Transcriptional Activation of the Ovine Follicle-Stimulating Hormone b-Subunit Gene by Gonadotropin-Releasing Hormone: Involvement of Two ActivatingProtein-1-Binding Sites and Protein Kinase C*

BRIAN D. STRAHL, HUEY-JING HUANG, JOSEPH SEBASTIAN,BASAVDUTTA R. GHOSH†, AND WILLIAM L. MILLER

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

ABSTRACTFSH is an a/b heterodimeric glycoprotein, the formation of which

is regulated primarily by expression of its b-subunit. Recent studieson transcriptional regulation of the ovine FSH b-subunit gene(oFSHb) have defined two functional activating protein-1 (AP-1) en-hancers in the proximal promoter (located at 2120 and 283 bp) thatare probably physiologically important for FSHb expression. AsGnRH is a major regulator of FSHb expression and is also known tostimulate the synthesis of Jun and Fos family members (AP-1), weinvestigated the possibility that oFSHb transcription may be regu-lated by GnRH through AP-1. Here we report the use of an in vitrocell system involving transient transfection of GnRH receptors (Gn-RHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHb. This system was used to show thatexpression of luciferase constructs containing either the 24741/1759region of the oFSHb gene (24741oFSHb-Luc) or the 2846/144 regionof the human a gene (a-Luc; a positive control) was stimulated 3.1 60.3- and 7.7 6 1.9-fold, respectively, by 100 nM GnRH. Another lu-ciferase expression plasmid containing the Rous sarcoma virus pro-moter (a negative control) showed no response to GnRH. Similarresults with these constructs were obtained in COS-7 cells. Studieswith progressive 59-deletion constructs and site-specific mutationsdemonstrated that this stimulation was dependent on each AP-1 site

in the proximal promoter of oFSHb. Gel shift assays demonstrated theability of GnRHR in HeLa cells to increase AP-1 binding activity.Responses in the HeLa cell system were dependent on GnRH (ED505 0.5 nM) and GnRHR, which was identified by photoaffinity labeling.In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as proteinkinase C (PKC) is a known target of GnRH action in gonadotropes, therole of PKC in transcriptional regulation of oFSHb and a-subunitgenes by GnRH in HeLa cells was investigated. Although 12-O-tet-radecanoyl 13-acetate induction of a-Luc and 2215oFSHb-Luc couldbe completely blocked in a dose-dependent manner by the specificPKC inhibitor bisindolylmaleimide I, only 57–65% of the GnRH-mediated stimulation of these promoters was blocked, demonstratingthe involvement of PKC as well as other signaling systems in GnRHinduction. These data define a molecular action of GnRH on oFSHbgene transcription that involves two proximal AP-1 enhancer ele-ments and PKC activation. Furthermore, these studies establish theusefulness of HeLa and COS-7 cells to investigate specific aspects ofGnRH action on gonadotropin subunit gene expression, as similarsignaling pathways and transcription factors that are activated byGnRH in gonadotropes (such as PKC, mitogen-activated protein ki-nase, Ca21, and AP-1) exist in these cells. (Endocrinology 139: 4455–4465, 1998)

FSH IS AN a/b heterodimeric glycoprotein hormone nec-essary for folliculogenesis and the female reproductive

cycle. It is produced, along with LH, in gonadotropes of theanterior pituitary. The a-subunit in FSH is also common toLH, but the biological activity of each hormone is defined byits unique b-subunit (1). As expression of the b-subunit forFSH is the primary rate-limiting step in overall FSH pro-duction, those factors controlling FSHb synthesis are the keyregulators of gonadal and reproductive function.

One primary regulator of FSHb expression in vivo isGnRH. This decapeptide hormone is released from the hy-pothalamus and binds a specific G protein-coupled receptor

(GnRHR) on plasma membranes of gonadotropes to activateintracellular signals that lead to gonadotropin secretion andincreased transcription of all gonadotropin subunits (1–3).Evidence is now emerging that GnRH induces and/or acti-vates components of the AP-1 transcriptional complex,which usually consists of Jun/Jun homodimers or Jun/Fosheterodimers that bind target genes to increase their expres-sion. It has been shown that GnRH administration to cul-tured rat pituitary or immortal aT3–1 gonadotrope cellscauses a rapid increase in the messenger RNAs (mRNAs) forthe protooncogenes c-jun, junB, and c-fos (4). Additionally,pulsatile administration of GnRH to ovariectomized hypogo-nadotropic (GnRH-deficient) lambs was shown to increasepituitary mRNAs for c-jun and c-fos (5). This same studydemonstrated that pituitary levels of c-jun mRNA increased2- to 3-fold just before the preovulatory LH surge, stronglysuggesting that dynamic changes in activating protein-1(AP-1) transcriptional complexes were occurring in gonado-tropes throughout the reproductive cycle. Finally, it has beenshown that GnRH administration to perifused rat pituitarycells activates protein kinase C (PKC), and PKC is a knownactivator of c-jun (6, 7). These data, taken together, imply an

Received June 5, 1998.Address all correspondence and requests for reprints to: Dr. William

L. Miller, Department of Biochemistry, Box 7622, North Carolina StateUniversity, Raleigh, North Carolina 27695-7622.

* This work was supported by the North Carolina State UniversityAgricultural Research Service, NICHD Grant 34863, and the MellonFoundation.

† Present address: Division of Reproductive Toxicology, U.S. Envi-ronmental Protection Agency, Research Triangle Park, North Carolina27711.

0013-7227/98/$03.00/0 Vol. 139, No. 11Endocrinology Printed in U.S.A.Copyright © 1998 by The Endocrine Society

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important role for AP-1 transcriptional complexes in gona-dotropes and, further, suggest the possibility that GnRHaction on gonadotropin subunit gene expression may includemechanisms involving AP-1.

Recent studies with the ovine FSHb gene (oFSHb) uncov-ered two functionally linked AP-1 sites in the proximal pro-moter, lending strength to a hypothesis that FSHb gene tran-scription in vivo is regulated at least in part through Jun andFos family members (8). Additionally, these studies providedthe first evidence that the proteins for Jun and Fos familymembers are directly expressed in a majority of gonado-tropes in vivo, demonstrating that AP-1 is present to poten-tially act on the oFSHb gene. Working on the hypothesis thatGnRH is an important regulator of FSHb, and that AP-1 maybe a component of GnRH action on FSHb gene transcription,we engineered an in vitro cell assay system to analyze reg-ulatory elements involved in GnRH action on the FSHb gene.In this report we use such a system to demonstrate thattranscriptional activation of the oFSHb gene by GnRH inHeLa cells involves each of the previously characterizedAP-1 sites in the oFSHb gene and, further, show that thisstimulation partially involves PKC activation. HeLa cellswere used as a model system because there are no FSHb-producing cell lines available and because all transformedpituitary cell lines known to date poorly express oFSHbpromoter/luciferase constructs. These data are the first tocharacterize a mechanism of action of GnRH at the level ofFSHb promoter sequence and provide a direct physiologicalrole for GnRH-induced AP-1 transcriptional complexes ingonadotropes.

Materials and MethodsPlasmid constructs

Generation of the deletion and site-directed mutation constructs con-taining 59-regions of the ovine FSHb promoter fused to the luciferasegene has been previously described (8). Luciferase expression plasmidscontaining either the 2846/144 region of the human a-subunit pro-moter or the Rous sarcoma virus (RSV) promoter (a-Luc and RSV-Luc,respectively) (9) were provided by Dr. J. Larry Jameson (Department ofMedicine, Northwestern University, Chicago, IL). The cytomegalovirus(CMV) mouse GnRHR expression plasmid (10) was a gift from Dr. StuartC. Sealfon (Mt. Sinai School of Medicine, New York, NY). The RSV-b-galactosidase expression construct (pRSVbgal), an internal control forthese studies, has been described previously (8).

Cell culture and transient transfection

HeLa and COS-7 cells were obtained from the American Type CultureCollection (Manassas, VA). aT3–1 cells were provided by Dr. Pamela L.Mellon (University of California-San Diego, La Jolla, CA). All trans-formed cells in this study were grown at 37 C in DMEM (Life Tech-nologies, Grand Island, NY) containing 10% FBS (HyClone Laboratories,Inc., Logan, UT) under 95% air-5% CO2. Cells used for transfection weregrown in 150-cm2 flasks until they were confluent and then were re-plated in six-multiwell plates (diameter, 35 mm/well) at a concentrationof 300,000 cells/well the day before transfection. Unless otherwise in-dicated, HeLa cells were transiently transfected in triplicate with 8 mgreporter construct, 1 mg GnRHR, and 10 mg pRSVbgal (total volume, 0.5ml) using the calcium phosphate method (11). COS-7 cells were trans-fected in the same way as HeLa cells, except that 6 mg reporter constructand 4 mg pRSVbgal were used. Eighteen hours after the start of trans-fection, the precipitates were removed and replaced with low serummedium (0.5% FBS). Six hours later, cells were treated with either 100nm GnRH (Sigma Chemical Co., St. Louis, MO) or 10 nm 12-O-tetra-decanoylphorbol 13-acetate (TPA; Calbiochem-Novabiochem Interna-

tional, La Jolla, CA). For transfection studies involving the PKC inhibitorbisindolylmaleimide I (BIM; also known as GF 109203X; Calbiochem-Novabiochem International), HeLa cells were pretreated with the indi-cated concentration of BIM 15 min before the addition of GnRH or TPA.Cells were harvested for determination of luciferase and b-galactosidaseactivities 12 h after GnRH or TPA treatment. Luciferase and b-galacto-sidase activity assays from transient transfections were performed aspreviously described (8).

Normalization and statistical analysis of the transienttransfection data

Activity in the internal control b-galactosidase expression construct,pRSVbgal, was used to normalize the transfection efficiency in HeLa orCOS-7 cell culture studies. The transfected gene for b-galactosidasetypically showed 5- to 10-fold higher activity than the endogenousb-galactosidase gene in all transfections. Variation in transfection effi-ciency was normalized by dividing the b-galactosidase values directlyinto the luciferase values from control and GnRH- or TPA-treated cul-tures. Expression of the pRSVbgal construct under GnRH treatment wasenhanced 1.5- to 1.8-fold in these experiments. Because the expressionsof RSV-Luc, 284oFSHb-Luc, and 2120/283 mut FSHb-Luc were in-creased to the same degree as that of pRSVbgal, this slight enhancementwas judged to be a nonspecific effect caused by GnRH. Thus, althoughthe negative control RSV-Luc and deletion/mutation oFSHb-Luc con-structs listed above were normalized to about 1.0 (no stimulation), atypical 5.0- to 6.0-fold increase in 2215oFSHb-Luc expression was nor-malized to 3.2- to 3.8-fold.

To combine data from independent experiments, the basal expressionfor each construct in each experiment was averaged and then assigneda relative value of 1 (see figures). Induction ratios (fold induction) wereobtained by dividing the average value for the GnRH-induced or TPA-induced reporter by the average basal expression of the same reporter.Differences between means obtained for basal expression, stimulatedGnRH or TPA expression, and induction ratios of all luciferase expres-sion constructs were determined using ANOVA followed by Tukey’smultiple range test for individual comparisons (12).

Measuring intracellular calcium ([Ca21]i)

HeLa cells were transiently transfected as described above, using 1 mgGnRHR and 18 mg Bluescript or Bluescript alone (19 mg). Eighteen hoursafter transfection, HeLa cells as well as aT3–1 cells were plated intofour-well Nunc chamber slides (Fisher Scientific International, Inc., Pitts-burgh, PA; catalog no. 1256517) and cultured 24 h before treatment with100 nm GnRH. According to protocols provided by Molecular Probes,Inc., cells were loaded with 4 mm indo-1/AM, washed in HBSS (1 mmCa21), and mounted on an ACAS 570 Interactive Laser Cytometer (Me-ridian Instruments, Inc., Ann Arbor, MI). Two-dimensional spatial flu-orescent 10-sec image scans were collected before and after GnRH treat-ment. Intracellular calcium concentrations ([Ca21]i) were determinedusing quantitative ratiometric fluorescence analysis. The total number oflines in each graph corresponds to the total number of cells in the fieldof analysis. Each line in the graph represents the change in [Ca21]i withina single cell.

Photoaffinity labeling of GnRHR

HeLa cells were transiently transfected as described above using 1 mgGnRHR and 18 mg Bluescript or Bluescript alone (19 mg). TransfectedHeLa and aT3–1 cells used for photoaffinity labeling studies were har-vested and stored at 280 C before analysis. HeLa cells (3 3 106) or aT3–1cells (9 3 106; equal cell pellet sizes) were incubated with 100,000 cpm[125I]GnRH-N-hydroxysuccinimidyl-4-azido-benzoate (13) at 4 C for 4 hand then exposed to a mercury lamp for 5 min to covalently link theradiolabeled ligand to the GnRHR. Radiolabeled GnRHR was fraction-ated using 10% SDS-PAGE. Gels were fixed, dried, and visualized by aPhosphorImager (model 445 SI, Molecular Dynamics, Inc., Sunnyvale,CA).

Electrophoretic mobility shift analysis

To analyze induction of nuclear proteins by GnRH, HeLa cells werestably transfected with the GnRHR expression plasmid. Twenty micro-

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grams of BglII-linearized CMV-GnRHR were transfected as describedabove, and HeLa cells expressing CMV-GnRHR were selected usingG418 (Mediatech, Herndon, VA). Individual HeLa cell colonies wereisolated, expanded, and analyzed for GnRHR expression by assaying forGnRH induction of transiently transfected 2215oFSHb-Luc.

Micropreparations of nuclear protein extracts from stably transfectedHeLa (HeLa-Rec) or aT3–1 cells were prepared as described by Andrewsand Faller (14), using 1 3 106 HeLa-Rec or 3 3 106 aT3–1 cells. Beforeharvesting, cells were incubated for 12 h in low serum medium and thenwere treated with or without 100 nm GnRH for 1 h. Binding reactionswith HeLa-Rec or aT3–1 nuclear extract were performed by mixing 8 mgnuclear 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], 0.75 mg poly(dI-dC) (Boehringer Mannheim, India-napolis, IN), and 0.4 ng (13,000–14,000 cpm) end-labeled AP-1 consensus(Promega Corp., Madison, WI) at room temperature in 20-ml reactionvolumes. All binding reactions (including those with competitor oligo-nucleotides or antibodies) were carried out simultaneously. For super-shift assays, 0.15 mg anti-Fos rabbit polyclonal antibody (provided by Dr.M. J. Iadarola, NIH, Bethesda, MD) (15), 0.5 mg anti-JunD rabbit poly-clonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 0.5mg nonspecific rabbit IgG (Sigma) were preincubated in the bindingreactions for 15 min at room temperature before the addition of radio-labeled probe. The anti-Fos antibody is directed to residues 128–152 ofhuman c-Fos and recognizes all of the Fos family members (c-Fos, FosB,Fra-1, and Fra-2). The affinity-purified rabbit anti-JunD polyclonal an-tibody was directed against residues 329–341 of mouse JunD and wasspecific for JunD only (confirmed by Santa Cruz Biotechnology, Inc.using Western blot analysis). For competition studies, unlabeled AP-1 orSP-1 competitor oligonucleotides (Promega Corp.) were preincubated inbinding reactions at a 75-fold molar excess for 15 min before the additionof radiolabeled probe. After the addition of probe, all binding reactionswere incubated for an additional 25 min at room temperature beforebeing loaded onto 5.6% nondenaturing polyacrylamide gels (1.5 mm),which were run at 200 V for 1.5 h at 4 C in 0.5 3 TBE (0.089 m Tris; 0.089m borate; 0.002 m EDTA). Gels were fixed, dried, and visualized by aPhosphorImager (model 445 SI, Molecular Dynamics, Inc.).

ResultsUsing HeLa cells to study transcriptional regulation ofoFSHb by GnRH

HeLa cells were chosen to study the effects of GnRH onoFSHb-Luc expression because this cell line is well charac-terized and readily expresses oFSHb-Luc constructs, andnone of the pituitary or GnRHR-expressing cell lines tested,such as aT3–1, GH3, RC-4B/C, and GT-1 cells, efficientlyexpresses oFSHb promoter/luciferase expression constructs.To investigate the ability of GnRHR to activate oFSHb genetranscription, increasing amounts of mouse GnRHR expres-sion plasmid were cotransfected along with a luciferase ex-pression construct containing the 2215/1759 region of theoFSHb gene (2215oFSHb-Luc) into HeLa cells that weresubsequently treated with 100 nm GnRH for 12 h. As shownin Fig. 1A, the promoter activity of 2215oFSHb-Luc in-creased with increasing amounts of cotransfected GnRHR. Amaximal response of 3.8 6 0.8-fold was obtained with 1 mgGnRHR and remained constant with up to 4 mg GnRHR(3.5 6 0.5-fold induction). Similar induction of 2215oFSHb-Luc by GnRH was observed as early as 6 h and as late as 18 h(data not shown). Stimulation by GnRH was dependentupon cotransfection of GnRHR. The ED50 of GnRH actionwas determined from a dose-response study using 1 mgGnRHR. As Fig. 1B shows, increasing amounts of GnRHresulted in a dose-dependent increase in 2215oFSHb-Lucpromoter activity with an ED50 of 0.5 nm. It is notable that this

response is in the range of normal GnRHR action in gona-dotropes (4, 16).

GnRHR action in gonadotropes results in a rapid andtransient mobilization of intracellular calcium. To determinewhether GnRHR-expressing HeLa cells would mobilize cal-cium as normally observed in gonadotropes, calcium mobi-lization studies were performed. As shown in Fig. 1C, ad-dition of 100 nm GnRH to HeLa cells transiently expressingGnRHR resulted in mobilization of intracellular calcium sim-ilar to that observed in aT3–1 cells. More than three inde-pendent experiments showed similar results with calciumresponses in aT3–1 and GnRH-responsive HeLa cells typi-cally ranging from 0.2–3.5 mm after GnRH treatment. Thelevel of calcium mobilized in GnRH-responsive HeLa cells issimilar to that reported for ovine pituitary gonadotropes (17).As only a small population of HeLa cells actually becametransfected with GnRHR using the CaPO4 method (15–30%),only a limited number of cells were expected to be responsiveto GnRH. HeLa cells transfected only with control plasmidDNA (Bluescript) lacked the ability to mobilize intracellularcalcium in response to GnRH. Ionomycin, a calcium iono-phore that permits calcium to flow freely into cells, was usedas a positive control in each study to confirm the integrity ofthe calcium detection system (data not shown).

To explore the physical nature of GnRHR expressed inHeLa cells, GnRHR from transiently transfected HeLa cellswere radiolabeled by photoaffinity labeling methods using[125I]azidobenzoyl-GnRH agonist ([125I]azidobenzoyl-GnRH-A; see Materials and Methods). Mouse GnRHR from aT3–1 cellswas radiolabeled as a positive control, and SDS-PAGE studiesrevealed the presence of a band corresponding to authenticGnRHR at approximately 46 kDa (Fig. 1D, lane 1). The pho-toaffinity labeling of [125I]azidobenzoyl-GnRH-A to GnRHR inaT3–1 cells was specific, as the addition of 100 nm GnRH-A(unlabeled competitor) to the binding reaction prevented for-mation of the radiolabeled band (Fig. 1D, lane 2). HeLa cellstransiently transfected with GnRHR contained a radiolabeledband with an apparent molecular mass of 63 kDa (Fig. 1D, lane4). This band was specific, as 100 nm GnRH-A prevented thephotoaffinity labeling with [125I]azidobenzoyl-GnRH-A (Fig.1D, lane 5). Without the cotransfection of GnRHR, no radiola-beled bands were observed (Fig. 1D, lane 3). To addresswhether the higher molecular mass form of GnRHR in HeLacells was due to differences in glycosylation, we used the re-combinant glycosidase peptide-N-glycosidase F (Oxford Gly-coSciences, Inc., Bedford, MA) to cleave all carbohydrates fromthe GnRHR and found that deglycosylated GnRHR from eithertransfected HeLa or aT3–1 cells exhibited a single major bandat 28 kDa (data not shown). It should be noted that other studieshave shown a similar apparent molecular mass of 57–65 kDafor mouse GnRHR expressed in COS-1 cells and that this re-ceptor binds GnRH with wild-type affinity (18).

GnRH induces 24741oFSHb-Luc and human a-Luc intransformed cell types

The a-subunit gene is known to be GnRH responsivethrough several characterized promoter elements (reviewedin Refs. 19 and 20). To determine whether GnRH-responsiveHeLa cells would support stimulation of the human a-sub-

TRANSCRIPTIONAL ACTIVATION OF oFSHb BY GnRH 4457

FIG. 1. Functional characteristics of a GnRH-responsive HeLa cell system. A luciferase expression construct containing region 2215/1759 ofthe ovine FSHb gene (2215oFSHb-Luc) was transiently transfected along with the CMV-mouse GnRHR (GnRHR) expression plasmid into HeLacells as described in Materials and Methods. A, GnRHR dose-response with the 2215oFSHb-Luc. HeLa cells were transiently transfected with2215oFSHb-Luc along with 0.1, 0.25, 0.5, 1, 2, or 4 mg GnRHR expression plasmid. DNA concentrations in precipitates were kept constant usingBluescript DNA. Upon removal of the DNA precipitates, cells were cultured in low serum (0.5% FBS) for 9 h followed by administration of 100nM GnRH for the final 12 h of incubation. B, GnRH dose response with the 2215oFSHb-Luc. HeLa cells transiently transfected with2215oFSHb-Luc and GnRHR were cultured as described above and treated with 0.01, 0.1, 1, 10, 100, or 1000 nM GnRH for 12 h before harvesting.In each case, basal expression of 2215oFSHb-Luc was assigned (normalized to) a relative value of 1. Values represent the mean 6 SEM from

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unit gene in addition to a 4.7-kb oFSHb promoter construct,luciferase expression constructs containing either the 2846/144 region of the human a-subunit gene (a-Luc) or the24741/1759 region of the oFSHb gene (24741oFSHb-Luc)were assayed for GnRH stimulation as described above. Asa negative control, a luciferase expression construct contain-ing the RSV promoter (RSV-Luc) was also assayed. As shownin Fig. 2, both 24741oFSHb-Luc and a-Luc were stimulated(3.1 6 0.3- and 7.7 6 1.9-fold, respectively) by 100 nm GnRH,whereas RSV-Luc expression was not changed. Similar re-sults with these constructs were observed in COS-7 cells (Fig.2), indicating that this regulation is not restricted to HeLacells. It should be noted that the level of stimulation causedby GnRH in these gonadotropin subunit promoters in HeLaand COS-7 cells are within the ranges observed by others forthese genes both in vivo and in vitro (2, 3, 21, 22). Several othercell lines tested for GnRH stimulation (JAR, T47-D, and NIH3T3), however, did not result in significant levels of24741oFSHb-Luc and a-Luc stimulation (data not shown),suggesting that either factors required for GnRH inductionare absent from these cell types or that GnRHR is not effi-ciently expressed.

Localization of the GnRH-responsive region in theoFSHb gene

A series of progressive 59-deletion constructs of the oFSHbgene fused to the luciferase gene were assayed in the HeLa

cell system to determine the promoter regions associatedwith GnRH induction. As shown in Fig. 3, constructs con-taining deletions from 24741 to 2215 bp were stimulatedbetween 2.8- to 3.2-fold by 100 nm GnRH. An additionaldeletion to 284 bp resulted in a complete loss of GnRHresponsiveness, demonstrating that sequences between2215 and 284 bp are involved in the GnRH-mediated in-duction of oFSHb-Luc. Basal expression of all deletion con-structs was statistically equivalent (P . 0.05) and did notvary by more than 15,000–30,000 relative light units/100 ml(1/10th of the total sample) between individual experiments.

The 2120 and 283 AP-1-binding sites each function toenhance oFSHb-Luc transcription by GnRH

The deletional data described above along with the knowl-edge that GnRH action leads to the induction of AP-1 sug-gested that the GnRH-mediated induction of oFSHb-Lucinvolved the AP-1 sites located at 2120 and 283 bp. Todetermine whether the 2120 and 283 sites played any rolein the induction of oFSHb by GnRH, each site was mutatedin 2215oFSHb-Luc (individually or collectively) and ana-lyzed under GnRH stimulatory conditions as describedabove. As shown in Fig. 4, mutations in either site signifi-cantly reduced (P , 0.05) the level of GnRH activation of2215oFSHb-Luc from 3.2-fold to about 2-fold (50% reduc-tion). Luciferase expression of a construct containing muta-tions at both sites (283/-120 mutFSHb-Luc) was not en-

FIG. 2. Transcriptional activation of the ovine FSHb and human a-subunit genes by GnRH. Luciferase expression constructs containing the24741/1759 region of the oFSHb gene, the 2864/144 region of the human a-subunit gene, or 524 bp of the RSV 39-long terminal repeat weretransiently transfected into HeLa or COS-7 cells along with the CMV-GnRHR expression plasmid as described in Fig. 1. Before harvesting,cells were treated with 100 nM GnRH for 12 h. Luciferase expression plasmids are shown on the left, and the relative luciferase activities observedwith the constructs are shown on the right. Basal expression of all constructs were assigned (normalized to) a relative value of 1. Values representthe mean 6 SEM from three independent transfection experiments (each assayed in triplicate). In parentheses are the induction ratios obtainedfrom those promoters stimulated by GnRH.

at least three independent transfection experiments (each assayed in duplicate). C, GnRH-stimulated [Ca21]i mobilization in aT3–1 andGnRHR-expressing HeLa cells. aT3–1 or HeLa cells transiently transfected with either Bluescript or GnRHR expression plasmid were platedin four-well chambers slides and analyzed for GnRH-induced mobilization of intracellular calcium as described in Materials and Methods. Eachline in each graph represents the change in [Ca21]i within a single cell. An arrow on the x-axis marks the time of GnRH addition (100 nM),and asterisks indicate those cells responding to GnRH. The results presented are representative of three independent experiments. D,Visualization of GnRHR in aT3–1 and GnRHR-expressing HeLa cells. aT3–1 and HeLa cells transiently transfected with either Bluescript orGnRHR expression plasmid were photoaffinity labeled with [125I]azidobenzoyl-GnRH-A and analyzed by SDS-PAGE as described in Materialsand Methods. Lanes 1 and 4 show the migration of photoaffinity-labeled GnRHR in either aT3–1 or GnRHR-expressing HeLa cells. Lanes 2and 5 show the effects of 100 nM unlabeled GnRH on the photoaffinity labeling of GnRHR in either aT3–1 or HeLa cells. Arrows on the leftof the data denote the calculated sizes (molecular masses) of photoaffinity-labeled GnRHR.

TRANSCRIPTIONAL ACTIVATION OF oFSHb BY GnRH 4459

hanced by GnRH, demonstrating that both sites are essentialfor GnRH induction. To determine whether any up-streamregions (.215 bp) in the oFSHb gene possessed functionalelements that might be contributing to the GnRH induction,the 2120 and 283 sites were mutated in 24741oFSHb-Lucby site-directed mutagenesis and assayed for GnRH respon-siveness as described. The results of these studies paralleledthose observed with the 2215oFSHb-Luc mutation con-structs, indicating that the two AP-1 sites in the oFSHb geneare the only GnRH-responsive elements that can be detectedin HeLa cells (data not shown). Basal level expression of allconstructs containing wild-type or mutated oFSHb se-

quences did not vary significantly between individualexperiments.

GnRH increases AP-1-binding activity in aT3–1 andGnRHR-expressing HeLa cells

To determine whether GnRH increased AP-1-associatedDNA binding in GnRHR-expressing HeLa cells, a stableHeLa cell line expressing GnRHR (HeLa-Rec) was produced,and nuclear extracts from control and GnRH-treated HeLa-Rec cultures were prepared for gel shift analyses employinga radiolabeled AP-1 consensus ([32P]conAP-1; see Materials

FIG. 3. Localization of the GnRH-responsive region in the oFSHb gene. Luciferase expression constructs containing 59-deletions of the oFSHbpromoter were transfected along with the CMV-GnRHR expression plasmid into HeLa cells as described in Fig. 1. Cells were treated with 100nM GnRH for 12 h before harvesting. oFSHb-Luc constructs are shown on the left, and the relative luciferase activities observed with theconstructs 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. Values represent the mean 6 SEM from three independent transfection experiments (each assayed in triplicate).

FIG. 4. Transcriptional activation of oFSHb by GnRH involves both the 2120 and 283 AP-1 enhancer elements. The 2120 and 283 sites in2215oFSHb-Luc were mutated, individually and collectively, and then transfected along with CMV-GnRHR into HeLa cells as described inFig. 1. Cells were treated with 100 nM GnRH for the final 12 h of incubation. Luciferase expression constructs containing wild-type or mutatedoFSHb sequences are shown on the left and the relative luciferase activities observed with these constructs are shown on the right. Basalexpression of all constructs tested did not vary significantly (P , 0.05) and were assigned (normalized to) a relative value of 1. Values representthe mean 6 SEM from three independent transfection experiments (each assayed in triplicate). Means that share asterisks are not significantlydifferent from each other (P . 0.05).

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and Methods). As a positive control for induction of AP-1binding, nuclear extracts of control or GnRH-treated aT3–1cells were prepared and analyzed along with the HeLa-Recnuclear extracts. As shown in Fig. 5, incubation of[32P]conAP-1 with nuclear proteins from control or GnRH-treated cells resulted in the formation of distinct DNA/protein complexes (lanes 2, 3, 9, and 10) that were not presentin the control lane (lane 1). Comparison of the control vs.GnRH-treated bands (lanes 2 vs. 3 and lanes 9 vs. 10) revealeda 4.5-fold increase in AP-1 binding in aT3–1 nuclear extractsand a 2.2-fold increase in AP-1 binding in HeLa-Rec nuclearextracts. Three independent experiments showed similar re-sults, with increases in AP-1 binding ranging from 2- to 3-foldin HeLa-Rec nuclear extract after 1 h of GnRH treatment.Similar increases in AP-1-associated DNA binding were alsoobserved when either the 2120 or 283 oFSHb AP-1 siteswere radiolabeled and analyzed by gel shift analysis usingthe extracts described above (data not shown). Increases in[32P]conAP-1 binding in GnRH-treated HeLa-Rec were seenas early as 30 min after GnRH treatment and lasted as longas 4 h before returning to control levels (data not shown). The[32P]conAP-1 shift observed with these extracts was specific,as competition with a 75-fold molar excess of unlabeled AP-1,but not a SP-1 competitor oligonucleotide, prevented theformation of DNA-protein complexes (compare lanes 4 vs. 5and lanes 11 vs. 12). Additional Western blot analyses em-ploying an anti-Fos antibody showed that the increase inAP-1 binding in both aT3–1 and GnRHR-expressing HeLacells paralleled increases in the proteins for various Fos fam-ily members, indicating that the increased AP-1 binding ac-tivity is due at least in part to induction of AP-1 familymembers (data not shown).

To address whether Jun and Fos family members wereassociated with the DNA-protein complexes formed underGnRH-treated cells, anti-Fos or anti-JunD antibodies were

incubated with the nuclear extracts from GnRH-treated cellsalong with [32P]conAP-1 before fractionation on a polyacryl-amide gel. As shown in Fig. 5 (lanes 6, 7, 13, and 14), theanti-Fos and anti-JunD antibodies were each able to super-shift the DNA-protein complexes formed under GnRH-treated aT3–1 and HeLa-Rec cells, whereas nonspecific rab-bit IgG could not (lanes 8 and 15). The Fos antibody, inaddition to supershifting, showed the ability to prevent theformation of DNA-protein complexes (Fig. 5, lanes 7 and 14).Additional supershift experiments showed that the DNA-protein complexes formed from control extracts also con-tained JunD and Fos proteins (data not shown).

Transcriptional activation of oFSHb-Luc and a-Luc ismediated in part through PKC

The role of PKC in GnRH-induced activation of2215oFSHb-Luc and a-Luc in HeLa cells was investigatedusing a highly specific inhibitor of PKC activation, BIM (alsoknown as GF 109203X). As a positive control of PKC inhi-bition, varying concentrations of BIM (from 1 nm to 2 mm)were administered to HeLa cells transfected with2215oFSHb-Luc or a-Luc before the addition of 10 nm TPAfor 12 h. As shown in Fig. 6, A and B, increasing concentra-tions of BIM completely blocked a TPA-mediated PKC re-sponse (in a dose-dependent fashion) for both the oFSHb anda-subunit genes. Under similar experimental conditions,GnRH induction of either a-Luc or 2215oFSHb-Luc wasreduced by 57% and 65%, respectively, by BIM (Fig. 6, C andD), suggesting that GnRH is targeting PKC in addition toother signaling pathways to stimulate gonadotropin subunitgene transcription in HeLa cells. Similar results were ob-tained when another highly specific and structurally differ-ent PKC inhibitor, chelerythrine chloride, was tested (datanot shown).

FIG. 5. Induction of AP-1-binding ac-tivity in GnRHR-expressing HeLa andaT3–1 cells. ConAP-1 was end labeledand incubated with nuclear proteinsisolated from either control or GnRH-treated aT3–1 or GnRHR-stably inte-grated HeLa cells (HeLa-Rec) alongwith poly(dI-dC) before fractionation on5.6% nondenaturing polyacrylamidegels. For competition and supershift as-says, competitor oligonucleotides orJun/Fos specific antibodies were prein-cubated in the binding reaction 15 minbefore the addition of radiolabeledprobe. Lane 1 shows the migration offree [32P]conAP-1, and lanes 2 and 9show the distinct DNA/protein shiftscaused by control aT3–1 or HeLa-Recnuclear proteins. Lanes 3 and 10 showthe increases in AP-1 binding after theaddition of 100 nM GnRH for 1 h. Lanes4–8 and 11–15 show the effects of var-ious competitor oligonucleotides or Jun/Fos antibodies on the formation or mi-gration of the [32P]conAP-1/proteincomplexes from GnRH-treated cul-tures. Arrows to the left of the data de-note the positions of the shifted, super-shifted, free, and bound [32P]conAP-1.

TRANSCRIPTIONAL ACTIVATION OF oFSHb BY GnRH 4461

Discussion

Recent studies have identified two functionally linked AP-1-like enhancer elements in the proximal promoter of theovine FSHb gene (8). These sites, located at 2120 and 283 bp,bind c-Jun and Fos proteins in vitro as well as mediate aphorbol ester (TPA) response in cell culture. Because 75–85%of ovine gonadotropes possess Jun/Fos family members invivo, the data strongly suggest that these AP-1 sites are im-portant for expression of the oFSH b-subunit (8). One reg-ulator of FSHb expression that may mediate its effectsthrough AP-1 is GnRH. Not only has it been shown thatGnRH can increase the rate of transcription of the FSHb genein rat pituitaries in vivo, but GnRH also induces AP-1 familymembers in both cultured rat pituitary cells and ovine pi-tuitaries in vivo (4, 5). These data, taken together, suggest thatthe regulation of FSHb gene transcription by GnRH is likelyto involve mechanisms including AP-1.

To determine whether GnRH regulation of oFSHb genetranscription involves the two oFSHb AP-1 sites, a GnRH-responsive HeLa cell system was established through tran-sient expression of mouse GnRHR along with oFSHb pro-

moter/luciferase constructs. These cells responded to GnRHby inducing oFSHb-Luc expression 2.8- to 3.8-fold when theconstructs contained both AP-1 sites. Site-directed mutationof either the 2120 or 283 sites resulted in a significantreduction of GnRH stimulation (50%), and a double muta-tion abolished all GnRH responsiveness. These results dem-onstrated that each site independently promotes GnRH-stimulated transcription.

Although the data presented here show that both sitesfunction independently under GnRH, previous data dem-onstrated that each site is required for c-Jun/c-Fos or TPAinduction (8). It is unclear why the mechanism of GnRHregulation differs from that of AP-1 regulation, but severaltheories are suggested. First, it is known that GnRH inducescertain Jun/Fos family members, so it may be that specificcombinations of Jun and Fos induced by GnRH function ateach site independently (4). Binding studies show, for in-stance, that each site has specific capacities to bind differentAP-1 family members (our unpublished data). Further, cer-tain combinations of Jun and Fos family members possessdistinct and separate affinities for an AP-1 consensus, and

FIG. 6. Involvement of PKC in GnRH induction of oFSHb-Luc and human a-Luc. 2215oFSHb-Luc or a-Luc was transiently transfected alongwith the CMV-GnRHR expression construct into HeLa cells. Before harvesting, cells were treated with vehicle, 10 nM TPA, or 100 nM GnRHfor 12 h as described in Materials and Methods. Fifteen minutes before GnRH or TPA treatment, cells were administered vehicle or 1 nM, 10nM, 100 nM, 1 mM, or 2 mM of the PKC inhibitor BIM. A–D show the effects of various concentrations of BIM on either TPA or GnRH inductionof 2215oFSHb-Luc or a-Luc. Values are reported as the fold increase over control and represent the mean 6 SEM from three independenttransfection experiments (each assayed in duplicate). Asterisks indicate means that differ significantly from means obtained from TPA- orGnRH-treated cells in the absence of BIM (P , 0.05).

4462 TRANSCRIPTIONAL ACTIVATION OF oFSHb BY GnRH Endo • 1998Vol 139 • No 11

these combinations possess unique biological activities dif-ferent from those of c-Jun/c-Fos (23, 24). A second hypothesisto explain the differences between AP-1 and GnRH regula-tion may be found in the ability of GnRH to induce non-AP-1proteins that cooperate with Jun or Fos to bind and trans-activate oFSHb through each site. In support of this, datashow that the 2120 and 283 sites bind proteins other thanAP-1 (8). It may be that these proteins, in conjunction withJun or Fos, bind to and activate transcription through eachsite. Finally, it is possible that additional GnRH-responsiveelements/regions exist between sequences 2215 and 1759,allowing each site to cooperate with a yet unknown factor.Theoretical support for this comes from the concept that acomposite region consisting of separate and distinct elementsparticipates in mediating normal GnRH regulation of thea-subunit promoter (reviewed in Refs. 19 and 20). Thus, it ispossible that other elements in the oFSHb gene may also beinvolved in mediating the observed GnRH regulation. Stud-ies are underway to determine which, if any, of the theoriesdescribed above are correct.

The 2120 AP-1 site is conserved in the FSHb genes of allspecies analyzed to date (sheep, cow, pig, human, rat, mouse,and rabbit). This suggests a universal involvement of the2120 site in GnRH induction of most, if not all, mammals. Forthe 283 site to be considered part of the normal GnRHresponse in vivo, one would predict that it should also be acommon feature of all FSHb genes, but the 283 site is con-served only among the ovine, bovine, and porcine species. Inaddition, oligonucleotides coding for the human, rabbit, andrat 283 sites show no ability to compete for AP-1 proteins(our unpublished results). As the rat FSHb gene has beenshown to be transcriptionally activated by GnRH, and tosimilar or higher levels than those reported here, these datasuggest that other sites, in addition to the 2120 site, con-tribute to the GnRH responsiveness in these FSHb genes(2, 21).

Given that the 2120 and 283 sites appear to be importantfor GnRH regulation of oFSHb gene transcription, a com-parison of these elements was made to those identified asbeing involved in GnRH regulation of other GnRH-respon-sive genes. Comparison of the 283 AP-1 site to these ele-ments reveals a striking sequence similarity between the 283site and the pituitary glycoprotein hormone basal element(PGBE) located in the mouse a-subunit promoter at 2342 bp(283: 59-CTTACTAAT-39; PGBE: 59-taCTTAgCTAATta-39;lower case letters in the PGBE represent sequence mismatchesto oFSHb sequences, and underlined letters highlight regionsof high homology) (22). The PGBE acts as a basal enhancerand is reported to bind members of the LIM homeodomainfamily of transcription factors, including LH-2 and mLim-3(22, 25). These factors are thought to bind to the PGBE andcooperate with other GnRH-responsive elements in thea-subunit promoter to promote GnRH induction. Mutatingthe PGBE severely disrupts GnRH responsiveness as well asLH-2 binding (22, 25). As gel shift studies with the 283 AP-1site using HeLa nuclear extract demonstrate the ability of the283 AP-1 site to bind proteins other than AP-1, it may alsobe that the 283 site of oFSHb binds LIM homeodomaintranscription factors. The ability of the 283 site in the oFSHb

gene to bind to and be trans-activated by LH-2 or mLim-3awaits confirmation.

A comparison of the 2120 and 283 AP-1 sites in oFSHbcan be made to the tandem cAMP response elements (CREs)located in the human a-subunit gene, which appear to playa role in GnRH-regulated a-subunit gene transcription(Jameson, J. L., personal communication). Consensus CREsdiffer by only one nucleotide from consensus AP-1 enhancerelements (CRE: TGACGTCA; AP-1: TGAC/GTCA) and it hasbeen demonstrated that both elements can be regulated byJun/Fos as well as CRE-binding factors (24). Because GnRHaction in gonadotropes is known to induce AP-1 transcrip-tional complexes, such regulation by GnRH at the tandemCREs may involve Jun and/or Fos family members. In sup-port of this, studies in our laboratory show that human a-Lucis stimulated 5-fold by c-Jun/c-Fos transcriptional complexesin HeLa cells (unpublished data). Therefore, AP-1 may be acommon regulatory mechanism used by GnRH to stimulatethe a- and FSH b-subunit genes.

Studies with GnRH-responsive HeLa and COS-7 cellsshowed that GnRH could stimulate the promoter activities ofhuman a-Luc and 24741oFSHb-Luc (but not RSV-Luc) tophysiologically relevant levels (approximately 7- and 3-fold,respectively). These data suggest that HeLa and COS-7 cellscontain the required components necessary for proper GnRHsignaling to these gonadotropin subunit genes. This is prob-ably due to the fact that these cell lines possess many of themajor signaling pathways and some of the transcription fac-tors known to be important for GnRH regulation of a-subunitgene transcription, including PKC, mitogen-activated pro-tein kinase members ERK1 and ERK2, as well as Ets familymembers (26–30). In addition, it is well established that HeLaand COS-7 cells contain PKC-inducible AP-1 transcriptioncomplexes, which appear to be important for FSHb tran-scription in gonadotropes. Thus, it is reasonable to concludethat the observed GnRH-induced regulation found in HeLaand COS-7 cells is probably physiologically relevant.

PKC inhibition studies demonstrated the involvement ofPKC in GnRH-mediated induction of both human a-Luc and2215oFSHb-Luc in HeLa cells (57–65% of the GnRH-medi-ated stimulation of these promoters was blocked by BIM).Because these same studies showed that a TPA-induced PKCresponse can be completely blocked by BIM, these data in-dicate that GnRH action in HeLa cells involves PKC as wellas additional signaling mechanisms. It is notable that othershave observed PKC involvement in GnRH-mediated a-Lucexpression in aT3–1 cells (31). It is not clear what additionalsignaling mechanisms are involved in this GnRH induction,although it may include mechanisms involving Ca21/cal-modulin, as an antagonist of calmodulin activity (W-7) com-pletely blocked the GnRH response to oFSHb-Luc (data notshown).

Given that GnRHR-expressing HeLa cells represent a newcell system to study the effects of GnRH, studies were per-formed to characterize their physical and functional nature.Cotransfection studies revealed the ability of GnRHR to ac-tivate 2215oFSHb-Luc transcription in both a dose- andreceptor-dependent manner, suggesting normal GnRHRfunction in these cells. Further investigations to determinewhether GnRHR was targeting gonadotrope-specific signal-

TRANSCRIPTIONAL ACTIVATION OF oFSHb BY GnRH 4463

ing pathways in HeLa cells showed that GnRHR was capableof mobilizing intracellular calcium similar to that observedin aT3–1 and ovine gonadotrope cells. In addition, gel shiftstudies demonstrated the ability of GnRHR in HeLa cells toactivate/induce components of AP-1 as is normally seen inaT3–1 cells, albeit to a lesser extent. These studies, takentogether, suggest that GnRHR is not only expressed at phys-iologically relevant levels in these HeLa cells, but is capableof signaling through pathways that are similar or commonto those used by gonadotropes.

Through photoaffinity labeling studies it was determinedthat mouse GnRHR expressed in HeLa cells was higher inmolecular mass compared with endogenous mouse GnRHRexpressed in aT3–1 cells. Subsequent glycosidase studies,however, revealed that this higher molecular mass was dueto differences in glycosylation between these receptors (datanot shown). Differences in the glycosylation patterns ob-served are probably due to the differences between HeLacells and gonadotropes in their protein secretion and mat-uration pathways. Nevertheless, additional glycoslyation onGnRHR has been shown to not alter normal GnRH bindingand, further, appears to not alter its normal function (18).

Regulation of gonadotropin subunit expression by GnRHin vivo normally requires pulsatile administration of GnRHfrom the hypothalamus, whereas continuous administrationof GnRH is known to prevent stimulation of the LHb andFSHb genes. The studies presented here as well as otherstudies involving cotransfection of GnRHR into GH3 cells(21) did not require pulsatile administration of GnRH forFSHb-Luc stimulation. This may be due in part to the fact thatthe GnRHR expression plasmid is driven by a CMV pro-moter, which continuously produces GnRHR and is notdown-regulated by GnRH. Additionally, it has been shownin rat pituitary cell cultures that continuous GnRH treatmentdramatically increases the level of follistatin, which wouldblock the essential function of activin for FSHb expression(32). The GnRH-responsive HeLa cells are not likely to pro-duce follistatin under GnRH treatment and may not evenproduce activin. In some aspects, the HeLa cell system seemsto be especially valuable in studying FSHb regulation, as itcan be engineered to respond to only one hormone withoutinterference of other pathways normally induced by thatsame hormone in gonadotrope cells.

In summary, we have used a GnRHR-expressing HeLa cellsystem to study the transcriptional regulation of the oFSHbgene and have shown that GnRH-mediated induction ofoFSHb gene transcription involves two AP-1 enhancers andPKC activation. In addition, these studies demonstrate theusefulness of GnRHR-expressing HeLa cells to study theeffects of GnRH on gonadotropin subunit gene expression,as GnRHR signaling in HeLa cells leads to normal mobili-zation of intracellular calcium, activation of PKC, activation/induction of AP-1, and stimulation of a and FSHb promoteractivities. The roles of these AP-1 sites in GnRH regulationof oFSHb gene transcription in vivo are currently under in-vestigation using transgenic mouse models.

Acknowledgments

We thank Drs. Stanko S. Stojilkovic and Robert C. Smart for valuableadvice throughout these studies, Dr. J. Larry Jameson for the human a

and RSV luciferase expression constructs, Dr. Stuart C. Sealfon for themouse GnRHR expression plasmid, Dr. Michael J. Iadorola for gener-ously providing the Fos antibody for the studies, Dr. William L. Flowersfor performing statistical analyses, and Dr. Jean Harry at the NIEHS(Research Triangle Park, NC) for providing access to the Meridian ACAS570 Interactive Laser Cytometer.

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