerization domain, a ligand-binding domain, and a conserved ligand-dependent activation function 2 (AF-2) in the distal C terminus essential for receptor transactivation (39).

Ueda et al. (40) demonstrated that SF-1 binds as a monomer to its recognition element, the hexameric AGGTCA motif, which is recognized by the two zinc fingers, leading to high-affinity binding. In this work, the binding of SF-1 to SLBS-3 (AGGaCt) appeared less effective than to a consensus SF-1 site. In addition, SLBS-3-Con (carrying consensus sequence) was a more potent competitor than SLBS-3. These data suggest a sequence- dependent binding ability of the putative SF-1 site to SF-1 transcription factor. The differences in SF-1 binding motifs observed in SLBS-1 (AGGaat) and SLBS-2 (TCCcaT) lead to near-totally or severely suppressed binding ability. However, mutation in SLBS-2 significantly affected the FSHR promoter activity. In the rat StAR promoter, Sandhoff et al. (41) described high- and low-affinity SF-1 binding sites, all possessing the core CAAGG domain but with differences at their 3'-ends. All the three SLBSs described in the FSHR promoter contain the core CAAGG sequence essential for SF-1 binding (42). However, the differences in function of SLBS-1, -2, and -3 suggest that the presence of this core sequence is not sufficient for full SF-1-mediated promoter activation. Thus, participation of additional nucleotides in the binding site and/or spatial arrangement of the SF-1 site may contribute to full regulatory function. In the present work, gel shift assays confirmed that the GG dinucleotide in the core sequence was critical for SF-1 binding ability and functional activity. Furthermore, we showed that cytosine at the 3'-end of the recognition sequence was also crucial for binding, while the TCA motif supports the ability for high-affinity binding, consistent with the canonical SF-1 sequence, 5'-YCAAGGYCR-3' (34, 42). Moreover, SF-1 binding and transcriptional activity have been demonstrated through other nonconsensus sequences (43, 44). Hence, the involvement of SLBS-2 or even SLBS-1 sites in the SF-1-induced FSHR promoter activation, as well as through other noncharacterized sequences, cannot be totally excluded. Additional sequences resembling the SF-1 recognition site have been reported in the 2.1-kb 5'-upstream region of the ovine FSHR gene (9), but their functions have not been explored.

Tissue-specific gene expression requires the combined action of tissue- and promoter-specific activators and repressors. Mutation or deletion of the functional SF-1-like motif (SLBS-3) significantly decreased, but did not abolish, the mFSHR promoter activity. The positive element located in the -1,548/-1,110 region of the mFSHR promoter was not able to evoke promoter activity without cooperation with the proximal minimal promoter, suggesting the presence of multiple response elements for cell-specific factors in addition to the ubiquitous regulatory sequences. We characterized various putative binding sites for ubiquitous and cell-specific transcription factors in the mFSHR promoter that could participate in the restricted FSHR expression specific for FSH target cells. Interestingly, functional interaction or direct heterodimerization has been shown between SF-1 and a number of these transcription factors, including Sp1 (45), cAMP-responsive element binding protein (44), estrogen receptor (46), and GATA-4 (47). These transcription factors act in synergy with SF-1 to potentiate the SF-1 regulatory function. Another regulatory factor, DAX-1, has been extensively described as a potent repressor of SF-1 action (48, 49, 50). Moreover, coactivators and corepressors, with distinct tissue-specific expression patterns and hormonal regulation, are also involved in the modulation of SF-1 action (51, 52).

Targeted disruption of the murine SF-1 gene resulted in adrenal and gonadal aplasia, male-to-female sex reversal of the internal and external genitalia, malformations of the ventromedial hypothalamus, and selective deficiency of GnRHR, LHß, and FSHß mRNA in the pituitary gland (22, 25, 53). Analysis of SF-1 expression in embryonic gonads did not only support a role for SF-1 in steroidogenesis, but also indicated that this transcription factor may play additional roles in development (54). The pattern of SF-1 expression correlates with the ontogeny of FSHR expression during fetal life, as SF-1 mRNA level was detected before the onset of FSHR expression in both sexes (25, 55). After birth, SF-1 is mainly expressed in seminiferous tubules, and high expression was observed in rat Sertoli cells during a limited period of pubertal maturation before the first cycle of spermatogenesis (56), coinciding with a peak in FSHR mRNA expression (57). SF-1 is a negative regulator of granulosa cell mitosis; thus, enhanced SF-1 expression is part of the molecular mechanism associated with granulosa cell differentiation. In addition, SF-1 knockout females fail to develop ovaries, suggesting that SF-1 is essential for ovarian organogenesis (58).

In conclusion, the SF-1-dependent regulation of FSHR expression, as demonstrated in the present study, is in line with functions demonstrated earlier for this transcription factor. The concomitant changes in FSHR promoter activities with the expression levels of SF-1 further supported SF-1 as an in vivo regulator of FSHR gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
An XbaI/XhoI genomic DNA fragment, containing a 1.5-kb fragment of the murine FSHR 5'-flanking region, was obtained by PCR methods using the pFSHRI plasmid (7) carrying 7.5 kb of the 5'-end and exon I of the mouse FSHR gene. Two endonuclease restriction sites were introduced in pFSHRI using primers FSHR1 and FSHR2 (Table 1Go), XbaI site at position -1,548 into the 5'-untranslated region, and XhoI site to substitute the ATG translation start site. The -1,548/-1 promoter fragment was subcloned into pBL-Luci plasmid in front of the luciferase reporter gene-coding sequence using XbaI and XhoI restriction endonucleases to obtain pBL-1548Luc. All mouse FSHR promoter deletion mutants were prepared using endonuclease restriction sites of the promoter region present in pBL-1548Luc. pBL-1110Luc and pBL-867Luc mutants were constructed by digestion with PstI and HindIII, respectively. With BamHI and BglII we obtained two fragments, -1,548/-555 and -555/-99, which we used to construct pBL-555Luc, pBL-99Luc, pBL-99–555Luc, and pBL-1548{Delta}-555–99Luc after random ligations. The identities of all FSHR promoter constructs were verified by restriction mapping and sequencing.


View this table:
[in this window]
[in a new window]
 
Table 1. Oligonucleotides Used in This Study

 
Cell Culture
The mouse Leydig tumor cell line (mLTC-1) (26) was cultured in HEPES (20 mM)-buffered Waymouth’s medium (Sigma, St Louis, MO), supplemented with 9% heat-inactivated horse serum (Life Technologies, Inc., Paisley, Scotland), and 4.5% heat-inactivated FCS (iFCS) (Bioclear, Wilts, UK) containing 0.1 ng/ml gentamycin (Life Technologies, Inc., Gaithersburg, MD). The lines of granulosa cells (KK-1) (17), mouse Sertoli cells (MSC-1) (18), and human embryonic kidney cells (HEK 293) were maintained in DMEM/Ham’s F-12 1:1 medium (Sigma), supplemented with 10% iFCS, containing 50 mIU/ml ampicillin and 0.5 µg/ml streptomycin (Sigma). The cells were allowed to grow on 10-cm diameter plates to 70–80% confluency under a humidified atmosphere of 95% and 5% CO2 at 37 C.

Transient Transfection of Cell Lines
The transient transfection method was optimized for each cell lines using pCMV-ß-galactosidase as the marker of transfection efficiency. Cells were seeded on six-well plates at a density of 0.5 x 106 cells per well, 16–20 h preceding transfection to achieve 60–70% confluence. Cells were transfected with 1.5 µg of one of the FSHR promoter constructs and 0.3 µg of pCMV-ß-galactosidase as control. The mLTC-1 cells were transiently transfected by the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) as described in the manufacturer’s protocol. After 15 min incubation of the FuGENE 6-DNA complex, it was distributed dropwise on cells grown overnight and cultured for a further 24 h. For HEK 293, KK-1, and MSC-1 cells, LipofectAMINE transfection reagents (Life Technologies, Inc.) were used, according to instructions of the manufacturer. The transfection solution was removed after 5 h incubation at 37 C and replaced by 2 ml of complete DMEM/Ham’s F-12 for a further 24-h culture.

For cotransfection experiments, we used plasmids expressing SF-1 cDNA in correct or reverse orientation, pCMV119+-SF-1 and pCMV119--SF-1, respectively (obtained from Dr. K. L. Parker, Durham, NC). The amount of transfected DNA was equalized using the empty pMT2 vector. The volume of both LipofectAMINE and FuGENE 6 reagents was increased and adjusted to the DNA amount according to the manufacturer’s instructions.

After 24 h, transfection media were replaced by 100 µl of cell lysis buffer [12.5 mM Tris-HCl, pH 7.8, 10 mM NaCl, 0.4 mM EDTA, 0.2 mM MgSO4, 1 mM dithiothreitol (DTT), and 0.2% Triton X-100] for 5 min. The cells were scraped off and centrifuged for 1 min at room temperature. Luciferase activity was measured from 10 µl of the lysate in a Victor multilabel counter (Wallac, Inc., Turku, Finland) by adding 100 µl of luciferase assay buffer (40 mM Tris-HCl, pH 7.8, 0.5 mM ATP, 10 mM MgSO4, 0.5 mM EDTA, 10 mM DTT, 0.5 mM coenzyme A, 0.5 mM luciferin). The ß-galactosidase activity was assessed from the same lysate in 100-mm phosphate buffer, pH 7.0, supplemented with 10 mM KCl, 1 mM MgSO4, and 50 mM ß-mercaptoethanol and incubated for 30 min at 37 C in the presence of O-nitrophenyl-ß-D-galactopyranoside (ONPG) at 0.8 mg/ml final concentration. Luciferase activity was normalized for transfection efficiency by dividing the luciferase activity by ß-galactosidase activity.

Northern Hybridization Analysis
Total RNA was isolated from cells using the single-step TRIZOL (Life Technologies, Inc.) method, according to instructions of the supplier. Twenty micrograms of RNA per lane were resolved on 1.2% denaturing agarose gel and transferred onto Hybond-XL nylon membranes (Amersham International , Aylesbury, Bucks, UK). Membranes were prehybridized overnight at 42 C in a solution containing 5 x SSC, 5 x Denhardt’s, 0.5% SDS, 50% formamide, and 5 mg/ml of denatured calf thymus DNA. An EcoRI/PstI fragment of SF-1 cDNA probe was cut out from pCMV119+-SF-1, labeled with Prime-a-Gene kit (Pharmacia Biotech, Uppsala, Sweden) using [{alpha}-32P]dCTP during 4 h at 37 C and purified with NickColumn (Pharmacia Biotech). Hybridization was carried out at 42 C for 20 h in the same prehybridization solution after addition of radioactively labeled probe. After hybridization, the membranes were washed twice in 2 x SSC and 0.1% SDS at room temperature for 10 min, followed by two washes in 0.1 x SSC and 0.1% SDS at 42 C to remove most of the background. Membranes were exposed to x-ray film (XAR-5; Eastman Kodak Co., Rochester, NY) at -70 C for 4–7 days or to phosphorimager (BAS-5000 film I{alpha}I, Fuji Photo Film Co., Ltd., Tokyo, Japan) for 4–24 h. The intensities of specific bands were quantified using Tina software (Raytest, Stranbenhardt, Germany) and related to those of the 28S rRNA, in the gel stained with ethidium bromide. The molecular sizes of the mRNA species were estimated by comparison with mobilities of the 18S and 28S rRNAs.

Immunoblotting
Nuclear extracts from various cell lines were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) by electroblotting, preincubated for 2 h in TBS, 1% Tween-20, and 5% nonfat dry milk, and washed three times for 10 min in the same buffer without milk. Incubation with rabbit anti-SF-1 polyclonal antibody (33) (1:5,000) (obtained from Dr. K. Morohashi, Fukuoka, Japan) was performed overnight at 4 C. The filter was washed and incubated for 1 h with 1:1,000 rabbit Ig, horseradish peroxidase-linked antibody. After washing three times, the membrane was subjected to chemiluminescent detection using ECL Western blotting detection kit (Amersham Pharmacia Biotech), and finally the membranes were exposed for 2–10 min to Kodak x-ray films. The immunospecific bands were quantified by Tina software.

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from confluent cell cultures as described previously (59). Complementary oligonucleotides, containing the respective SF-1 like sequences, and competitors (Table 1Go) were annealed in 10 mM Tris HCl, pH 7.5, 1 mM EDTA, 25 mM NaCl, 10 mM MgCl2, 1 mM DTT. 5'-GG overhangs present in the double-stranded oligonucleotide were filled with [{alpha}-32P]dCTP for 2 h at 30 C using the Klenow DNA polymerase. The labeled oligonucleotide probes were purified in Nick Column. Nuclear extracts (10 µg) were incubated with 6 fmol (~40,000 cpm) radiolabeled, double-stranded oligonucleotides for 1 h at 4 C in a reaction buffer containing 12 mM HEPES, pH 7.9, 4 mM Tris HCl, 12% glycerol, 1 mM EDTA, 60 mM KCl, 1 mM DTT, and 300 µg/ml BSA in the presence of 2 µg poly (dI:dC). For competition experiments, the competitor was first incubated with nuclear extract at 4 C for 1 h in reaction buffer before addition of labeled probe. In antibody-abrogation gel shifts, nuclear extracts were incubated with a rabbit polyclonal antibody directed against the full-length bovine SF-1 protein (33) or an equal dilution (1:10 and 1:100) of nonimmunized rabbit serum for 45 min at 4 C before the addition of radiolabeled probe (34). Protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide gels using 0.25x Tris-borate-EDTA buffer, and the gel was dried and exposed to Kodak x-ray film at –50 C for 1–3 days.

Site-Directed Mutagenesis
Site-directed mutants of the FSHR promoter sequence were prepared using QuickChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The same complementary oligonucleotides were used as competitors in gel shift experiments and as primers for mutagenesis (Table 1Go). One hundred nanograms of the plasmid DNA template were incubated with 125 ng of appropriate primers, 25 mM deoxynucleoside triphosphates, and 50 µl of 1x reaction buffer in the presence of 2.5 IU of Pfu DNA polymerase. The PCR conditions included 16 cycles with denaturing step at 95 C for 30 sec, annealing at 55 C for 1 min, and extension at 68 C for 13 min. The parental DNA template was digested by adding 10 IU of DpnI restriction endonuclease for 1 h at 37 C. One to 5 µl of PCR reaction were used to transform XL-1 Blue Supercompetent cells. The mutations were verified by restriction mapping using Mfe I, Vsp I, XmaI, and/or Tsp 45I followed by sequencing of both strands.

Statistical Analysis
All results presented are from two to six independent experiments performed in triplicate. The data are expressed as mean ± SEM. Statistically significant differences between groups were determined by one-way ANOVA, followed by Duncan’s test; P < 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. K. L. Parker (Departments of Medicine and Pharmacology and Howard Hughes Medical Institute, Durham, NC) for the generous gift of pCMV119--SF-1 and pCMV119+-SF-1 plasmids, and to Dr. K. Morohashi (Department of Molecular Biology, Kyushu University, Fukuoka, Japan) for providing the SF-1 antibody. We thank Dr. Matti Poutanen for the helpful discussions during preparation of this manuscript. The invaluable help of Dr. Pirjo Pakarinen and technical assistance of Ms. Riikka Kytömaa are gratefully acknowledged.


    FOOTNOTES
 
Address requests for reprints to: Ilpo Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi

This study was supported by grants from the Academy of Finland and the Sigrid Juselius Foundation.

Received for publication March 13, 2000. Revision received September 19, 2000. Accepted for publication September 25, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gromoll J, Pekel E, Nieschlag E 1996 The structure and organization of the human follicle-stimulating hormone receptor (FSHR) gene. Genomics 35:308–311[CrossRef][Medline]
  2. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[Medline]
  3. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci USA 95:13612–13617[Abstract/Free Full Text]
  4. Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG, Charlton HM 2000 The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction. Endocrinology 141:1795–1803[Abstract/Free Full Text]
  5. Gromoll J, Dankbar B, Gudermann T 1994 Characterization of the 5' flanking region of the human follicle-stimulating hormone receptor gene. Mol Cell Endocrinol 102:93–102[CrossRef][Medline]
  6. Heckert LL, Daley IJ, Griswold MD 1992 Structural organization of the follicle-stimulating hormone receptor gene. Mol Endocrinol 6:70–80[Abstract]
  7. Huhtaniemi IT, Eskola V, Pakarinen P, Matikainen T, Sprengel R 1992 The murine luteinizing hormone and follicle-stimulating hormone receptor genes: transcription initiation sites, putative promoter sequences and promoter activity. Mol Cell Endocrinol 88:55–66[CrossRef][Medline]
  8. Houde A, Lambert A, Saumande J, Silversides DW, Lussier JG 1994 Structure of the bovine follicle-stimulating hormone receptor complementary DNA and expression in bovine tissues. Mol Reprod Dev 39:127–135[Medline]
  9. Sairam MR, Subbarayan VS 1997 Characterization of the 5' flanking region and potential control elements of the ovine follitropin receptor gene. Mol Reprod Dev 48:480–487[CrossRef][Medline]
  10. You S, Bridgham JT, Foster DN, Johnson AL 1996 Characterization of the chicken follicle-stimulating hormone receptor (cFSH-R) complementary deoxyribonucleic acid, and expression of cFSH-R messenger ribonucleic acid in the ovary. Biol Reprod 55:1055–1062[Abstract]
  11. Goetz TL, Lloyd TL, Griswold MD 1996 Role of E box and initiator region in the expression of the rat follicle-stimulating hormone receptor. J Biol Chem 271:33317–33324[Abstract/Free Full Text]
  12. Heckert LL, Daggett MA, Chen J 1998 Multiple promoter elements contribute to activity of the follicle-stimulating hormone receptor (FSHR) gene in testicular Sertoli cells. Mol Endocrinol 12:1499–1512[Abstract/Free Full Text]
  13. Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor. Proc Natl Acad Sci USA 92:10673–10677[Abstract]
  14. Tilly JL, LaPolt PS, Hsueh AJ 1992 Hormonal regulation of follicle-stimulating hormone receptor messenger ribonucleic acid levels in cultured rat granulosa cells. Endocrinology 130:1296–1302[Abstract]
  15. Themmen AP, Blok LJ, Post M, Baarends WM, Hoogerbrugge JW, Parmentier M, Vassart G, Grootegoed JA 1991 Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression. Mol Cell Endocrinol 78:R7–13
  16. LaPolt PS, Tilly JL, Aihara T, Nishimori K, Hsueh AJ 1992 Gonadotropin-induced up- and down-regulation of ovarian follicle-stimulating hormone (FSH) receptor gene expression in immature rats: effects of pregnant mare’s serum gonadotropin, human chorionic gonadotropin, and recombinant FSH. Endocrinology 130:1289–1295[Abstract]
  17. Kananen K, Markkula M, Rainio E, Su JG, Hsueh AJ, Huhtaniemi IT 1995 Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin {alpha}-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616–627[Abstract]
  18. Peschon JJ, Behringer RR, Cate RL, Harwood KA, Idzerda RL, Brinster RL, Palmiter RD 1992 Directed expression of an oncogene to Sertoli cells in transgenic mice using Mullerian inhibiting substance regulatory sequences. Mol Endocrinol 6:1403–1411[Abstract]
  19. 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]
  20. Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:5794–5804[Abstract]
  21. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
  22. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, 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]
  23. Roselli CE, Jorgensen EZ, Doyle MW, Ronnekleiv OK 1997 Expression of the orphan receptor steroidogenic factor-1 mRNA in the rat medial basal hypothalamus. Brain Res Mol Brain Res 44:66–72[Medline]
  24. Asa SL, Bamberger AM, Cao B, Wong M, Parker KL, Ezzat S 1996 The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 81:2165–2170[Abstract]
  25. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[Medline]
  26. Rebois RV 1982 Establishment of gonadotropin-responsive murine Leydig tumor cell line. J Cell Biol 94:70–76[Abstract]
  27. Xin JH, Cowie A, Lachance P, Hassell JA 1992 Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev 6:481–496[Abstract]
  28. Faisst S, Meyer S 1992 Compilation of vertebrate- encoded transcription factors. Nucleic Acids Res 20:3–26[Medline]
  29. Quintana J, Hipkin RW, Sanchez-Yague J, Ascoli M 1994 Follitropin (FSH) and a phorbol ester stimulate the phosphorylation of the FSH receptor in intact cells. J Biol Chem 269:8772–8779[Abstract/Free Full Text]
  30. Maizels ET, Cottom J, Jones JC, Hunzicker-Dunn M 1998 Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein kinase pathway, inducing small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulosa cells. Endocrinology 139:3353–3356[Abstract/Free Full Text]
  31. Saffer JD, Jackson SP, Annarella MB 1991 Developmental expression of Sp1 in the mouse. Mol Cell Biol 11:2189–2199[Medline]
  32. Tena-Sempere M, Manna PR, Huhtaniemi I 1999 Molecular cloning of the mouse follicle-stimulating hormone receptor complementary deoxyribonucleic acid: functional expression of alternatively spliced variants and receptor inactivation by a C566T transition in exon 7 of the coding sequence. Biol Reprod 60:1515–1527[Abstract/Free Full Text]
  33. Morohashi K, Zanger UM, Honda S, Hara M, Waterman MR, Omura T 1993 Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Mol Endocrinol 7:1196–1204[Abstract]
  34. Duval DL, Nelson SE, Clay CM 1997 A binding site for steroidogenic factor-1 is part of a complex enhancer that mediates expression of the murine gonadotropin-releasing hormone receptor gene. Biol Reprod 56:160–168[Abstract]
  35. Zimmermann S, Schwarzler A, Buth S, Engel W, Adham IM 1998 Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. Mol Endocrinol 12:706–713[Abstract/Free Full Text]
  36. Fornasari D, Battaglioli E, Flora A, Terzano S, Clementi F 1997 Structural and functional characterization of the human {alpha}3 nicotinic subunit gene promoter. Mol Pharmacol 51:250–261[Abstract/Free Full Text]
  37. Mariman E, Wieringa B 1991 Expression of the gene encoding human brain creatine kinase depends on sequences immediately following the transcription start point. Gene 102:205–212[CrossRef][Medline]
  38. Hammer GD, Ingraham HA 1999 Steroidogenic factor-1: its role in endocrine organ development and differentiation. Front Neuroendocrinol 20:199–223[CrossRef][Medline]
  39. Honda S, Morohashi K, Nomura M, Takeya H, Kitajima M, Omura T 1993 Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J Biol Chem 268:7494–7502[Abstract/Free Full Text]
  40. Ueda H, Sun GC, Murata T, Hirose S 1992 A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein. Mol Cell Biol 12:5667–5672[Abstract]
  41. Sandhoff TW, Hales DB, Hales KH, McLean MP 1998 Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1. Endocrinology 139:4820–4831[Abstract/Free Full Text]
  42. Le Drean Y, Liu D, Xiong F, Hew CL 1997 Presence of distinct cis-acting elements on gonadotropin gene promoters in diverse species dictates the selective recruitment of different transcription factors by steroidogenic factor-1. Mol Cell Endocrinol 135:31–40[CrossRef][Medline]
  43. Kawabe K, Shikayama T, Tsuboi H, Oka S, Oba K, Yanase T, Nawata H, Morohashi K 1999 Dax-1 as one of the target genes of Ad4BP/SF-1. Mol Endocrinol 13:1267–1284[Abstract/Free Full Text]
  44. Ito M, Park Y, Weck J, Mayo KE, Jameson JL 2000 Synergistic activation of the inhibin {alpha}-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate. Mol Endocrinol 14:66–81[Abstract/Free Full Text]
  45. Liu Z, Simpson ER 1999 Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression. Mol Cell Endocrinol 153:183–196[CrossRef][Medline]
  46. Drean YL, Liu D, Wong AO, Xiong F, Hew CL 1996 Steroidogenic factor 1 and estradiol receptor act in synergism to regulate the expression of the salmon gonadotropin II ß subunit gene. Mol Endocrinol 10:217–229[Abstract]
  47. Tremblay JJ, Viger RS 1999 Transcription factor GATA-4 enhances Mullerian inhibiting substance gene transcription through a direct interaction with the nuclear receptor SF-1. Mol Endocrinol 13:1388–1401[Abstract/Free Full Text]
  48. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
  49. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  50. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J 1998 Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 18:2949–2956[Abstract/Free Full Text]
  51. Misiti S, Schomburg L, Yen PM, Chin WW 1998 Expression and hormonal regulation of coactivator and corepressor genes. Endocrinology 139:2493–2500[Abstract/Free Full Text]
  52. Ito M, Yu RN, Jameson JL 1998 Steroidogenic factor-1 contains a carboxy-terminal transcriptional activation domain that interacts with steroid receptor coactivator-1. Mol Endocrinol 12:290–301[Abstract/Free Full Text]
  53. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478–486[Abstract]
  54. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
  55. Rannikki AS, Zhang FP, Huhtaniemi IT 1995 Ontogeny of follicle-stimulating hormone receptor gene expression in the rat testis and ovary. Mol Cell Endocrinol 107:199–208[CrossRef][Medline]
  56. Hatano O, Takayama K, Imai T, Waterman MR, Takakusu A, Omura T, Morohashi K 1994 Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120:2787–2797[Abstract/Free Full Text]
  57. Ito I, Minegishi T, Hasegawa Y, Shinozaki H, Nakamura K, Igarashi S, Nakamura M, Miyamoto K, Ibuki Y 1993 Developmental changes of testicular activin and FSH receptor mRNA and plasma FSH and inhibin levels in the rat. Life Sci 53:1299–1307[CrossRef][Medline]
  58. Shapiro DB, Pappalardo A, White BA, Peluso JJ 1996 Steroidogenic factor-1 as a positive regulator of rat granulosa cell differentiation and a negative regulator of mitosis. Endocrinology 137:1187–1195[Abstract]
  59. Hurst HC, Masson N, Jones NC, Lee KA 1990 The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43. Mol Cell Biol 10:6192–6203[Medline]