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Endocrinology Vol. 138, No. 10 4324-4329
Copyright © 1997 by The Endocrine Society

ARTICLES

Evidence That Pituitary Adenylate Cyclase Activating Polypeptide Suppresses Follicle-Stimulating Hormone-ß Messenger Ribonucleic Acid Levels by Stimulating Follistatin Gene Transcription1

Stephen J. Winters, Alan C. Dalkin and Toshihiko Tsujii2

Department of Medicine (S.J.W., T.T.), University of Pittsburgh, Pittsburgh, Pennsylvania 15213; and Department of Medicine (A.C.D.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Stephen J. Winters, M.D., Department of Medicine, University of Pittsburgh Medical Center, Montefiore N-919, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. E-mail: winters{at}med1.dept-med.pitt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator of gonadotrope function. One of the actions of PACAP identified previously is to decrease FSHß messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHß mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived {alpha}T3–1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in {alpha}T3–1 cells PACAP activation of the follistatin promoter appears to be via the cAMP- dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHß mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pronounced effects on gonadotrope function (1, 2). PACAP administered iv increases LH secretion in rats several fold (3). In cultured rat pituitary cells, PACAP, although a weak and transient stimulator of gonadotropin secretion when administered alone, substantially enhances GnRH-stimulated gonadotropin release by decreasing the EC50 for GnRH (4, 5). PACAP also regulates each of the gonadotropin subunit genes. PACAP increases {alpha}-subunit messenger RNA (mRNA) levels by stimulating {alpha}-subunit gene transcription (5, 6), lengthens LHß mRNA and presumably prolongs its half-life (5), and reduces FSHß mRNA levels in pituitary cells perifused with pulses of GnRH (5).

The follistatin-activin-inhibin system appears to play a pivotal role in the control of FSHß gene expression and thereby FSH production (7). Previous studies provide evidence that PACAP might regulate FSHß mRNA tran-scripts through a follistatin-activin mechanism. Specifically, PACAP receptors are present on most pituitary folliculostellate cells and on a subpopulation of gonadotropes (8), and both cell types produce follistatin (9). Follistatin mRNA levels are increased when adenylate cyclase is activated by forskolin or by the phorbol ester phorbol-12-myristate-13-acetate (PMA) (10, 11), and follistatin suppresses FSH secretion (12) and FSHß mRNA levels (13). The {alpha}T3–1 gonadotrope cell line expresses PACAP type 1 (PVR1) receptors (14), which are coupled to adenylate cyclase and, at slightly higher ligand concentration, to phospholipase C (15). The experiments presented herein were conducted in primary pituitary cell cultures and in {alpha}T3–1 cells to begin to test the hypothesis that PACAP suppresses FSHß mRNA levels by stimulating follistatin gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pituitary cell cultures
Pituitary cells were prepared from 7-week-old male Sprague-Dawley rats (Zivic-Miller Laboratories, Inc., Allison Park, PA) that were castrated at age 5 weeks according to a protocol approved by the Animal Welfare and Use Committee of the University of Pittsburgh. Pituitary cells from castrated rats were used to produce an increase in the level of FSHß mRNA to better demonstrate suppression by PACAP. Pituitaries were minced and treated with 0.4% collagenase as described previously (16). Cells were cultured in DMEM-Ham’s F-12 nutrient mixture (DMEM/F-12) supplemented with 10% charcoal-stripped FCS in 6-cm petri dishes at a density of 3 x 106 cells per dish. After 3 days of preculture, the medium was changed to include 10 nM PACAP38 or control medium. The incubation was continued for 8 h or 24 h, and cells were removed, quick-frozen in 4 M guanidinium thiocyanate solution on an ethanol dry-ice slurry, and stored at -70 C for RNA extraction.

RNA extraction and Northern hybridization
RNA was extracted by the guanidinium thiocyanate-phenol-chloroform procedure (17). The concentration of total RNA was determined by reading the OD at 260 nm. FSHß mRNA levels were determined by Northern analysis. Aliquots of pituitary RNA samples were subjected to electrophoresis in 1.2% agarose-formaldehyde gels. RNAs were transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) and cross-linked to the membranes by baking for 2 h at 80–90 C followed by irradiation for 2 min with UV light. A purified complementary DNA (cDNA) for rat FSHß (from Dr. Richard Maurer, Oregon Health Sciences University, Portland, OR) was labeled by the random primer method with [{alpha}32P]deoxycytosine triphosphate (3000 Ci/mmol; New England Nuclear Research Products, Boston, MA) to a specific activity of 6–8 x 108 dpm/µg. Labeled probes were added to the hybridization solutions at a concentration of approximately 5 ng/ml for 48–72 h. The membranes were washed and autoradiographed, and slices corresponding to full-length mRNAs were excised and counted in a Packard 4530 scintillation counter (Packard, Downers Grove, IL). Residual membranes were rehybridized without stripping for normalization to cyclophilin cDNA.

Measurement of follistatin mRNA
A previously described quantitative RT-PCR assay was used to quantitate follistatin mRNA levels in pituitary cell cultures (18). RT of total pituitary RNA was performed from control and PACAP-treated cell cultures and analyzed in the same PCR assay. The follistatin cDNA (from Dr. Kelley Mayo, Northwestern University, Evanston, IL) was size-altered by substituting a 163-bp fragment of unrelated DNA for the 72 bp SplI/AccIII segment to create a competitive template that was used as an internal standard. The method therefore allows the same oligonucleotide primers to be used to amplify the native and competitive template cDNAs. Ten nanograms of RNA were used for each PCR reaction, and hence the results are expressed as femtograms of mRNA/10 ng total pituitary RNA.

Construction of follistatin-LUC fusion genes
A rat follistatin-LUC fusion construct was prepared by blunt end ligation of the -757/+139 fragment of the rat follistatin gene into the luciferase (LUC) reporter plasmid Luc-Link (derived from pBR-322 by Dr. Richard Maurer). The -757/+139 Pst-1/EcoRI fragment of the rat follistatin gene was prepared from a follistatin-chloramphenicol acetyltransferase (follistatin-CAT) plasmid from Dr. S. Shimasaki (Whittier Institute, La Jolla, CA) (pFSCAT64) containing the -2,600/+139 fragment of rat follistatin in the pCAT basic vector (Promega Corp, Madison, WI). The plasmid was purified by two runs of cesium chloride density gradient centrifugation.

To confirm results with the above construct, a second follistatin-LUC construct was prepared by PCR. Oligonucleotide primers (upstream 5'-TTGTGAAGACATCCAGTGC-GGRGGT-3' and downstream 5'-CTCTTCCTCCGTTTCTTCCGAGATG-3', designed according to the published rat follistatin sequence (19), were synthesized and used to amplify the 5' portion of the rat follistatin gene (bp -717 to +183) from genomic DNA (Promega) using Pfu DNA-dependent DNA polymerase (Stratagene, La Jolla, CA) in the PCR reaction for 35 cycles. The DNA product was subcloned, sequenced for verification, ligated to the firefly luciferase coding sequence in a plasmid derived from PUC19 (Dr. Richard Day, University of Virginia, Charlottesville, VA), and propagated for use in JM-109 cells.

{alpha}T3–1 Cell culture and transfection
{alpha}T3–1 cells (provided by Dr. Pamela Mellon (UC San Diego, La Jolla, CA) were maintained in monolayer culture in MEM containing HEPES (25 mM), NaHCO3 (26 mM) glucose (4.5 g/liter), 5% bovine serum, and 5% FBS, penicillin, streptomycin, and fluconazole. Transfections were performed in cells plated in 60-mm petri dishes at a density of 1.75 x 106 cells per dish and cultured in 3 ml media (containing dextran-coated charcoal-treated bovine serum and FBS) for 2 days to 50–75% confluence. Media were removed, and the cells were washed twice. Then 3 ml fresh medium containing 10% dextran-coated charcoal-treated FBS were added for 3 h, and the cells were transfected with 10 µg DNA using the calcium phosphate procedure (Life Technologies, Gaithersburg, MD). The precipitate was dispensed to all control and treated cultures in triplicate so that luciferase activity could be compared within an experiment without correction for efficiency of transfection. After 16 h of incubation, cells were washed with fresh medium and treated with medium alone or medium containing test substances. Cells were lysed in Cell Culture Lysis Reagent (Promega) that was microfuged twice to remove debris. The extract was analyzed for protein by the Bradford dye-binding procedure using the Bio-rad (Hercules, CA) protein assay kit. Luciferase activity was determined at room temperature using the Luciferase Assay System (Promega) in aliquots containing 20 µg protein (less than 10 µl cell extract) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).

Hormones
PACAP38 was obtained from Peninsula Labs. (Belmont, CA). The GnRH analog des-Gly10, [D-Trp6]-LHRH ethylamide was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human (rh) follistatin was kindly provided by the National Hormone and Pituitary Program, NIDDK. Rh inhibin A was a gift from Biotech Australia (Sydney, Australia), and rh activin A was a gift from Genentech Inc, (South San Francisco, CA).

Data analysis
Data are presented as mean ± SEM FSHß mRNA levels were transformed for statistical analysis because of between-experiment variation in the specific activity of the radiolabeled cDNA. In each replicate experiment, the results for PACAP-treated cultures were expressed as a percentage of the value for cells treated with control medium, which was set at 100%. Data for each replicate experiments were analyzed independently and then compiled for statistical analysis. mRNA levels in PACAP-treated and control cultures were compared by Student’s t test after log transformation because of unequal variance. The results of the transfection experiments were analyzed by ANOVA and posthoc Dunnett’s test for comparison with media-treated controls. Multiple comparisons were performed by ANOVA and Tukey’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PACAP decreases FSHß and increases follistatin mRNA levels in primary pituitary cell cultures
Pituitary cell monolayer cultures were treated with 10 nM PACAP38 or with control medium for 8 h or for 24 h. As illustrated in Fig. 1Go, FSHß mRNA levels were reduced to 36.3 ± 1.8% (P < 0.01) of the value for medium-treated controls after 8 h of PACAP treatment. When the incubation was conducted for 24 h, FSHß mRNA levels were 72.5 ± 14.9% (P < 0.05) of control. Follistatin mRNA levels were increased by PACAP at each time point. The increase at 8 h was to 420 ± 217% of control (P < 0.05), and the rise after 24 h treatment was to 197 ± 39.6% (P < 0.05) of control.



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  		Figure 1. Effects of PACAP on FSHß and follistatin mRNA levels in primary pituitary cell cultures. The values represent the mean ± SEM of three separate experiments at 8 h and six replicate experiments at 24 h. All experiments were conducted independently. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
The follistatin promoter is stimulated by PACAP in {alpha}T3–1 cells
To examine the regulation of the follistatin promoter by PACAP, we used transient transfection assays of {alpha}T3–1 cells since these cells express PACAP type 1 (VPR-1) receptors coupled to adenylate cyclase and to phospholipase C (15). The hormonal control of the follistatin promoter by PACAP and by the GnRH analog des-Gly10, [D-Trp6]-LHRH ethylamide, rh follistatin, rh inhibin A, and rh activin A was compared with follistatin-LUC activity in cells exposed to control medium (Fig. 2Go). Treatment of transfected cultures with PACAP increased (P < 0.01) follistatin-LUC activity 3.3-fold relative to medium-treated controls. No change in LUC activity occurred after treatment with the GnRH analog, follistatin, or activin, whereas treatment with inhibin reduced (P < 0.05) basal follistatin-LUC activity to 72% of control. Similar results were obtained with the construct containing the -717 to +183 region of the PACAP promoter (data not shown). For comparison, {alpha}T3–1 cells were also transfected with 10 µg of a promoterless luciferase expression plasmid (Luc-Link) or a thymidine kinase-luciferase fusion gene (TK-Luc, each from Dr. Richard Maurer). Both luciferase activity from the TK promoter and the promoterless reporter were unaffected by PACAP (data not shown).



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  		Figure 2. Hormonal activation of the follistatin promoter. {alpha}T3–1 cells were transfected with -757/+139 follistatin-luciferase and treated with control medium, 10 nM PACAP, 3 ng/ml activin, 3 ng/ml inhibin, 12 ng/ml follistatin, or 10 nM of the GnRH analog, des-Gly10, [D-Trp6]-LHRH ethylamide. Cells were collected after 8 h of treatment, and luciferase activity was determined. The values shown are the mean and SEM of three replicate dishes for each treatment. The experiment was repeated two to six times with similar results. *, P < 0.05 vs. control; **, P < 0.01 vs. control, ANOVA and Dunnett’s test.

 
The stimulatory effect of PACAP38 on follistatin-LUC transcription was examined over a wide range of doses as shown in Fig. 3Go. Maximum stimulation was observed with the lowest dose tested (1.0 nM). Table 1Go reveals that significant activation was observed at the earliest time point examined (2 h), and that fold-induction over basal activity increased with time over 24 h.



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  		Figure 3. Responsiveness of the follistatin promoter to increasing doses of PACAP38. For each dose, triplicate dishes were transfected, and the mean ± SEM luciferase activity is plotted. All doses of PACAP increased LUC activity (P < 0.01).

 

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  		Table 1. Time course of transcriptional activation of the follistatin gene by PACAP

 
Because PACAP is known to stimulate cAMP as well as inositol triphosphate production in {alpha}T3–1 cells (15), we next examined the different signal transduction pathways by which PACAP may activate the follistatin promoter (Fig. 4Go). Cells were stimulated with the adenylate cyclase stimulator, forskolin, or with the phorbol ester, PMA. Forskolin produced near-maximum LUC stimulation whereas PMA had no effect. Adding PMA to forskolin was no more effective than forskolin alone. To verify that PMA was indeed active in our model, we confirmed (20) that PMA was an effective stimulator of the human {alpha}-subunit promoter in the {alpha}T3–1 cells (data not shown). In addition, PMA inhibited follistatin-LUC stimulation by PACAP (Fig. 4Go).



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  		Figure 4. Stimulation of the follistatin promoter by PACAP, forskolin, or PMA. For each stimulator, triplicate dishes were transfected, and the mean ± SEM luciferase activity is plotted. The doses used were 10 nM PACAP38, 10 µM forskolin, and 100 nM PMA, each for 8 h. The experiment was replicated three times with similar results. Bars with same letter are not different from each other.

 
If the actions of PACAP on the follistatin promoter were via cAMP/PKA, we predicted that addition of PACAP to forskolin would be similar to forskolin alone. Figure 5Go reveals that forskolin was maximally effective at a dose of 10 µM and that forskolin together with PACAP was no more effective than forskolin alone.



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  		Figure 5. PACAP and forskolin are not additive in stimulating the follistatin promoter. {alpha}T3–1 cells were transfected with -757/+139 follistatin-luciferase, and then incubated with increasing concentrations of forskolin, or with 10 µM forskolin together with 10 nM PACAP for 8 h. The bars represent the mean ± SEM of triplicate dishes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although GnRH is considered to be the primary regulator of gonadotropin synthesis and secretion, evidence is accumulating implicating the pituitary and gonadal peptides activin, follistatin, and inhibin as key factors in the differential regulation of FSH. Activin and follistatin are produced in the pituitary where they appear to constitute a regulatory system (21, 22, 23, 24, 25, 26, 27), whereas in vivo studies suggest that inhibin control of FSH is primarily of gonadal origin (28). Activins stimulate FSH secretion and increase FSHß mRNA levels (13, 29, 30), whereas follistatins and inhibins produce the opposite effect (12, 13, 31, 32). Follistatin is believed to decrease FSH by binding to and neutralizing activin (33), and inhibin may be a competitive antagonist of the activin receptor (34).

The established regulators of pituitary follistatin include GnRH (18, 35) and activin (11, 23, 26), which up-regulate, and inhibin, follistatin, and testosterone (36, 11, 25), which decrease follistatin. FSH itself increases follistatin mRNA levels in granulosa cells (37). The present results indicate that PACAP also rapidly increases follistatin mRNA levels. Although a cause-and-effect relationship was not proven, the magnitude of the reciprocal changes in follistatin and FSHß mRNA levels observed as a function of time of incubation in PACAP-treated pituitary cell cultures (Fig. 1Go) suggests that increased follistatin production accounts for the decrease in FSHß mRNA levels. PACAP receptors are found on folliculostelate cells and on a subpopulation of gonadotropes (38), and both cell types appear to produce follistatin (9). Further studies are needed to identify the cell type(s) that contribute to FSHß mRNA suppression by PACAP.

The reason PACAP was more effective at 8 h than at 24 h is unknown but could relate to less activin when the medium was changed in the static cultures (39), to degradation of PACAP during incubation, or to changes in PACAP receptors or signaling with prolonged stimulation (desensitization). In this regard, treatment of ovine pituitary cell cultures with PACAP for 3 days was without effect on follistatin protein production (40).

This is the first report to examine the transcriptional regulation of the follistatin promoter by hormones. We found that this gene is stimulated in {alpha}T3–1 cells by PACAP, but not by activin or by GnRH, two other peptides that are known to increase follistatin mRNA levels (11, 18, 23, 26, 35). The low EC50 for PACAP-stimulated follistatin transcription is a consequence of the high affinity binding of PACAP to PVR1 (41). Dow et al. (42) reported that the concentration of PACAP38 in rat hypophysial portal plasma is 0.05 to 0.1 nM. Thus, the doses of PACAP38 used in this study are physiolgically relevant.

Miyanaga and Shimasaki (43) have sequenced the proximal 5' flanking region of the rat follistatin gene prepared by PCR from ovarian RNA (43). Among the motifs potentially recognized by established transcription factors is one imperfect palindromic cAMP response element (CRE)-like response element (TGACGTCC) positioned at -4/+4, a sequence similar to an AP-1 site (TGATTCA) at -159/-153, and four potential SP-1 sites. In their studies (43) both forskolin and TPA activated the follistatin promoter in granulosa cells and were additive stimulators, suggesting that the protein kinase A and protein kinase C signal transduction pathways were operative. Forskolin and TPA also increase follistatin mRNA levels in granulosa cells (44), renal mesangial cells (45), and in pituitary cell cultures (11).

In light of these findings, PACAP, which increases cAMP and inositol triphosphate production in {alpha}T3–1 cells (15), was predicted to transcriptionally regulate the follistatin gene. Indeed we confirmed this hypothesis but found that only the cAMP-dependent PKA pathway reproduced the transcriptional response of the follistatin gene to PACAP in {alpha}T3–1 cells. Our results differ from those of Miyanaga and Shimasaki (43) who identified 2-fold increases in promoter activity with both forskolin and PMA in primary granulosa cell cultures transfected with plasmids containing the rat follistatin promoter sequences fused to a CAT reporter gene. A recent study of the gonadotropin {alpha}-subunit promoter in {alpha}T3–1 cells (46) has suggested that the transcriptional actions of PACAP on this gene are also mediated largely by the cAMP/PKA pathway in the gonadotrope-derived cell line. Further studies in normal gonadotropes are needed.

The variant monomeric CRE located at -4/+4 of the follistatin promoter may mediate PACAP-stimulated transcription through a cAMP-responsive element binding protein (47), although the location of this motif overlaps a potential transcription start site. An alternative possibility is that PACAP-stimulated cAMP production regulates other factors that, in turn, activate the follistatin promoter. For example, the genes encoding cFos and cJun are increased by PACAP in several cell types (48, 49, 50), and the AP-1 motif (Fos/Jun binding site) of the human interstitial collagenase gene functions as a CRE as well as a phorbol ester response element in PACAP-responsive PC12 pheochromocytoma cells (51). Mutagenesis of these elements should begin to clarify these various possibilities.

PKC activation can potentiate or inhibit adenylate cyclase activity in various cells (52). In the present study, the phorbol ester PMA was found to antagonize PACAP activation of the follistatin promoter in {alpha}T3–1 cells. This result is consistent with the finding by McArdle et al. (53) that PMA inhibits PACAP-stimulated cAMP accumulation in {alpha}T3–1 cells and may result from lack of expression of {gamma}-PKC (54), a PKC isoform that is directly coupled to the potentiation of cAMP formation (55). Inhibition of follistatin transcription by PMA was PACAP receptor-specific since PMA did not block forskolin activation of the follistatin promoter. The detailed mechanism for, and significance of, this signal cross-talk in the control of follistatin gene expression by PACAP remains to be determined.

The mechanism(s) by which GnRH and activin increase follistatin mRNA in the rat pituitary are uncertain and may be interrelated (27). A promoter containing the sequences -757 to +139 was employed in the present studies in which neither hormone stimulated transcription. It is possible that absence of PKC isozymes ({gamma}-PKC) or transcription factors in {alpha}T3–1 cells explains the lack of promoter activity, that upstream sequences not present in the construct tested mediate transcriptional actions of GnRH and activin, that other pituitary cells are necessary for the effects of GnRH or activin, or that the actions of GnRH and activin are nontranscriptional. Regardless of the explanation, the mechanism by which GnRH and activin up-regulate follistatin mRNA appears to be distinct from that of PACAP.

The physiological importance of PACAP in the regulation of gonadotropin synthesis and secretion is presently unknown. PACAP is expressed in parvocellular neurons of the paraventricular nucleus (1) and is a presumed hypophysiotropic factor (1, 2). Because PACAP increases the sensitivity of gonadotropes to GnRH (4, 5), stimulates {alpha}-subunit transcription, and lengthens and presumably prolongs the half-life of LHß mRNA (5) while suppressing FSHß mRNA (Ref. 5 and present findings), it is tempting to propose that PACAP plays a role in the differential control of LH and FSH. PACAP could also contribute to the spike in follistatin mRNA that occurs in proestrus in the rat (24). Finally, the PACAP gene is expressed in various tissues including the brain, adrenals, and gonads (1), which also express follistatin (56, 57), and it is intriguing to propose that PACAP stimulates follistatin transcription and thereby neutralizes activin in tissues other than the anterior pituitary.


   Acknowledgments
 
The authors wish to acknowledge the expert technical assistance provided by Ms. Joyce Szczepanski and Mr. Dushan Ghooray. We thank Dr. Barbara Attardi for technical advice, and Dr. William H. Walker for his comments on the manuscript.


   Footnotes
 
1 This research was supported in part by NIH Grant RO1-HD-19546. A portion of the data was presented at the 10th International Congress of Endocrinology, San Francisco, June 1996, Abstract P1–399. Back

2 Present address: Department of Urology, Tokyo Medical and Dental University, Tokyo, Japan. Back

Received April 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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