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A Novel Role for Bone Morphogenetic Proteins in the Synthesis of Follicle-Stimulating Hormone¹

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. E-mail: wlmiller{at}bchserver.bch.ncsu.edu

	Abstract

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FSH is produced in pituitary gonadotropes as an /ß heterodimer, and synthesis of the ß-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHß can be regulated by activin and inhibin, both of which are members of the transforming growth factor-ß superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-ß family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHß promoter linked to a luciferase reporter gene (oFSHßLuc), BMP-7 or BMP-6 was found to stimulate oFSHßLuc expression by 6-fold. Transient expression of the oFSHßLuc in a transformed gonadotrope cell line, LßT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LßT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHßLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83–88% and 47–48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHßLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LßT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
FSH IS PRODUCED in pituitary gonadotropes as an /ß heterodimer, and synthesis of the ß-subunit is the rate-limiting step in overall FSH production. In females, FSH is required for ovarian follicle maturation, because female mice lacking FSH are infertile due to a block in folliculogenesis (1).

Bone morphogenetic proteins (BMPs) were originally identified by their activity to induce bone and cartilage formation (2). However, recent studies have revealed that BMPs have a wide variety of effects on many cell types, including monocytes and epithelial, mesenchymal, and neuronal cells. The BMPs regulate growth, differentiation, chemotaxis, and apoptosis of these cells and play pivotal roles in the morphogenesis of various tissues and organs (3). More than 15 BMPs have been identified to date. More recently, some of the BMPs, BMP-7 and BMP-4, were shown to be functional in the ovary (4), suggesting that BMPs also play a role in the reproductive system. BMPs belong to the transforming growth factor-ß (TGFß) superfamily. Members of this family transduce their signals through binding two serine/threonine kinase receptors (types I and II). Upon ligand binding, a heteromeric receptor complex is formed. Within this complex, the type I receptor is phosphorylated by the type II receptor, and subsequent activation of the type I receptor is essential for signaling (5). Two BMP type I receptors, BMPR-IA and BMPR-IB, and one type II receptor, BMPR-II, have been identified (3).

Activin, another TGFß family member, was originally identified as a factor in ovarian fluid that stimulated the secretion of FSH from pituitary cells (6). Subsequent studies showed that activin A can increase FSHß gene expression at the transcriptional level (7). Activin is a dimer of two highly related ß-subunits (ß_A and/or ß_B), resulting in three possible molecular species: activin A (ß_Aß_A), activin B (ß_Bß_B), and activin AB (ß_Aß_B). Two activin type II receptors (ActR-II and ActR-IIB) and two activin type I receptors (ActR-I and ActR-IB) have been isolated (8, 9, 10, 11). Inhibin also belongs to the TGFß family and is a heterodimer composed of one -subunit and one of the ß-subunits (ß_A or ß_B), which are the same ß-subunits found in the activins. Despite the structural similarity with activin, inhibin is a negative regulator of FSHß synthesis (12). It was shown that activin/inhibin ß_B-subunit protein was found uniquely in pituitary gonadotropes, suggesting that activin B (ß_Bß_B) might act as an autocrine stimulator of FSH production (13). Strengthening this perception was the fact that bioneutralizing antibodies to activin B (but not to activin A) inhibited FSH production in rat pituitary cultures, so it was concluded that activin B was particularly important for FSH production (14). Moreover, it was found that in mice lacking ActR-II receptors, serum FSH levels were reduced by 60%, suggesting that activins were necessary and primary inducers of FSHß expression. However, in activin/inhibin ß_B knockout mice, serum FSH levels did not decrease but actually showed a modest elevation (20%) (15). The reason for elevated FSH was possibly explained by the increase in levels of ß_A-subunit produced in the ovary in these mice (which could increase activin A), the lack of inhibin B (ß_B), or both. However, levels of BMP-7 were not examined in these mice, and as BMP-7 has been shown to bind ActR-II and transduce an activin-like signal (16), it could also be the ligand that induces FSHß in the absence of activin B and AB to maintain FSH production.

Follistatin has been shown to suppress FSHß messenger RNA (mRNA) levels and FSH secretion in vitro (12) and in vivo (17). The negative effect of follistatin was thought to be due entirely to binding and neutralization of activins (18). However, recently it has been shown that follistatin can also bind BMP-7 and antagonize BMP activities in early Xenopus embryos and in Mv1Lu cells (16, 19), presenting the possibility that follistatin may decrease FSH synthesis by incapacitating a number of TGFß family members other than activin.

Having developed a transgenic pituitary culture system that expresses luciferase specifically in gonadotropes under control of the ovine FSHß promoter (19A ), we set out to confirm that activins control the activity of the ovine FSHß promoter linked to a luciferase reporter gene (oFSHßLuc) in mouse pituitary cultures. Surprisingly, bioneutralizing antibodies to neither activin A nor activin B had any significant effect on oFSHßLuc expression in mouse pituitary cultures, although follistatin routinely inhibited oFSHßLuc expression up to 90%. As follistatin had recently been shown to inactivate BMP-7 (19), and BMP-7 had also been shown to bind ActR-II receptors (16) (necessary for FSH production; see above), we decided to test the hypothesis that BMP-7 is a major inducer of FSH production in mouse pituitary cultures. In this report we document major effects of anti-BMP7 bioneutralizing antibodies on oFSHßLuc in transgenic pituitary cultures and FSH production in primary mouse, rat, and sheep pituitary cell cultures. Furthermore, we show the effects of BMP-7 and -6 on oFSHßLuc and mFSH production in primary mouse pituitary cells and transformed mouse gonadotropes (LßT2 cells).

	Materials and Methods

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Transgenic mouse pituitary cell cultures
The preparation of pituitary cell cultures derived from oFSHßLuc transgenic mice has been described previously (19A ). Transgenic mice from line 7152 (carrying the ovine FSHß promoter-luciferase construct) were used in this study. Briefly, the pituitaries were dispersed into single cells by digestion with collagenase (Sigma, St. Louis, MO) and pancreatin (Life Technologies, Inc., Grand Island, NY). The dispersed cells were then washed and resuspended in medium 199 containing 10% charcoal-treated sheep serum and antibiotics. Charcoal-treated sheep serum was used because estradiol and progesterone have been shown to have some inhibitory effect on oFSHßLuc transgene expression or mouse FSH secretion (19A ), although cells cultured in nontreated FBS with DMEM showed similar basal oFSHßLuc expression (data not shown), suggesting that the effect of steroids in FBS is negligible. Cells were plated in 96-well plates at 30,000–60,000 cells/well and allowed to attach for 1–2 days. For activin or BMP treatments, mouse pituitary cell cultures were first treated with 250 ng/ml follistatin for 16 h, which reduced luciferase activity by 80–90% (19B ). Follistatin was then withdrawn, cells were washed, and medium (containing 1% serum) with or without activin or BMPs was added. To assay for luciferase activity, cells were harvested after 6 h of treatment. To assay for FSH, cells were treated with hormone or antibody for 24 or 48 h, and culture media were collected for RIA. The data shown in this study used either male or female transgenic mice, as we performed our experiments using exclusively male or female pituitary cultures and found no differences in their responses to hormones or antibodies.

Rat and sheep pituitary cell cultures
Rat and sheep pituitaries were dispersed in the same way as the mouse pituitaries described above. Cells were plated in 24-well plates at 200,000 cells/well, allowed to attach for 1–2 days, and treated with anti-BMP7 for 48 h, then culture media were collected to assay for FSH.

Luciferase assay
For mouse pituitary cultures, 6 h after addition of hormone media were removed, and 50 µl Passive Lysis Buffer (Promega Corp., Madison, WI) were added. Thirty-five microliters of lysate were assayed for luciferase activity using a Luciferase Assay System (Promega Corp.). Luminescence was measured as relative light units (RLU) for 20 sec using the Monolight 2010 Luminometer (BD PharMingen, San Diego, CA). For LßT2 cells, after cells were treated with hormones for 24 h media were removed, 100 µl Passive Lysis Buffer were added, and 20 µl lysate were assayed for luciferase activity.

Cell culture and transient transfection of LßT2
LßT2 cells were grown at 37 C in DMEM (Life Technologies, Inc.) containing 10% FBS (HyClone Laboratories, Inc., Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin under 95% air-5% CO₂. Cells were grown in 150-cm² flasks until they were confluent and then were replated in 24-multiwell plates (diameter, 15 mm/well) at a concentration of 200,000 cells/well. Cells were transfected 24 h later in triplicate with 0.44 µg of -4741FSHßLuc(pGL3) and 0.06 µg Rous sarcoma virus-ß-galactosidase (RSV-ßgal) using Fugene6 (Roche Molecular Biochemicals, Basel, Switzerland). -4741FSHßLuc(pGL3) and RSV-ßgal have been described previously (19A, 20). At the time of transfection, media containing 10% FBS and 250 ng/ml follistatin were added to the cells. Sixteen hours after transfection, cells were washed, and media with or without activin (or BMP) were added. Twenty-four hours after addition of hormones, media were removed, and 100 µl lysis buffer were added. Twenty microliters and 15 µl lysate were assayed for luciferase and ß-galactosidase activities, respectively. Luciferase activity was measured as RLU as described above. ß-Galactosidase assays were performed as previously described (20).

FSH secretion
Culture media from either mouse pituitary cells or LßT2 cells were collected after the indicated time of treatment. FSH levels were measured with reagents provided by the National Pituitary and Hormone Program of the NIDDK. FSH was determined in triplicate with mouse FSH Reference Preparation as standard, antimouse FSH antiserum as primary antibody, and rat FSH-I-8 as trace. Rat FSH was assayed as with mouse FSH. Sheep FSH was assayed using a USDA antibody to FSH (DJBR5-4P) and 95% pure ovine FSH for standard and tracer. Intraassay variation was less than 10% for all assays and all samples from one experiment were assayed together in one assay.

Radioiodination
Activin A, BMP-7, and BMP-6 were iodinated using the lactoperoxidase method, as previously described (21), except that 10 µg activin A (carrier-free from R & D Systems, Minneapolis, MN), 20 µg BMP-7 (Creative BioMolecules, Hopkinton, MA), or 20 µg BMP-6 (Creative BioMolecules) were used. Hormones were separated from free iodine by PAGE and tested for follistatin binding, as previously described (21). Fractions containing the highest specific binding for follistatin were pooled and tested for binding to different antibodies.

Solid phase ligand binding assay
Protein A (Sigma; 5 µg/ml) was plated onto Immulon-4 microtiter strips (Dynatech Laboratories, Inc., Chantilly, VA) in carbonate buffer (pH 9.2) overnight at 4 C in a volume of 100 µl. After three rinses in wash buffer (0.5% Tween-20 in PBS), plates were incubated with 150 µl blocking solution (0.05% gelatin and 0.05% Tween-20 in PBS) for 2 h at room temperature. Plates were then washed once, and 100 µl antibody (1:100) were added and incubated overnight at 4 C. Plates were then washed three times, and 100 µl iodinated hormone were added. After incubation at 4 C overnight, plates were washed three times and counted. Nonspecific binding was determined by plating normal rabbit serum or normal sheep serum and adding iodinated hormone.

RNA isolation and RT-PCR
Total RNA was isolated from mouse pituitary or cultured LßT2 cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Complementary DNA was synthesized from 2 µg RNA in a volume of 20 µl containing 450 ng random primer (Roche, Indianapolis, IN), 10 mM depxy-NTPs (Roche), 1 x PCR buffer [from 10 x assay buffer B (100 mM Tris-HCl (pH 8.3) and 500 mM KCl); Fisher Scientific, Pittsburgh, PA], 5 mM MgCl₂, and 12 U AMV reverse transcriptase (Promega Corp.). A portion of the total RNA was treated identically as that subjected to complementary DNA synthesis, except for the omission of reverse transcriptase to control for the possible amplification of genomic DNA. One additional reaction contained RT, but no RNA, to control for possible contamination of solutions or pipettors. All tubes were incubated in a PTC-100 programmable thermal controller (MJ Research, Inc., Cambridge, MA) for 10 min at 21 C, 15 min at 42 C, and 10 min at 99 C, then cooled rapidly to 4 C.

PCR was carried out in a volume of 50 µl containing 20 µl RT reaction mixture, 1 x PCR buffer, 500 nM each of 5'- and 3'-primers, 1 µCi [-³²P]deoxy-CTP (3000 Ci/mmol; NEN Life Science Products), and 2.5 U Taq polymerase (Fisher Scientific). Reactions were then overlaid with 100 µl mineral oil and amplified. For BMP-7, PCR was performed for 30 cycles of 1 min at 94 C, 1 min at 58 C, and 1 min at 72 C. For BMP-6, PCR was performed for 35 cycles of 1 min at 94 C, 1 min at 64 C, and 1 min at 72 C. Twenty-five microliters of each reaction were electrophoresed through 6% nondenaturing acrylamide gels for 1.5 h at 300 V. Gels were dried onto blotting paper, and bands were visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Primer sequences for BMP-7 (forward, 5'-GCAGAGCATCAACCCCAAGT-3'; reverse, 5'-GGACAGAGATGGCGTTGAGC-3') were suggested by Dr. Donald Jin (Creative BioMolecules). Primer sequences for BMP-6 (forward, 5'-AGACCTGGGATGGCAGG-3'; reverse, 5-ACCATCCCGCTTCGCTGTGC-3') were cited from Ref. 22 .

Hormones and antibodies
Recombinant human follistatin was provided by the National Pituitary and Hormone Program of the NIDDK. Recombinant human activin A and BMP-6 were obtained from R & D Systems. BMP-7, BMP-6, and anti-BMP7 were gifts from Drs. Dattatreyamurty Bosukonda and Paul L. Kaplan (Creative BioMolecules). Sheep antiactivin A (no. 841), which bioneutralizes activin A, was a gift from Dr. Wylie Vale (The Salk Institute, La Jolla, CA). Monoclonal antiactivin B, which bioneutralizes activin B, was a gift from Dr. Ralph Schwall (Genentech, Inc., South San Francisco, CA).

Statistical analysis
Statistical calculations were performed using the Prism computer program (GraphPad Software, Inc., San Diego, CA). One-way ANOVA was used to test whether differences between groups were significant. If differences were significant (P < 0.05), Dunnett’s multiple comparison test was then used for post-hoc evaluation of differences between various hormone- or antibody-treated groups and the control groups. To compare the difference between antiserum and control serum groups at the same concentration, a t test was performed.

	Results

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BMP-7 and BMP-6 induce oFSHßLuc expression in pituitary cultures
To study the role of BMPs in regulating FSHß expression, pituitary cultures derived from oFSHßLuc transgenic mice were used. This in vitro system was described previously (19A ) as a model suitable for studying transcriptional regulation of the oFSHß gene in pituitary gonadotropes. It has been shown that follistatin pretreatment lowered basal oFSHßLuc expression, presumably by removing FSH-stimulating factors secreted by these pituitary cells [either by forming inactive complexes with activins (18) or by accelerating degradation of activin (23)], thus allowing the observation of a more dramatic transgene stimulation by activin (19B ). In this study follistatin-pretreated transgenic mouse pituitary cultures were used to test the ability of BMP-7 and BMP-6 to induce FSHß gene transcription. After 16 h of follistatin pretreatment, pituitary cells were washed, and BMP-7 (1 µg/ml) or BMP-6 (1 µg/ml) was added to the culture. Activin A (300 ng/ml) was also added for comparison. After 6 h of treatment, BMP-7 and BMP-6 increased oFSHßLuc transgene activity by 5.6- and 6.1-fold, respectively (Fig. 1), similar to the stimulation by activin A (5.3-fold). BMP-3 (1 µg/ml) or TGFß-1 (200 ng/ml) did not increase transgene activity (data not shown). The dose of follistatin (250 ng/ml) used for pretreatment was determined based on a dose-response study presented previously (19B ). It should be noted that luciferase activity in control cells in Fig. 1 represents the activity of the follistatin-pretreated control. As follistatin routinely decreases basal luciferase expression by 80–90%, as shown previously (19A ), the control value in Fig. 1 is only 10–20% of the control value shown in Fig. 4A or 5 (see below).

View larger version (16K): [in this window] [in a new window]   Figure 1. Stimulation of oFSHßLuc expression by activin and BMPs in transgenic mouse pituitary cell cultures. Cultured pituitary cells were pretreated with 250 ng/ml follistatin for 16 h; follistatin was then withdrawn, and cells were washed before activin or BMP was added. Cells were treated with activin A (300 ng/ml), BMP7 (1 µg/ml), or BMP6 (1 µg/ml) for 6 h before being harvested for determination of luciferase activity. One-way ANOVA demonstrated significant treatment effects. **, P < 0.01 (by Dunnett’s post-hoc test). Values are the mean ± SEM from one representative experiment with triplicate determinations. These experiments were performed three times with similar results.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of anti-BMP7 on oFSHßLuc expression (A) and FSH secretion (B) in transgenic mouse pituitary cell cultures. Cells were treated with follistatin (FS; 250 ng/ml), rabbit anti-BMP7 [BMP-Ab(r); 1:100], sheep anti-BMP7 [BMP-Ab(s); 1:100], antiactivin A [ActA-Ab; 1:100], or antiactivin B [ActB-Ab; 500 µg/ml] for 24 h and harvested for determination of luciferase activity. Culture media were collected to assay for FSH. One-way ANOVA demonstrated significant treatment effects. **, P < 0.01 (by Dunnett’s post-hoc test). ns, Not significant. Values are the mean ± SEM from one representative experiment with triplicate determinations. These experiments were performed three times with similar results.

View larger version (20K): [in this window] [in a new window]   Figure 2. FSH secretion from LßT2 cells in response to activin A, BMP-7, or BMP-6. LßT2 cells were plated in six-well plates (diameter, 35 mm/well) at a concentration of 1 million cells/well. Twenty-four hours after plating, the media were changed to fresh media containing activin A (), BMP-7 (•), or BMP-6 (). Cells were incubated for another 3 days before the media were collected to assay for FSH by RIA. One-way ANOVA demonstrated significant treatment effects. *, P < 0.05; **, P < 0.01 (by Dunnett’s post-hoc test). Each point represents the mean ± SEM from three independent experiments.

View larger version (16K): [in this window] [in a new window]   Figure 3. Activation of the oFSHßLuc construct by activin A, BMP-7, or BMP-6 in LßT2 cells. Luciferase expression vector containing the ovine FSHß promoter [-4741FSHßLuc(pGL3)] was transfected along with an internal control RSV-ßgal into LßT2 cells. Follistatin (250 ng/ml) was added to the cells at the time of transfection. Sixteen hours after transfection, cells were washed; treated with activin A (100 ng/ml), BMP-7 (1 µg/ml), or BMP-6 (1 µg/ml); and cultured for an additional 24 h before being harvested for determination of luciferase and ß-galactosidase activities. Luciferase activity was normalized against the cotransfected ß-galactosidase activity. Results were shown as the fold induction compared with the control cultures in the absence of hormone treatment. One-way ANOVA demonstrated significant treatment effects. **, P < 0.01 (by Dunnett’s post-hoc test). Values are the mean ± SEM from one representative experiment with triplicate determinations. These experiments were performed three times with similar results.

To demonstrate that the effect of rabbit or sheep anti-BMP7 antibody was dose dependent and specific, transgenic mouse pituitary cultures were treated with increasing concentrations of either control serum or anti-BMP7. Neither rabbit control serum nor sheep control serum significantly altered luciferase expression (Fig. 5). Rabbit anti-BMP7 significantly decreased luciferase expression by 31% at 1:10,000, 74% at 1:1,000, and 80% at 1:100 dilution (Fig. 5A). Sheep anti-BMP7 did not significantly decrease luciferase expression at 1:10,000. At 1:1,000 dilution, sheep anti-BMP7 decreased luciferase expression by 28%, although this suppressive effect did not reach statistical significance. Sheep anti-BMP7 significantly decreased luciferase expression by 71% at 1:100 dilution (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   Figure 5. Dose-dependent inhibition of oFSHßLuc in mouse pituitary cell cultures by rabbit and sheep anti-BMP7. Cells were treated with different concentrations (1:10,000, 1:1,000, or 1:100) of either the control serum or the anti-BMP7 for 24 h. Cells were then harvested for luciferase activity. One-way ANOVA demonstrated no significant treatment effects with either rabbit control serum or sheep control serum. A t test was used to determine whether the effect of anti-BMP7 is significant compared with the effect of control serum at the same concentration (*, P < 0.05; **, P < 0.01). Each point represents the mean ± SEM from one representative experiment with triplicate determinations. These experiments were performed three times with similar results.

View larger version (16K): [in this window] [in a new window]   Figure 6. Dose-dependent inhibition of FSH secretion by rabbit anti-BMP7 in mouse (A), rat (B), or sheep (C) pituitary cell cultures. Cells were treated with different concentrations (1:3000, 1:500, or 1:100 for mouse and rat cultures; 1:450, 1:150, or 1:50 for sheep cultures) of either the control serum or the anti-BMP7 for 48 h. Culture media were then collected to assay for FSH. One-way ANOVA demonstrated no significant treatment effect with rabbit control serum in all three cultures. A t test was used to determine whether the effect of anti-BMP7 is significant compared with the effect of the control serum at the same concentration (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Each point represents the mean ± SEM from three independent experiments.

Characterization of binding specificity of anti-BMP7
Although rabbit and sheep anti-BMP7 decreased basal oFSHßLuc expression and FSH secretion, as shown above, it was not known whether these antibodies cross-react with activins. To address this question, solid ligand binding assays were used to test the binding specificities of the anti-BMP7 antibodies. Activin A, BMP-7, and BMP-6 were radiolabeled with ¹²⁵I and purified as described in Materials and Methods. Rabbit anti-BMP7 or sheep anti-BMP7 was immobilized on protein A plates, and different concentrations of [¹²⁵I]activin A, [¹²⁵I]BMP-7, or [¹²⁵I]BMP-6 were incubated with anti-BMP7-coated plates. Normal rabbit serum was immobilized and incubated with ¹²⁵I-labeled hormones to determine nonspecific binding. Figure 7A shows that BMP-7 binds to rabbit anti-BMP7 with highest affinity. BMP-6 showed significant binding, although the affinity of BMP-6 was lower than that of BMP-7. Absolutely no binding between anti-BMP7 and activin A was observed. Similar results were obtained when sheep anti-BMP7 was used (Fig. 7B). The specificity of the antiactivin A was also tested for cross-reactivity with BMP-7 and BMP-6. Figure 7C showed that antiactivin A bound with high affinity to activin A, but also bound to BMP-7 or BMP-6 at a lower affinity.

View larger version (17K): [in this window] [in a new window]   Figure 7. Specificity of rabbit anti-BMP7 (A), sheep anti-BMP7 (B), and sheep antiactivin A (C). Increasing amounts of iodinated activin A, BMP-7, or BMP-6 were incubated with different antibodies immobilized on protein binding plates to test the specific binding of each antibody to these hormones. Each point represents the mean ± SEM (n = 3 for A; n = 2 for B and C).

View larger version (18K): [in this window] [in a new window]   Figure 8. Detection of mRNAs for BMP-7 and BMP-6 by RT-PCR amplification. Total RNA from mouse pituitary (lanes 1 and 3) or LßT2 cells (lanes 2 and 4) was reverse transcribed and PCR amplified. The expected product sizes are indicated at the right of the gels. Negative controls of nonreverse transcribed RNA are shown in lanes 5 and 7 (for pituitary cells) and lanes 6 and 8 (for LßT2 cells). Lanes 9 and 10 are water controls with no RNA input. M, One-kilobase DNA marker; P, pituitary cells; L, LßT2 cells; B7, BMP-7; B6, BMP-6; RT, reverse transcribed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The abilities of BMP-7 and BMP-6 to stimulate FSHß synthesis were first discovered using pituitary cultures derived from transgenic mice that carry oFSHßLuc, a transgene composed of an ovine FSHß promoter (-4741 to +759 bp) linked to a luciferase reporter gene. This transgene has been shown previously to be expressed specifically in pituitary gonadotropes at very high levels in vivo (100,000–20,000,000 RLU/pituitary) and in pituitary cultures (10,000–2,000,000 RLU/50,000 cells) (19A ). Thus, the transgenic mice and their pituitary cultures provided a convenient and sensitive system to screen for factors that regulate FSHß gene transcription. The finding of a functional BMP signaling system in gonadotropes, which has not been detected previously, further demonstrated the usefulness of this transgenic mouse model.

The unique quality of oFSHßLuc-expressing pituitary cultures that permitted detection of FSH induction by activins and BMPs was the ability of activins and BMPs to rapidly induce luciferase expression (2–6 h) after follistatin pretreatment. After 6 h, endogenous factors build up in static primary pituitary cultures that induce FSH synthesis (previously thought to be activin only) and rapidly obscure the effects of exogenously added activin and BMP. The 6-h time frame is too short to accurately measure FSH production itself due partly to assay sensitivity problems, but also to nonregulated secretion of FSH from storage granules and intracellular time delays needed to process newly synthesized FSH (26). The measurement of luciferase activity avoided the technical problems involved with assaying FSH.

To show that BMP-7 and BMP-6 can induce the secretion of FSH, which is the ultimate functional molecule in vivo, a transformed gonadotrope cell line, LßT2, was used. LßT2 cells express very low levels of FSHß mRNA and secrete very little FSH, presumably because there is not much endogenous induction of FSH produced in these cells. However, after activin A treatment, FSHß mRNA expression and FSH secretion from LßT2 cells are significantly increased, suggesting that these cells are fully differentiated gonadotropes (24). In this study BMP-7 and BMP-6 were shown to be bona fide FSH stimulators, as both BMPs can induce FSH secretion from LßT2 cells. It has been shown in one report that 300 ng/ml BMP-7 was ineffective in increasing FSH secretion from rat pituitary cultures (16). However, in the current report BMP-7 caused a 6-fold increase in FSH secretion from LßT2 cells at 1 µg/ml and a more dramatic 10-fold increase at 3 µg/ml. It is possible that LßT2 cells provide a more sensitive cell model to observe stimulation of FSH production by activin or BMPs because endogenous FSH-stimulating factors secreted by LßT2 cells are more limited compared with those in primary rat pituitary cultures. For example, the same study that reported no response of rat pituitary cultures to BMP-7 showed only a 2-fold induction of FSH secretion in response to 30 ng/ml activin A (16), whereas in our current study LßT2 cells showed a 15-fold increase in FSH secretion in response to 10 ng/ml activin A.

In this study activin A was found to be more effective and more potent than either BMP in increasing FSHß gene expression and FSH secretion. It should be noted, however, that heterodimers of BMP-2 or BMP-4 with BMP-7 have been shown to be more effective and potent (by 20-fold) than the homodimer of BMP-7 in other cell systems (27, 28). As the mRNAs for BMP-2, BMP-4, and BMP-7 were all detected in mouse pituitary (BMP-2 and BMP-4 RT-PCR data are preliminary), it is possible that BMP-2/7 or BMP-4/7 heterodimers may be the natural and more potent FSH inducer present in vivo, although identification of endogenous BMP heterodimers in pituitary tissue awaits further studies.

Interestingly, activin A (at 100 ng/ml) increased oFSHßLuc expression by 8-fold, but increased FSH secretion by 26-fold from LßT2 cells, whereas effects of BMP-7 on oFSHßLuc expression and FSH secretion were similar (4- and 6-fold, respectively, at 1 µg/ml). Expression of the oFSHßLuc transgene in primary pituitary cells was also similarly increased by activin A (5-fold at 300 ng/ml) and BMP-7 (6-fold at 1 µg/ml). These data suggest that activin may have both transcriptional and posttranscriptional effects on FSH synthesis, although it is possible that the greater response of FSH secretion to activin A in LßT2 cells is caused by use of the oFSHß promoter in a mouse cell or by aberrant behavior of the transformed LßT2 cells.

Follistatin is generally thought to decrease basal FSH secretion in pituitary cultures by incapacitating activin produced endogenously by these cultures. In the current report, two anti-BMP7 antibodies that have been shown to neutralize the activity of BMP-7 decreased basal oFSHßLuc transgene activity and FSH secretion from transgenic mouse pituitary cultures similarly to follistatin, whereas neutralizing antibodies to activin A or activin B had no effect. These data suggested that BMP-7, but not activin A or activin B, may be the primary FSHß-stimulating factors produced by mouse pituitary cultures. Thus, the effects of follistatin down-regulation of basal oFSHßLuc expression and FSH secretion in mouse pituitary cultures were probably due to neutralization of the activity for BMP-7, rather than that for activin A or activin B. It is not clear why the antiactivin A or antiactivin B is ineffective in decreasing basal oFSHßLuc transgene expression or mFSH secretion from transgenic mouse pituitary cultures. Bioneutralizing activities of the antiactivin A or antiactivin B used in this study have been demonstrated previously (14, 25, 29). In the current study antiactivin antibodies were used at concentrations 10 times greater than (for antiactivin A) or equivalent to (for antiactivin B) the concentration used in the previous studies (14, 25). Therefore, the lack of neutralizing effect of antiactivin A or antiactivin B is probably not due to insufficient amount of antibodies used. We have also tested the bioneutralizing activity of antiactivin A using our transgenic mouse pituitary cultures and found that the ability of activin A to increase oFSHßLuc expression is blocked by preincubation of activin A with antiactivin A (data not shown), indicating that the antibody did, in fact, bioneutralize activin A under the conditions used in our laboratory. We did not test the activity of antiactivin B, because there is no purified activin B available currently, but two separate preparations of antiactivin B were used, and neither preparation inhibited oFSHßLuc expression or mFSH secretion. As activin A has never been demonstrated to be an important endogenous activator of FSH in rat pituitary cultures, it is not surprising that basal mouse FSH secretion or oFSHßLuc expression is not decreased by antiactivin A. However, antiactivin B was shown to decrease FSHß RNA levels and FSH secretion from rat pituitary cultures (14), suggesting that activin B is made by rat pituitary cells and is important for FSH production. One explanation for the absence of a bioneutralizing effect of antiactivin B on oFSHßLuc expression or mFSH secretion from transgenic mouse pituitary cultures in the current study is that different factors are being made by mouse and rat pituitaries to maintain basal FSH expression. For example, BMPs may be more abundant than activin in the mouse pituitary, so that BMPs play a more important role in an autocrine or paracrine fashion, whereas activins from the ovary play a more important role in an endocrine fashion. Another possibility is that the antiactivins used in rat pituitary cultures do not cross-react with mouse activins (antiactivin A was raised against human activin A and antiactivin B was raised against rat activin B), although this is unlikely because homologies are high for activins across species.

Using solid ligand binding assays we showed that the anti-BMP7 used in this study also binds BMP-6 with high affinity, suggesting that BMP-6 may be produced in pituitary cultures, although the ability of two BMP7 antibodies to bioneutralize the activity of BMP-6 needs to be confirmed. It was also shown that anti-BMP7 did not cross-react with activin A, indicating that the neutralizing effect of anti-BMP7 on basal oFSHßLuc or FSH secretion was not due to inactivation of endogenous activin A. However, antiactivin A can bind BMP-7 and BMP-6 with lower affinity. Due to the lack of purified activin B, we were unable to test the cross-reactivity of anti-BMP7 with activin B. It should be noted that the evidence supporting the concept of functional activin B production in gonadotropes is that bioneutralizing antiactivin B decreases FSH secretion from rat pituitary culture (14). However, it is not known whether antiactivin B cross-reacts with BMPs. As the production of BMPs in the pituitaries of different species has not been examined previously, it will be important in future studies to compare the abundance of BMPs and activins produced to determine the relative importance of activins and BMPs in the pituitary as autocrine/paracrine inducers of FSH.

Rat and sheep pituitary cultures were used in this study to further strengthen the concept that BMP7 is produced in pituitary cells. It should be noted that oFSHßLuc seems to be more sensitive to the bioneutralizing effects of BMP-7 antibodies than FSH secretion. It is possible that the smaller change in FSH secretion is due to constitutive secretion of stored FSH from secretory granules, which is not regulated by BMPs. It is also possible that use of the oFSHß promoter in mouse pituitary cells in some way exaggerates the transcriptional effect of BMPs, as FSH secretion from sheep pituitary cultures seems to be more sensitive to anti-BMP7 treatment than that from mouse cultures, although this effect is expected to be small. More experimentation will be needed to establish the physiological importance of BMPs in vivo. However, as FSH secretion from mouse, rat, and sheep pituitary cultures was decreased by bioneutralizing anti-BMP7, and BMP-7 is capable of inducing FSH from LßT2 gonadotrope cells, BMP-7 seems to be an important FSH stimulator and should be taken into account when studying the regulation of FSH synthesis in the future.

In the past, due to the lack of a gonadotrope cell line that produces FSH endogenously, it has been difficult to analyze the sequences of the FSHß gene that are responsible for hormone regulation. The recent discovery that LßT2 cells express FSHß mRNA and secrete FSH in response to activin A suggests that this cell line could be used to study transcriptional regulation of the FSHß gene. It has been shown in this report that the oFSHß promoter is expressed in LßT2 cells, and the expression is up-regulated by activin A, BMP-7, or BMP-6. These cells thus provide an in vitro tool to rapidly localize the response elements of the FSHß gene for activin, BMP-7, or BMP-6. However, the activin or BMP response elements identified in vitro should ultimately be tested in vivo in transgenic mice to verify the physiological importance of these sequences.

In conclusion, BMP-7 and BMP-6 were shown to increase oFSHß gene transcription and FSH secretion in primary pituitary cultures and transformed gonadotropes (LßT2 cells). Based on bioneutralization studies using anti-BMP7, it appeared that basal FSHß expression in mouse pituitary cultures was maintained by BMP-7. These results combined with the detection of mRNAs for BMP-7 and BMP-6 in mouse pituitaries suggest that BMP-7 or BMP-6 may be significant FSH stimulators, and their physiological importance awaits confirmation in vivo.

	Acknowledgments

 
We thank Drs. QiFa Wang and Patrick M. Sluss (Massachusetts General Hospital, Boston, MA) for technical assistance with solid phase ligand binding assay. We also thank Drs. Dattatreyamurty Bosukonda and Paul L. Kaplan (Creative BioMolecules) for the generous supply of BMP-7 and anti-BMP7, Dr. Wylie Vale for antiactivin A, and Dr. Ralph Schwall for antiactivin B. LßT2 cells were generously provided by Dr. Pamela L. Mellon (University of California, San Diego, CA).

	Footnotes

 
¹ This work was supported by NICHD Grant 34863 and the Mellon Foundation.

Received September 11, 2000.

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