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Two Proximal Activating Protein-1-Binding Sites Are Sufficient to Stimulate Transcription of the Ovine Follicle-Stimulating Hormone-ß Gene¹

Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Address all correspondence and requests for reprints to: Dr. William L. Miller, Department of Biochemistry, Box 7622, North Carolina State University, Raleigh, North Carolina 27695-7622.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the ß-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHß promoter has revealed a number of potential activating protein-1 (AP-1; Jun/Fos)-binding sites. To determine whether the gene encoding the ß-subunit of ovine FSH (oFSHß) is responsive to AP-1 transcriptional complexes, chimeric constructs containing deleted portions of the oFSHß promoter fused to a luciferase reporter were transiently transfected along with c-Jun and c-Fos expression constructs into JAR cells. Analysis of these deletion constructs revealed that the proximal promoter of oFSHß is highly stimulated by c-Jun and c-Fos proteins (typically 20-fold with a reporter construct containing oFSHß sequences from -215 to +759 bp). This stimulation was lost when a similar construct containing sequences from -84 to +759 bp was tested. Transcriptional start site analysis using reverse transcription-PCR verified that the transcriptional initiation of the -215-bp deletion construct, with or without cotransfected c-Jun/c-Fos, was the same as that observed in vivo. Computer analysis of oFSHß sequences from -215 to +1 bp identified four putative AP-1-like elements, located at -155, -120, -83, and -10 bp. Gel retardation experiments using oligonucleotides corresponding to the four putative AP-1-like sites revealed that only -120 and -83 sites in oFSHß bound AP-1 proteins in vitro. Site-directed mutagenesis of the -120 and -83 sites showed that each element was required for stimulation by c-Jun and c-Fos proteins as well as 12-O-tetradecanoyl phorbol-13-acetate in transient transfection assays. Finally, immunocytochemical dual labeling was used to show that more than 75% of all FSHß-containing cells in ovine pituitary sections from cycling ewes contained nuclear c-Jun, JunB, JunD, and Fos proteins. These data, taken together, show that oFSHß transcription can be stimulated by c-Jun and c-Fos proteins via two functionally linked AP-1-like sites in the oFSHß proximal promoter and that these sites are likely to be important regulators of FSH production in vivo.

	Introduction

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FSH, AN /ß heterodimeric glycoprotein absolutely essential for egg maturation in mammals, is produced along with LH in gonadotropes of the anterior pituitary. The -subunit in FSH is common to LH, and the biological activity of each hormone is defined by its unique ß-subunit (1). As the synthesis of FSHß is the primary rate-limiting step in overall FSH production, those factors that influence its production are important regulators of gonadal function and the female reproductive cycle.

The FSHß gene is a single copy gene in all mammals studied to date (2, 3), and currently, very little is known about the regulation of this gene at the transcriptional level. Sequence analysis of the ovine FSHß promoter (-4741 bp), however, has revealed a multitude of putative activating protein-1 (AP-1) sites with near or identical homology to the consensus AP-1 site (TGA^G/_CTCA), suggesting that the ovine FSHß gene may be a target of AP-1 action. As AP-1 transcriptional complexes are known to be important third messengers for target genes regulated by extracellular mediators, it was considered possible that the ovine FSHß (oFSHß) gene would be responsive to AP-1.

Here we describe the use of a series of deletion constructs containing decreasing amounts of the ovine 5'-flanking region fused to the luciferase gene to show that expression of the oFSHß gene is enhanced by c-Jun and c-Fos proteins in transiently transfected mammalian cells. This enhancement is localized to two functionally linked, AP-1-like enhancer elements in the proximal promoter of oFSHß that are each capable of binding AP-1 proteins. These same elements are necessary for the induction of oFSHß transcription by 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C activator known to induce gene transcription through activation/induction of AP-1 proteins (4). Immunocytochemical staining of pituitaries from mature cycling ewes establishes the presence of Jun/Fos family members in ovine gonadotropes in vivo. Thus, we present here the first data showing regulation of a gonadotropin subunit gene by AP-1 and suggest that such regulation may be the target of one or more known physiological regulators of FSH synthesis such as GnRH, activin, estrogen, and/or progesterone.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid constructs and mutagenesis
Generation of the deletion constructs containing 5'-lengths of the ovine FSHß promoter fused to luciferase or human ß-hemoglobin have been previously described (5, 6). The -69oFSHß-galactosidase expression construct, a new vector for this study, was derived from -69oFSHß-Luc and made by excising the luciferase-coding sequence using restriction endonucleases BamHI and HindIII and exchanging this sequence with a BamHI and HindIII fragment containing the ß-galactosidase-coding sequence from pCH110 (Pharmacia Biotech, Piscataway, NJ). The cytomegalovirus (CMV), CMV-c-Jun, and CMV-c-Fos expression plasmids (7) were gifts from Dr. T. Curran (St. Jude Children’s Research Hospital, Memphis, TN).

Mutagenesis of the -120 AP-1-like site in the oFSHß promoter was made according to procedures described by Kunkel (8). Specifically, oFSHß sequence from -215 to +759 plus an additional 5'-flanking pXP1 sequence, were excised from the -215oFSHß-Luc expression with NdeI and SalI and subcloned into the phagemid vector pNSB69, from which single stranded DNA was made (9). The complementary oligonucleotide 5'-GCTTGCTGTAAGTAGATCTGTGTTTGGATAGAC-3', spanning oFSHß sequences from -134 to -102 bp, was used to create the desired mutation at the -120 site. This oligonucleotide was synthesized by the Department of Genetics (North Carolina State University, Raleigh, NC); italicized nucleotides represent mismatches with the actual oFSHß sequence. Identified mutants were subcloned back into the -215oFSHß-Luc expression construct, generating the -120 mut FSHß-Luc expression construct. oFSHß promoter sequences from all plasmids containing either deletions or mutations (see below) were verified for accuracy using the Sequenase version 2.0 kit (U.S. Biochemical Corp., Cleveland, OH).

Site-directed mutagensis of the -83 AP-1-like site in oFSHß was performed according to the manufacturer’s instructions, using the Sculptor in vitro mutagenesis system (Amersham Life Science, Arlington Heights, IL). oFSHß sequence from -105 to +759 was excised from the wild-type -215oFSHß-Luc expression construct using the restriction endonuclease HindIII and was subcloned into the pGEM-3Zf(±) phagemid (Promega, Madison, WI) for single strand plasmid DNA generation and isolation. The complementary oligonucleotide 5'-GTTGGGTATTCGAAAGAGCGGTGTAGCC-3', spanning oFSHß sequences from -97 to -69 bp, was synthesized by Cruachem (Dulles, VA); italicized nucleotides represent mismatches with the actual oFSHß sequence. The mutated oFSHß sequence from -105 to +759 was excised with HindIII and placed back into both wild-type -215oFSHß-Luc and -120 mut FSHß-Luc expression plasmids, generating -83 mut FSHß-Luc and -83/-120 double mutated FSHß-Luc expression constructs, respectively.

Cell culture and transient transfection
Human choriocarcinoma (JAR) and HeLa cells were obtained from the American Type Culture Collection (Rockville, MD) and grown at 37 C in DMEM (Life Technologies, Grand Island, NY) containing 10% FBS (HyClone, Logan, UT) under 95% air-5% CO₂. Cells were grown in 150-cm² flasks until they were confluent and then were replated in six-multiwell plates (diameter, 35 mm/well) at a concentration of 500,000 JAR cells/well or 400,000 HeLa cells/well. Plated cells were transfected 24–48 h later. For transient transfections involving cotransfection of c-Jun/c-Fos expression constructs into JAR cells, cells were washed with medium and transfected in triplicate with 5 µg reporter construct, 5 µg each of CMV-c-Jun and CMV-c-Fos or 10 µg of control CMV plasmid, and 2 µg of the Rous sarcoma virus-ß-galactosidase construct (pRSV-ß-Gal; total volume, 0.5 ml) (10) using the calcium phosphate method as previously described (6). Cells were harvested for luciferase and ß-galactosidase activity 48 h after the start of transfection. For transient transfection studies involving TPA treatment of HeLa cells, cells were transfected in triplicate with 7 µg reporter construct and 10 µg pRSV-ß-Gal or 20 µg -69oFSHß-Gal (total volume, 0.5 ml) as described above. However, 18 h after the start of transfection, the precipitates were removed and then replaced with low serum medium (0.5% FBS). Six hours later, medium was removed and replaced with low serum medium containing either vehicle or 100 nM TPA (Calbiochem-Novabiochem International, La Jolla, CA). HeLa cells were harvested for luciferase and ß-galactosidase activity 12 h after the addition of TPA.

Luciferase and ß-galactosidase assays
Luciferase assays from transient transfections were performed as previously described (6). ß-Galactosidase activity was assayed using a modified version of the Galacto-light assay system (Tropix, Bedford, MA). Briefly, 20 µl cell lysate were mixed with 100 µl diluted substrate reaction buffer followed by a 100-µl automatic injection of accelerator by the Monolight 2010 Luminometer after a 1-h incubation at room temperature. Chemiluminescence was measured for 5 sec, and the results were expressed as the mean relative light units from three individual transfections. The transfected gene for ß-galactosidase typically showed 5- to 10-fold higher activity than the endogenous ß-galatosidase gene in all transfections.

Normalization and statistical analysis of the transient transfection data
Transfection efficiency in control and c-Jun/c-Fos-treated JAR cell cultures was normalized to ß-galactosidase activity. All transfections contained equal amounts of pRSV-ß-Gal as an internal control, which was used because its expression was the least affected by c-Jun/c-Fos cotransfection of all ß-galactosidase vectors tested. Expression of the ß-galactosidase internal control was always enhanced by c-Jun and c-Fos proteins 2- to 3-fold over basal expression. Although it is difficult to know why c-Jun/c-Fos cotransfection altered the expression of ß-galactosidase, the enhancement was judged to be unique to either the RSV promoter or the ß-galactosidase reporter in pRSV-ß-Gal because c-Jun/c-Fos did not alter luciferase values for the -84oFSHß-Luc or every mutant FSHß-Luc construct. Therefore, ß-galactosidase values from control cultures were used only to correct the transfection efficiencies in control cultures, and those from c-Jun/c-Fos-treated cultures were used only to correct the transfection efficiencies in c-Jun/c-Fos-treated cultures. In both cases, ß-galactosidase activities within each experiment were averaged, and a mean value was used as a correction factor to adjust individual luciferase values whose ß-galactosidase activity deviated from the average ß-galactosidase value. Corrections were generally small and had no affect on the overall conclusions, but did decrease variation within treatments.

For studies involving TPA treatment of HeLa cells, variation in transfection efficiency was normalized by dividing the ß-galactosidase values directly into the luciferase values from control and TPA-treated cultures. The expression of the pRSV-ß-Gal construct was not changed by TPA treatment in these experiments. Data obtained with -69oFSHß-Luc were not normalized in these studies, as the -69oFSHß-Gal expression construct was cotransfected with the -69oFSHß-Luc for direct comparison of stimulation during TPA treatment.

To combine data from completely independent experiments, the basal expression for each construct in each experiment was assigned a relative value of 1 (see Figs. 1, 4, and 5). Induction ratios (fold induction) were obtained by dividing the average value for the c-Jun/c-Fos-induced or TPA-induced reporter by the average basal expression of the same reporter. Statistical differences between means were determined for basal, TPA-stimulated, or c-Jun/c-Fos expression, and induction ratios of all oFSHß expression constructs were determined using ANOVA followed by Tukey’s multiple range test for individual comparisons (11).

View larger version (18K): [in this window] [in a new window]   Figure 1. Trans-activation of the ovine FSHß gene by c-Jun and c-Fos proteins. Luciferase expression vectors containing 5'-deletions of the oFSHß promoter were cotransfected into JAR cells along with CMV-c-Jun/CMV-c-Fos or the "empty" CMV expression plasmid. oFSHß-Luc constructs are shown on the left, and the relative luciferase activities observed with the constructs are shown on the right. Basal expression of all constructs did not vary significantly (P < 0.05) and were assigned (normalized to) a relative value of 1. All values are presented as the mean ± SEM from three independent transfection experiments (each assayed in triplicate). Means that do not share letters (superscripts) are significantly different from each other (P > 0.05).

View larger version (20K): [in this window] [in a new window]   Figure 4. c-Jun/c-Fos trans-activation of the oFSHß gene through the -120 and -83 sites. The -120 and -83 sites in -215oFSHß-Luc were mutated, individually and collectively, and then cotransfected along with CMV-c-Jun/CMV-c-Fos or CMV alone into JAR cells. Expression data from the -215oFSHß-Luc (see Fig. 1) were analyzed with the data from the mutated oFSHß-Luc expression constructs to compare differences in basal and induction ratios. Luciferase expression constructs containing wild-type or mutated oFSHß sequences are shown on the left, and the relative luciferase activities observed with these constructs are shown on the right. Basal expression of all constructs tested did not vary significantly (P < 0.05) and was assigned (normalized to) a relative value of 1. All values are presented as the mean ± SEM from three independent transfection experiments (each assayed in triplicate).

View larger version (23K): [in this window] [in a new window]   Figure 5. TPA induction of oFSHß gene expression through the -120 and -83 sites. Luciferase and ß-galactosidase expression plasmids containing either deletions or mutations of the oFSHß promoter were prepared and transfected into HeLa cells. Upon removal of the DNA precipitates, cells were cultured in low serum (0.5% FBS) for 9 h, followed by the addition of low serum medium containing vehicle or 100 nM TPA for the final 12 h of incubation. Luciferase and ß-galactosidase expression constructs containing wild-type or mutated oFSHß sequences are shown on the left, and the relative luciferase or ß-galactosidase activities observed with these constructs are shown on the right. Basal expression of all constructs was normalized to a relative value of 1. All values are presented as the mean ± SEM from four independent transfection experiments (each assayed in triplicate). Means that do not share letters (superscripts) are significantly different from each other (P > 0.05).

Start site initiation experiments involving total RNA from wether pituitaries were performed as described above, except that 6 µg wether RNA plus a complementary oligonucleotide corresponding to the coding sequence of oFSHß (primer C_FSHß; +222/+245 bp) were substituted for JAR cell RNA and the ß-globin primer. The sequence of the antisense oFSHß oligonucleotide was 5'-TCCTTGTACACCAAGTCCCGGGT-3'.

Oligonucleotides used in electrophoretic mobility shift assays
All oligonucleotides used in the electrophoretic mobility shift assays are defined in Table 1. Synthetic oligonucleotides, corresponding to the coding and noncoding strands of the four putative AP-1 sites in oFSHß, were synthesized by Cruachem (XhoI restriction site 5'-overhangs, not shown in the table, were incorporated for radiolabeling and future cloning purposes). Double stranded (ds) oligonucleotides were made by boiling the appropriate single stranded oligonucleotides in annealing buffer (50 mM NaCl and 20 mM Tris-Cl, pH 7.4) for 5 min, followed by slow cooling to room temperature over 2 h. The consensus AP-1 oligonucleotide was purchased from Promega in ds form.

Electrophoretic mobility shift assays
Nuclear protein extract from HeLa cells was prepared as described by Abmayr and Workman (14), using 5 x 10⁸ HeLa cells from a Spinner culture purchased from the University of North Carolina Lineberger Cancer Center (Chapel Hill, NC). Binding reactions with HeLa nuclear extract were generally performed by mixing 11 µg nuclear protein with binding buffer [final concentrations, 1 mM MgCl₂, 2 mM EDTA, 2 mM dithiothreitol, 25 mM NaCl, 10 mM Tris-Cl (pH 7.5), and 4% glycerol], 1.0 µg poly(dI-dC) (Boehringer Mannheim), and 0.4 ng radiolabeled probe at room temperature in 20-µl reaction volumes. All binding reactions (including those with competitor oligonucleotides or antibodies) were carried out simultaneously. For supershift assays, 0.15 µg anti-Fos rabbit polyclonal antibody (provided by Dr. M. J. Iadarola, NIH, Bethesda, MD) (15) or 0.5 µg nonspecific rabbit IgG (Sigma) was preincubated in the binding reactions for 30 min at room temperature before the addition of radiolabeled probe. The anti-Fos antibody is directed to residues 128–152 of human c-Fos and recognizes all Fos family members (c-Fos, FosB, Fra-1, and Fra-2). For competition studies, unlabeled competitor oligonucleotides (at 60-fold molar excess in HeLa extract incubations and 200-fold molar excess in c-Jun binding reactions) were preincubated in binding reactions for 10 min at 23 C before the addition of radiolabeled probe. After the addition of probe, all binding reactions were incubated for an additional 20 min at room temperature before being loaded onto 5.6% nondenaturing polyacrylamide gels (1.5 mm), which were run at 200 V for 3 h at 15 C. Gels were fixed, dried, and analyzed by a PhosphorImager (model 445 SI, Molecular Dynamics).

For studies involving purified c-Jun homodimer, binding reactions were assembled as described for HeLa nuclear extract, except that 0.25 µg purified c-Jun homodimer (Promega) was used instead of nuclear extract, 0.75 µg poly(dI-dC) was used instead of 1.0 µg poly(dI-dC), and 3 µg BSA were included in the binding reaction.

Immunocytochemistry
Ovine pituitaries were obtained from mature cycling ewes and immediately fixed in 4% paraformaldehyde for 24–48 h at 4 C. These pituitaries were dehydrated, embedded in paraffin, cut into 5-µm cross-sections and rehydrated before use using standard procedures. Various pretreatments with the tissue sections were necessary to either enhance or allow antibody recognition of the Jun or Fos antigens. Pituitary sections receiving anti-c-Jun, anti-JunB, or anti-JunD antibody were pretreated with a 0.1% pepsin solution in 0.01 M HCl (pH 2.3) for 20 min. Tissue sections receiving anti-Fos antibody were boiled for 5 min in citrate buffer (pH 6.0) and allowed to cool for 30 min in the same buffer before further use. The peroxidase-labeled streptavidin-biotin method (16), with diaminobenzidine (DAB) chromagen as a detection agent, was used to reveal the Jun/Fos antigens and was performed using secondary reagents and procedures from Zymed Laboratories (San Francisco, CA). Affinity-purified rabbit anti-c-Jun and anti-JunB polyclonal antibodies were made against N-terminal residues 73–87 of v-Jun and 45–61 of human JunB, respectively; both antibodies have been shown to be specific and without significant cross-reactivity to other Jun family members (determined by Oncogene Science using Western blot analysis, Cambridge, MA). The affinity-purified rabbit anti-JunD polyclonal antibody was directed against residues 329–341 of mouse JunD and was specific for JunD only (confirmed by Santa Cruz Biotechnology using Western blot analysis, Santa Cruz, CA). The Jun and Fos antibodies were used at 2–4 µg/ml. For neutralization studies, primary antibodies were preincubated with a 10-fold excess by weight of competing peptide obtained from Oncogene Science and Santa Cruz Biotechnology (between 350–500 molar excess per IgG-binding site based on peptide size).

The immunocytochemical dual labeling procedure involved detection of the Jun/Fos antigens before detection of the FSHß antigen. To block cross-reactivity between detection methods, tissues were incubated for 1 h at room temperature in double staining enhancer (Zymed Laboratories). The Immunogold silver-staining method (17) was used to detect the oFSHß antigen and was performed using secondary reagents and procedures from Zymed Laboratories. Antihuman FSH polyclonal antibody was purchased from Bimeda (Foster City, CA) and was used at a dilution of 1/100. This antibody was produced against the ß-chain of human FSH and shows less than 5% cross-reactivity with other pituitary glycoproteins (determined by BiØmeda using Western blot analysis). For FSH neutralization studies, anti-FSH antibody was preincubated with an excess (60 µg/ml) of highly purified ovine FSH (125 x NIH FSH S-1) (18).

The percentages of FSH cells containing c-Jun, JunB, JunD, or Fos proteins were determined from three pituitary slices (each slice from a separate pituitary). Over 200 FSH-positive cells were counted in each pituitary section, and the percentages of FSH cells containing Jun or Fos protein were determined. Results are expressed as the mean ± SEM of data obtained from the three sections assayed for each colocalization.

	Results

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Transcriptional activation of the oFSHß gene can be mediated by c-Jun and c-Fos proteins
Initial sequence analysis of the oFSHß promoter (-4741 bp) revealed a number of potential AP-1-binding sites with near or identical homology to the published AP-1 consensus (19) (data not shown). To determine whether the oFSHß gene is responsive to c-Jun and c-Fos proteins, the chimeric construct -4741oFSHß-Luc was transiently transfected into JAR cells along with either CMV-c-Jun/CMV-c-Fos or CMV control expression constructs. As shown in Figure 1, expression of -4741oFSHß-Luc was typically stimulated 3-fold by c-Jun/c-Fos proteins compared to that of the truncated -84oFSHß-Luc construct, which was not stimulated. A series of deletion constructs containing decreasing amounts of oFSHß 5'-flanking region was employed to show that as the oFSHß promoter was shortened, higher levels of induction were observed (see Fig. 1). Increased induction over -4741oFSHß-Luc was first noted (statistically significant) in -450oFSHß-Luc (6.5 ± 0.6-fold) and was maximal with -215oFSHß-Luc (20.3 ± 1.0-fold). Similar or identical results were also obtained with greater statistical variability using less c-Jun/c-Fos (2–4 µg instead of 10 µg c-Jun/c-Fos constructs; data not shown). Basal expression of all deletion constructs was statistically equivalent (P > 0.05) and did not vary by more than 7,505–13,411 relative light units/100 µl cell lysate between individual experiments.

Putative AP-1-like sites -83 and -120, but not -155 or -10, effectively compete for AP-1 proteins
Sequence analysis of the oFSHß proximal promoter (-215 to +1 bp) had identified four putative AP-1-binding sites (located at -155, -120, -83, and -10 bp in the oFSHß gene). Table 1 shows an alignment of these sites compared to the consensus AP-1 site. Comparison of the AP-1-like sites at -155, -120, and -83 showed that although each contained a single base pair mismatch to the published AP-1 consensus (19), three retained the palindromic nature of the element. The -120 AP-1-like site was identical to the consensus AP-1 site, except for a central T substitution instead of C or G. The only putative AP-1-like site that was not palindromic was at -10.

To determine whether any of the AP-1-likes sites identified in the oFSHß proximal promoter possessed AP-1-like binding characteristics, nuclear proteins from HeLa cells were isolated, and electrophoretic mobility shift assays were performed using a radiolabeled AP-1 consensus oligonucleotide ([³²P]conAP-1) with or without the unlabeled oFSHß oligonucleotides as competitors. As shown in Fig. 2, incubation of [³²P]conAP-1 with HeLa nuclear proteins resulted in the formation of a distinct DNA/protein complex (lane 2) that was not present in the control (lane 1). The [³²P]conAP-1 shift was specific, as competition with a 60-fold molar excess of the same unlabeled oligonucleotide resulted in a 92% reduction of the band (Fig. 2, lane 3). When the four AP-1-like sites found in oFSHß were tested as competitors in the same assay, only the -120 and -83 sites effectively competed for proteins that bound to the [³²P]conAP-1 (84% and 72% competed, respectively; lanes 4 and 5). Neither the -155 nor -10 putative AP-1 sites showed significant ability to compete AP-1 proteins and were not considered as being AP-1-like enhancer elements (Fig. 2, lanes 6 and 7). An oligonucleotide containing mutations in the -83 AP-1-like site (no AP-1 characteristic remaining) reduced only 10% of the [³²P]conAP-1 shift and verified that the competition observed in lane 5 was specific to that in the core -83 AP-1-like sequence TTACTAA (Fig. 2, lane 8).

View larger version (58K): [in this window] [in a new window]   Figure 2. Electrophoretic mobility shift competition analysis of four putative AP-1 sites in the oFSHß gene. conAP-1 was end labeled and incubated with HeLa nuclear protein and poly(dI-dC) in the absence or presence (60-fold molar excess) of the four putative AP-1-like sites found in the oFSHß proximal promoter. Reactions were fractionated by electrophoresis on 5.6% nondenaturing polyacrylamide gels. All oligonucleotides used in the analysis are defined in Table 1. Lane 1 shows the migration of free [32P]conAP-1, and lane 2 shows the distinct DNA/protein shift caused by HeLa nuclear proteins. Lanes 3–7 show the effects of various competitor oligonucleotides on formation of the [32P]conAP-1/protein complex. Arrows to the left of the data denote the locations of free and bound [32P]conAP-1.

View larger version (43K): [in this window] [in a new window]   Figure 3. Binding of c-Jun and Fos proteins to the -83 and -120 AP-1-like sites in the oFSHß gene. The ds oligonucleotides corresponding to the -83 and -120 oFSHß AP-1-like sites (see Table 1) were end labeled for electrophoretic mobility shift assays. A, Binding of c-Jun protein to both the -83 and -120 sites. 32P-labeled -83 or -120 sites were incubated with recombinant c-Jun protein and poly(dI-dC) before fractionation on 5.6% nondenaturing polyacrylamide gels. Lanes 1 and 6 show the free 32P-labeled -83 or -120 oligonucleotide, and lanes 2 and 7 show the specific -83 or -120 DNA/c-Jun shift. For competition analysis (lanes 3–5 and 8–10), a 200-fold molar excess of competitor oligonucleotide, as indicated, was added to the reactions before the addition of radiolabeled probe. B, Binding of Fos protein to both the -83 and -120 sites. HeLa nuclear extract was first incubated with anti-Fos antibody (or rabbit IgG) followed by the addition of either the 32P-labeled -83 or -120 site before fractionation by electrophoresis. Lanes 1 and 7 show the specific DNA/protein shift for each site. Lanes 5, 6, 11, and 12 show the effects on migration of the DNA/protein shifts when either anti-Fos or rabbit IgG was included in the binding reaction. For competition analysis (lanes 2–4 and 8–10), a 60-fold molar excess of unlabeled competitor oligonucleotide was added to the reaction before the addition of radiolabeled probe. Arrows to the left of the data denote the positions of the shifted/supershifted and free probes in the lanes receiving c-Jun protein, Fos antibody, nonspecific IgG, and competitor oligonucleotides.

To determine whether proteins other than Jun and Fos were binding to the -83 and -120 sites, conAP-1 was added as a competitor in the same assays, as described above. As shown in Fig. 3B, conAP-1 at a 60-fold molar excess competed 88% and 49% of the proteins from the -83 and -120 sites, respectively (lanes 3 and 9). The addition of conAP-1 with the -83 site prevented the formation of the lower, major DNA-protein complex, but did not affect formation of the upper complex. Thus, the -83 AP-1-like site can bind specific proteins other than Jun and Fos. Finally, conAP-1 could not compete with the proteins bound to the -120 site as efficiently as self-oligonucleotide (27% more protein remaining compared to the competition with self-oligonucleotide), suggesting that proteins besides Jun and Fos bind to the -120 AP-1-like site.

The -120 and -83 sites work cooperatively to enhance expression of the oFSHß gene by c-Jun and c-Fos
To determine the functional roles of each AP-1-like site in transcriptional activation of the oFSHß gene by c-Jun/c-Fos, the -120 and -83 sites were destroyed, individually and collectively, by mutation. As shown in Fig. 4, mutation of the -120 AP-1-like site abolished the 20-fold c-Jun/c-Fos enhancement that was normally observed when using the wild-type expression construct. Mutations disrupting the -83 AP-1-like site also destroyed the ability of c-Jun/c-Fos proteins to transcriptionally activate the oFSHß promoter. The 2.0 ± 0.1-fold increase observed with the -83 mutated FSHß-Luc was not significant (P > 0.05). Elimination of both AP-1-like sites from -215oFSHß-Luc abolished the induction normally observed by c-Jun/c-Fos proteins. Finally, basal level expression of all constructs containing wild-type or mutated oFSHß sequences did not vary significantly within individual experiments.

The -120 and -83 sites work cooperatively to enhance expression of the oFSHß gene by TPA
HeLa cells were chosen for studies with TPA-mediated induction of oFSHß-Luc because TPA action on AP-1-responsive genes is well established in these cells, and TPA treatment had no apparent effect on oFSHß-Luc expression in JAR cells. As shown in Fig. 5, the addition of 100 nM TPA for 12 h enhanced expression of -215oFSHß-Luc 10.4 ± 0.8-fold compared to basal expression of the same construct. Mutations in one or both AP-1-like sites significantly reduced the activation by TPA to less than half that observed by the wild-type construct, indicating that both AP-1 sites worked cooperatively to enhance the transcription of -215oFSHß-Luc.

Unexpectedly, both the single and double oFSHß mutant expression constructs were significantly activated by TPA (between 4.3- and 4.7-fold). To determine whether this activation might be associated with the luciferase reporter gene rather than the oFSHß promoter in the oFSHß-Luc constructs, the -69oFSHß-Luc and -69oFSHß-Gal expression constructs were prepared and analyzed under TPA treatment as described above. Figure 5 shows that expression of the -69oFSHß luciferase-containing construct was stimulated 4.0 ± 0.1-fold by the addition of TPA, whereas expression of the ß-galactosidase construct was not increased. These results suggest that TPA treatment selectively increased Luc expression by a posttranscriptional mechanism.

Transcriptional start site analysis of the -215oFSHß-globin construct under c-Jun/c-Fos treatment
To verify that transcriptional induction by c-Jun and c-Fos proteins did not change the initiation site of -215oFSHß-globin, RT-PCR was used to analyze transcriptional initiation of -215oFSHß-globin during c-Jun/c-Fos treatment. ß-Globin was substituted for luciferase because its longer messenger RNA (mRNA) half-life allowed enough accumulation of ß-globin mRNA to be detected by RT-PCR. Start sites were determined using RT-PCR to make fragments that correspond to products starting either before or after the start site. Oligonucleotides corresponding to oFSHß sequences before and after the start site (primer A, -23/-1 bp; primer B, +11/+34 bp) and an antisense oligonucleotide corresponding to a portion of the coding sequence of human ß-globin (primer C_ßglo; +292/+315 bp) were reverse transcribed and then amplified by PCR using total RNA isolated from control or c-Jun/c-Fos-treated cultures. Figure 6A shows the radiolabeled RT-PCR products from control and c-Jun/c-Fos-treated cultures after fractionation on a polyacrylamide gel (the figure above the data depicts the oFSHß-globin construct, including the predicted start site and oligonucleotides A, B, and C_ßglo). A comparison between control and treated cultures using primers B/C_ßglo demonstrated that c-Jun/c-Fos proteins increased the amount of RT-PCR product of the expected size (Fig. 6A, lane 1 vs. 3). In this experiment, cotransfection with c-Jun/c-Fos increased oFSHß/globin mRNA RT-PCR product (304 bp) 10-fold compared to RT-PCR product from the controls. Unexpectedly, use of the oFSHß oligonucleotide preceding the start site revealed a minor RT-PCR product in both control and c-Jun/c-Fos-treated cultures (Fig. 6A, lanes 2 and 4). Quantitation of the data from c-Jun/c-Fos-treated cultures showed that 90% of the oFSHß/globin mRNA initiated transcription at the predicted start site. Thus, the majority of transcription initiated at the predicted start site for both control and c-Jun/c-Fos-treated cultures transfected with -215oFSHß-globin.

View larger version (20K): [in this window] [in a new window]   Figure 6. Transcriptional start site analysis of the -215oFSHß-globin expression construct and oFSHß in vivo. The diagrams above the data depict a schematic representation of the mRNA for either the oFSHß/ß-globin construct or the oFSHß gene. The schematics indicate the locations of the primers used for RT-PCR (primers A, B, and Cßglo or CFSHß). A, Start site analysis of -215oFSHß-globin with or without c-Jun/c-Fos treatment. Total RNA was isolated from control and c-Jun/c-Fos-treated cultures, and RT-PCR analyses were performed using the oligonucleotides depicted in the diagram described in Materials and Methods. RT-PCR products were fractionated on 6% nondenaturing polyacrylamide gels, and for reference, the sizes of the RT-PCR products are indicated. B, Start site analysis using oFSHß mRNA isolated from wether pituitaries (5). RT-PCR was performed as described for culture analysis, except that an appropriate oligonucleotide for the oFSHß-coding sequence was substituted for the human ß-globin oligonucleotide. RT-PCR products were fractionated on 6% nondenaturing polyacrylamide gels, and the sizes of RT-PCR products are indicated.

Jun and Fos proteins are expressed in the nuclei of ovine gonadotropes in vivo
Ovine pituitary sections were first immunostained using peroxidase-DAB to visualize either Jun or Fos proteins and then immunostained for FSHß protein using an Immunogold silver-staining technique as described in Materials and Methods. As shown in Fig. 7 (B, E, H, and K), a majority of the FSHß-containing cells (black precipitate) showed nuclear immunoreactivity for Fos family members, c-Jun, JunB, and JunD protein (brownish orange). It was determined that 80 ± 9% of the cells immunostained for FSH contained Fos, 75 ± 6% contained c-Jun, 85 ± 3% contained JunB, and 78 ± 9% contained JunD. To verify that the observed staining was specific for the antigens in question, primary antibodies were preincubated with an excess of competitor peptide before the staining procedure. As shown in Fig. 7 (A, D, G, and J), the appropriate peptide at a 10-fold excess by weight completely blocked Jun and Fos staining. To show specificity of the FSH stain, anti-FSHß antibody was preincubated with highly purified oFSH protein at 60 µg/ml; immunostaining after such pretreatment blocked FSH staining (Fig. 7, C, F, I, and L).

View larger version (141K): [in this window] [in a new window]   Figure 7. Immunocytochemical localization of Jun and Fos proteins in ovine gonadotropes in vivo. Ovine pituitary sections were dual labeled for AP-1 family members and FSH. Jun/Fos antigens were revealed with peroxidase-DAB, and FSH was revealed with Immunogold. B, E, H, and K show, respectively, nuclear Fos, c-Jun, JunB or JunD, and FSHß immunoreactivity in ovine pituitary cells (Jun/Fos staining is orange/brown; FSH staining is black). A, D, G, and J show dual labeling experiments in which the Jun/Fos antibodies were competed with the appropriate peptide. C, F, I, and L show dual labeling experiments in which the FSH antibody was competed with excess ovine FSH (>95% pure). The percentage of FSH cells containing Jun/Fos protein was determined from three separate pituitaries. From more then 600 FSH-positive cells counted in total, 80 ± 9% contained Fos proteins, 75 ± 6% contained c-Jun, 85 ± 3% contained JunB, and 78 ± 9% contained JunD. Magnification, x132. Arrows define the Fos, Jun, and FSHß immunoreactivities.

View larger version (24K): [in this window] [in a new window]   Figure 8. Sequence comparison of the -120 and -83 sites in the oFSHß gene with five other mammalian species. Nucleotide sequences of the ovine, bovine, porcine, human, rabbit, and rat FSHß genes are shown as aligned to ovine sequences from -129 to -68 bp. Boxes define the two AP-1-like sites. Underlined lowercase letters refer to those nucleotides that deviate from the nucleotide sequences of the ovine -120 and -83 AP-1-like sites.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Even though FSHß plays a major role in mammalian gametogenesis, very little is known about its regulation at the molecular level. Difficulties in studying its regulation center on the lack of cell lines that express it and to the limited success experienced in expressing constructs containing 5'-flanking regions of the FSHß gene in heterologous cultured cells (13). Our laboratory has been able, however, to transiently express ovine FSHß-Luc constructs with relatively high basal activity in a multitude of cell lines, including HeLa, Chinese hamster ovary, JAR, Bewo, T47-D, and COS-7 (5, 6). It is important to note that both basal and AP-1-stimulated transcription (20-fold increase) begin at the correct start site that is observed in vivo. These results indicate that the oFSHß gene, at least up to 4.7 kilobases of 5'-flanking region, does not absolutely require gonadotrope-specific factors to direct significant basal expression of the luciferase reporter gene. Choriocarcinoma cells (JAR) were chosen for the transfection studies presented here because they are known to express two glycoprotein hormone subunits similar to FSHß ( and hCGß) and because they are readily transfected (5, 27).

Basal luciferase expression from the oFSHß-Luc deletion constructs in JAR cells was 15- to 30-fold above background and was not significantly altered as the promoter length decreased from -4741 to -84 bp (see Fig. 1). As promoter deletions usually change the basal rate of transcription (from a loss of enhancers and/or silencers), these results suggested that JAR cells contain sufficient basal transcription factors to permit expression of oFSHß-Luc, but lack factors that might bind to the 5'-flanking region to interfere with or stimulate basal transcription. By contrast, upstream oFSHß sequences were found to influence the rate of transcription activated by c-Jun and c-Fos. Although deletion from -4741 to -750 bp had no significant effect on the level of AP-1 stimulation, deletions between -750 and -215 bp revealed an abrupt 4.2-fold increase in the rate of c-Jun/c-Fos-stimulated transcription. These results suggest that sequences between -750 and -215 bp bind factors that interfere with the ability of AP-1 to stimulate oFSHß gene transcription. One alternative explanation for these results is the concept that cryptic AP-1-binding sites may reside in the backbone of the pXP plasmid and that gene deletion brings these AP-1 sites closer to those in the oFSHß promoter, resulting in increased stimulation (28). However, this explanation is unlikely because progressive deletions did not lead to progressive induction, but, rather, a sharp increase in induction due to small deletions in the proximal promoter. Thus, deletion analysis uncovered no DNA sequences critical for basal expression, but suggested the existence of one or more factors in JAR cells that modulate AP-1 stimulation of oFSHß-Luc transcription.

The AP-1 trans-activation studies presented here involved the continuous high expression of c-Jun and c-Fos proteins to obtain the 20-fold induction observed with the -215oFSHß-Luc expression construct. Although this procedure is routinely used to characterize AP-1-binding sites in genes known to be AP-1 responsive, the possibility remains that oFSHß-Luc induction is due to an interaction of c-Jun/c-Fos that normally does not occur at physiological levels of Jun/Fos. Therefore, we also tested the ability of the phorbol ester, TPA, to stimulate oFSHß expression in HeLa cells. This model has been used to analyze AP-1-responsive genes, but it has the advantage of creating high, but physiologically relevant, levels of Jun/Fos family members. Results from the TPA studies matched those obtained using cotransfection analysis with Jun/Fos constructs and reinforced the concept that each of the two AP-1-like sites at -120 and -83 was absolutely necessary for transcriptional activation. As a number of AP-1-responsive genes require two functional AP-1 sites for trans-activation by Jun and Fos, our data support the concept that the oFSHß gene is another member in a growing class of genes responsive to AP-1 (29, 30, 31, 32).

While using TPA, it was discovered that oFSHß-Luc constructs that were responsive to c-Jun/c-Fos were stimulated 10- to 13-fold by TPA, but that oFSHß-Luc constructs that were not responsive to c-Jun/c-Fos were also stimulated 4-fold with TPA. As TPA increased the expression of RSV-Luc 4-fold (data not shown) but did not affect the expression of RSV-ß-Gal, it appeared that TPA might be affecting a reporter-specific characteristic of luciferase. We then made -69oFSHß-Gal and compared its activation to that of -69oFSHß-Luc; again, TPA increased expression of the oFSHß luciferase-containing construct, but not that of the oFSHß ß-galactosidase-containing construct. Therefore, TPA action in Fig. 5 appears to comprise two types: 1) a 4-fold induction specific to the luciferase reporter gene, and 2) stimulation that is greater than 4-fold, which is based on transcriptional activation from the oFSHß 5'-flanking region.

Both the -83 and -120 AP-1-like sites deviate from the canonical AP-1 consensus. The -120 site deviates by only one nucleotide in the central position, but the -83 site contains deviations in the core sequence that would appear to destroy Jun/Fos binding. As it is known that certain combinations of Jun/Fos family members possess distinct and separate affinities for a consensus AP-1 site, and that deviations in the core and flanking regions of an AP-1 site alters Jun/Fos binding, it may be that deviations in the -120 and -83 sites from the consensus AP-1 site permit specific binding of only certain types of Jun/Fos complexes (33, 34). Evidence for this was recently provided by additional supershift assays using a JunD-specific antibody, which revealed that the -83 site, but not the -120 site, contained JunD protein in the major DNA-protein complex (data not shown). In addition, these deviations appear to be important for specifying proteins other than Jun/Fos, as competition studies using the consensus AP-1 site as a competitor demonstrated the potential for each site to bind proteins other than Jun/Fos. This observation suggests that these sites might be targets for a number of different transcription factors in addition to AP-1. As many AP-1-responsive genes possess functional noncanonical AP-1 sites, it may be that such diversity permits a greater range of specificities for certain AP-1 members as well as the binding of other transcription factors that are important for cell-specific and/or gene-specific regulation.

Because our studies have implicated Jun and Fos family members in the regulation of oFSHß expression, it became important to determine whether Jun and Fos proteins are normally present in significant amounts in pituitary gonadotropes in vivo. Indeed, strong staining was found in the majority (>75%) of FSH-producing cells for Fos family members as well as c-Jun, JunB, and JunD proteins. Demonstration that the -120 and -83 sites bind AP-1 in vivo or that GnRH and/or activin increase oFSHß transcription through Jun/Fos awaits confirmation, but the clear presence of Jun/Fos family members in ovine gonadotropes lends strength to the concept that the -120/-83 AP-1-like sites are physiologically important in oFSHß expression.

If the -120 and -83 sites are so important to FSHß gene expression in the sheep, it might be expected that FSHß genes from other mammals would contain similar sequences. Comparison of the -120 oFSHß AP-1-like sites in four other species (bovine, porcine, human, and rabbit) shows an absolute sequence conservation of the -120 site. Even in the rat FSHß sequence (which contains a one-nucleotide deviation in the -120 site from the ovine sequence), we have used studies such as those shown in Fig. 2 to prove that the rat -120 AP-1-like site competes for AP-1 complexes (data not shown). This functional conservation across six species lends considerable strength to the idea that the -120 AP-1-like site is critical for FSHß expression in pituitary gonadotropes. The fact that the -83 AP-1-like site is absolutely conserved in sheep, pigs, and cows suggests that it has major importance in these species. However, our inability to find similar sites in humans, rabbits, and rats suggests that alternative mechanisms may play a role in activating oFSHß transcription by AP-1 in these and many more species. It may be that alternative AP-1 sites work cooperatively with the -120 site, or that only a single AP-1 site is needed to activate FSHß transcription in these latter species.

In summary, we have demonstrated that transcription of the ovine FSHß gene is activated by c-Jun and c-Fos proteins in JAR cells as well as by phorbol esters in HeLa cells. Deletion and mutational analyses have shown that two AP-1-like sites in the proximal promoter (-120 and -83 bp) cooperate to mediate this response and that neither site can act individually. Further, both AP-1-like sites were shown to bind c-Jun and Fos proteins directly, but it appears that each site has different preferences for specific AP-1 family members and proteins not related to AP-1. Finally, it was established that Jun/Fos proteins are abundant in ovine gonadotropes known to synthesize FSHß. These are the first data to show regulation of a gonadotropin gene by AP-1, and they suggest that FSHß may be regulated in vivo through these two functional AP-1-like sites.

	Acknowledgments

 
We thank Michael J. Iadorola for generously providing the Fos antibody for the studies, Tom Curran for providing the c-Jun and c-Fos expression vectors, William L. Flowers for performing the statistical analysis, Sandra Horton and the staff of the Histopathology Laboratory (College of Veterinary Medicine, Raleigh, NC) for expert assistance with the tissue preparation for immunocytochemistry, and Ike Cotten for excellent sheep handling.

	Footnotes

 
¹ This work was supported by the North Carolina State University Agricultural Research Service, NICHD Grants 10773 and 34863, and the National Center for Infertility Research through the University of Michigan (Grant U-54-HD-29184).

² Present address: Division of Reproductive Toxicology, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.

Received December 5, 1996.

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