ORIGINAL PAPER (JJRE Outstanding Young Investigator Award in 2005)

Jpn J Reprod Endocrinol ⓒ2005 by
Japan Society of Reproductive Endocrinology

Prop-1 Activation of Porcine FSHβ Subunit Gene
Requires AT-rich Recognition Sites
Satoko AIKAWA, Takao SUSA, Kousuke KITAHARA, Takanobu SATO, Tetsuo ONO, Kenta SUZUKI,
Michie NAKAYAMA, Takako KATO and Yukio KATO
Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, School of Agriculture, Meiji University,
Kawasaki 214-8571, Japan

Abstract. We have recently cloned the pituitary specific transcription factor Prop-1 as a regulator of the porcine follicle-stimulating hormoneβ (pFSHβ) gene [1]. This study aimed to establish the characteristics of porcine Prop-1. The
deduced amino acid sequence showed considerable diversity in the N-terminal and moderate change in the C-terminal
regions among mammalian Prop-1s except for the homeodomain in the middle region. Prop-1 binds to two thirds of the
5 region of Fd2 located between the −852 and −746 base upstream of the pFSHβ gene, which shows a high AT-content (80%). The transfection assay of the pituitary tumor-derived cell line, LβT2, using the chimera genes fused the
upstream of the pFSHβ gene as a reporter vector, showed that Prop-1 activates FSHβ promoter but not SV40 promoter. The gene expression of Prop-1 during porcine pituitary ontogeny was observed from fetal days 40 through 110, andits postnatal expression increased even further. Thus, this study demonstrated that Prop-1 definitely stimulates the
porcine FSHβ gene by acting on the AT-rich Fd2 region and plays a role in the fetal and postnatal pituitary.

Key words: FSH, Transcription factor, Prop-1, Porcine anterior pituitary, Gonadotropin, Gene regulation

Introduction

itary specific transcription factor, Prophet of Pit-1 (Prop-1)[1]. Since this factor is the gene responsible for an inherited

The synthesis and secretion of gonadotropins (follicle

defect in the dwarf mouse, Ames [14] and a combined pitu-

stimulating hormone, FSH, and luteinizing hormone, LH),

itary hormone deficiency (CPHD) with hypogonadism [15,are restricted to the same cells called gonadotropes in re-16], our findings provide a novel approach and breakthroughsponse to several extracellular signals and physiological re-to a better understanding of the control mechanisms notquirements. Various investigations have revealed that the

only for FSHβ gene expression but also for the etiology ofgene expression of three subunits of gonadotropin express-

dwarf/CPHD.

ing common regulatory factors are involved under different

In this study, we demonstrated that Prop-1 binds to thecontrols. Several approaches have so far succeeded in con-

AT-rich sequence of Fd2, resulting in transactivation of thefirming the molecular mechanisms and transcription factors

FSHβ gene. RT-PCR of pituitary ontogeny demonstrated

governing basal and cell-specific expressions of the glyco-that the transcripts of Prop-1 gene persist throughout theprotein hormone α subunit (α-GSU) and LHβ genes

fetal and postnatal periods[2-6], but relatively little knowledge has been accumulated
about the molecular mechanisms regulating FSHβ gene expression [7-12]. More recently, we have found for the firsttime that one of the Fd2 binding proteins [13] is the pitu-

Materials and methods
１）Sequence analysis
Previously cloned porcine Prop-1 cDNA [1] was prepared

Accepted: August 21, 2005
Correspondence: Yukio KATO, Department of Life Science,
Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki,
Kanagawa 214-8571, Japan
TEL: +81-44-934-7035
FAX: +81-44-934-7035
E-mail: yukato@isc.meiji.ac.jp

from E. coli by the alkaline mini-preparation method. The
fluorescence labeled dye-terminator reaction was employedusing the Big Dye terminator system (Applied Biosystems,
Foster City, CA), according to the instruction manual.

Japanese Journal of Reproductive Endocrinology（2005）10：43-48

43Satoko AIKAWA et al.

２）Electrophoretic mobility shift assay (EMSA)LβT2 cells, which were established from the mouse pituitary tumor [17] and kindly provided by Dr. P. Mellon, were

Construction of porcine Prop-1 cDNA in the pET32a

cultured and used for transfection assay as described previ-

vector (Novagen, Darmstadt, Germany) and the production

ously [1]. Each 5 μl of cultured medium was assayed forand isolation of recombinant Prop-1 are described in our

secreted alkaline phosphatase activity using the Phospha-previous paper [1]. FAM-labeled DNA fragments were pro-Light Reporter Gene Assay System (Applied Biosystems).

duced by PCR using FAM-labeled oligonucleotide 5 -primer. The binding reaction mixture included 100 fmol of FAMlabeled probe DNA (10 ng) and 100 ng of porcine

５）RT-PCR analysis of Prop-1 expression during
porcine ontogeny

recombinant Prop-1 with 250 or 2000 ng poly (dI-dC) in 10

Total RNAs were extracted from the porcine anterior pi-

μl of 10 mM HEPES buffer, pH 7.9, containing 0.4 mM

tuitaries of German Landrace pigs with intact gonads of

MgCl2, 0.4 mM DTT, and 50 mM NaCl, and 4% glycerol wasboth sexes collected at fetal days (f40, f50, f65, f82, f95 andincubated at 30℃ for 30 min. Samples were then subjectedf110), postnatal days (p8, p60, p160 (prepubertal), andto electrophoresis on a 4% polyacrylamide gel as described

p230 (sexually mature)) [18].

in our previous paper [13].

Specific primer sets for porcine Prop-1 and cyclophilin A

The consensus sequence of the paired homeodomain

were synthesized as follows; Prop-1 forward 5 -CTA-

with Q9 in the third α-helix (PRDQ9), ACTAATT-CAGAAACCTCTCTGGCGTAGG-3 , and reverse 5 -TT-

GAATTAGC [14], was used as a control for the EMSA of

GCTTCCTCTGCTTAGCTCTGCG-3 , and cyclophilin A

Prop-1.

forward 5 -TGGTGACTTYACACGCCATAATG-3 , and re-３）Expression vector and reporter vector constructs
Porcine Prop-1 cDNA was ligated into the EcoRI andverse 5 -ATTCCTGGACCCRAAACGCTCC-3 . Amplifications and analysis on agarose gel (2%) were described previously [18].

X h o I s i t e s o f a m a m m a l i a n e x p r e s s i o n v e c t o r,
pcDNA3.1Zeo+ (Invitrogen, Carlsbad, CA). Reporter geneconstructs were generated by ligation of the upstream regions of the porcine FSHβ gene (Fβ3-Basic: −985/+10, ∆Results
１）Nucleotide sequence of Prop-1

Fβ3-Basic: deletion mutant of Fβ3-Basic from −745 to

The nucleotide sequence of the clone revealed the entire−104 bp, Fβ6-Basic: −103/+10) into pSEAP2-Basic (BDsequence of 966 bp, including the open reading frames of

Bioscience Clontech) and Fd2, −852 /−746, into pSEAP2-681 bases of 226 amino acids (DDBJ accession no.

Promoter (BD Bioscience Clontech) (Fd2-promoter).

AB187272). Fig. 1 shows the predicted amino acid se-４）Cell culture, DNA transfections, and reporter
gene assays

quence of porcine Prop-1, consisting of 226 amino acids byalignment with the known amino acid sequences of othermammalian Prop-1s. The central DNA-binding HD and

Fig. 1. Comparison of amino acid sequences of Prop-1. Amino acid sequence of porcine Prop-1 was compared with thoseof human, rat and mouse. Each amino acid identical to that of porcine Prop-1 is indicated by a hyphen (-), andopen spaces represent gaps to optimize homology. Homeodomain is underlined.

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Japanese Journal of Reproductive Endocrinology Vol.10 2005

Prop-1 Activation of Porcine FSHβ Subunit Gene Requires AT-rich Recognition Sites

carboxyl terminus of the activation domain [14] were re-activation by unexpected regulatory elements present inspectively well and moderately conserved, while the amino

the vector construct. The expression level did not increase

terminus of the inhibitory domain showed a considerable

following the transfection of Fd2-Promoter, indicating thatdivergence.

the SV40 promoter did not work properly in combination
with Fd2. Reporter vectors of Fβ3-Basic, ΔFβ3-Basic,

２）In vitro binding assay by EMSA

and Fβ6-Basic as well as the transcriptional activity of

To confirm the binding of Prop-1 to Fd2, EMSA was car-

Prop-1 were examined. Unlike Fβ6-Basic, Fβ3-Basic andried out using 5 subfragments of Fd2 (Fd2-1~Fd2-5) de-

ΔFβ3-Basic showed a significant activation (P<0.01) ofsigned to overlap each other. As seen in Fig. 2, PRDQ9

2.2-fold and 2.5-fold, respectively, by cotransfection withshowed a single shift band even in the presence of 250 ng of

the expression vector of porcine Prop-1.

poly (dI-dC). Fd2-1, -2, and -3 exhibited shift bands and４）RT-PCR analysis of Prop-1 gene expression

binding was not prevented by 2000 ng of poly (dI-dC). In

during porcine pituitary ontogeny

contrast, Fd2-4 showed a shift band that was lost following
2000 ng of poly (dI-dC) (data not shown). In addition,

RNAs prepared from fetal and postnatal porcine anterior

Fd2-1, -2, and -3 mainly showed two shift bands, andpituitaries of both sexes were observed during all periods

Fd2-4 a single one, while no binding at all was observed

examined for both genders, though some changes and sex

only in Fd2-5, which is not AT-rich.

differences were detected (Fig. 4). The level of Prop-1 expression in male fetuses decreased temporally at f82 and３）Transcriptional activation of porcine FSHβ genef110, and showed a slight postnatal increase. In females, theby Prop-1 in LβT2 cells

amounts of Prop-1 transcript kept almost similar levels

Transfection of pSEAP2-Basic and pSEAP2-Promoterwith no marked change during the fetal period. Interesting-with or without expression vectors of porcine Prop-1

ly, the postnatal expression level in females increased mark-showed similar expression level of SEAP gene as that inedly.

LβT2 cells (Fig. 3), indicating the absence of endogenous

CTCAGTTTC CATGGAGCTCATAGTC TACTGGGAGACTCCAGCCAAGGAGCTTAATTAATTAATAAGCTTA
Fd2-5: -780/-746
-746
-852
Fd2-1: -852/-818
ATTAATTGCTCAATTAATAAATATTTG TTAAATCAAT
Fd2-3: -817/-781

TGTTAAATCAATCTCAGTTTCCATGGAGCT
Fd2-4: -792/-763

TAATTAATAAGCTTAATTAATTGC TCAATT
Fd2-2: -832/-803

Fd2-1
Prop-1poly(dIdC) (ng)

250

+
250

+
2000

Fd2-2
250

+
250

+
2000

Fd2-3
250

+
250

+
2000

Fd2-4
250

+
250

Fd2-5
250

+
250

PRDQ9
250

+
250

Fig. 2. Electrophoretic mobility shift assay. Binding between recombinant Prop-1 and FAM-labeled Fd2 subfragments was
separated on a 4% polyacrylamide gel followed by visualization with a fluorescence viewer. Composition of eachbinding mixture is listed above the electrophoretic pattern. Paired homeodomain response element (PRDQ9), ACTAATTGAATTAGC [14], was used as a control for EMSA.

ORIGINAL PAPER

45

Satoko AIKAWA et al.

*

*

*

x2.5

*

x2.2

0.0

1.0

2.0

3.0

4.0

5.0

Relat ive SEAP activity
Fig.3.

f40 f 5 0

MaleTranscriptional activation of FSHβ gene by Prop-1 in LβT2 cells. Porcine Prop-1 cDNA in pcDNA3.1+ vector (closed bar) or pcDNA3.1+ vector alone (open bar) was cotransfected with
pSEAP2-Basic, pSEAP-Promoter, Fd2-Promoter, Fβ6-Basic, ∆Fβ3-Basic, and Fβ3-Basic vectors into LβT2 cells. After 48-h incubation, an aliquot of cultured medium was assayed for secreted alkaline phosphatase activity. Each activity was normalized using that obtained from cotransfection of pcDNA3.1+ vector alone with pSEAP2-Basic. Asterisks show a significant
difference (P<0.01) compared with the values with Prop-1-pcDNA3.1 and pcDNA3.1+ vector
alone.

f65

f82

f95

f110

p8 p60 p160 p230

itary organogenesis, and an elevated expression postnatally,
indicating that Prop-1 participates in pituitary developmentby acquiring a capacity for hormone production and maintaining the mature pituitary function.
Our examination of sub-fragments of Fd2 showed that
Prop-1 binds to four of five of them (Fig. 2). It is noteworthy that the binding of Prop-1 showed two shift bands, oneof which corresponded to the shift band of PRDQ9 known

Femalef40 f 5 0

f65

f82

f95

f110

p8 p60 p160 p230

to be bound with two molecules [14], indicating a binding of
one or two Prop-1 molecules. In addition, these sub-fragments are AT-rich and contain a core motif TAAT/ATTA ofthe binding site for homeodomain transcription factor [19],except for Fd2-4, which has no core motif and shows only aweak binding affinity.
Transfection assay of LβT2 cells showed a significant
transcriptional activation of the SEAP gene (Fig. 3). It is

Fig. 4. Ontogeny of porcine Prop-1. Total RNAs prepared from fetal
and postnatal porcine pituitaries of both sexes were analyzed
by RT-PCR. Amounts of cDNA sample, which produce thesame amounts of PCR product for cyclophilin A (shown inlower panel), were subjected to PCR reaction for Prop-1.

noteworthy, that the fusion of Fd2 to the SV40 promoter did
not work, whereas the region −100 bp upstream of porcine
FSHβ gene clearly acted as a promoter for transcriptional
activation by Fd2, indicating the importance of the combination of the endogenous promoter with Fd2. In addition, Fd2

Discussionfunctioned as a cis-acting element in both Fβ3-Basic and ∆Fβ3-Basic, indicating its enhancer activity.

In this study we demonstrated that Prop-1 acts as a tran-

Prop-1 has been an indispensable transcription factor for

scription factor for the FSHβ gene by binding to the multi-

the development of Pit-1 lineage hormone-producing cells

ple binding sites of an AT-rich region about −800 bp up-[14]. During the ontogeny of murine Prop-1 gene expres-stream of the porcine FSHβ gene. In addition, RT-PCR

sion, the Prop-1 transcript could first be detected as early

revealed a continuous Prop-1 gene expression during pitu-

as embryonic day 10 (e10) when Rathke s pouch is devel-

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Japanese Journal of Reproductive Endocrinology Vol.10 2005

Prop-1 Activation of Porcine FSHβ Subunit Gene Requires AT-rich Recognition Sites

oping. Prop-1 gene expression reached its maximum levelby e12, followed by a decline to an extremely low level by
e14.5 with the distribution of the dorsal/ventral gradient[14]. During anterior pituitary development, the differentiation of hormone-producing cell types takes place in a temporal sequence in distinct regions. In the murine anterior
pituitary, both LH- and FSH-producing cells finally appear
last of all at e16.5 and e17.5 in the ventral region where theexpression level of Prop-1 is low [20]. Thus, a spatially andtemporally unique expression of Prop-1 at an early stage isessential for the determination and differentiation of pituitary hormone-producing cells. However, we demonstrated
by RT-PCR of porcine pituitary ontogeny that the Prop-1
gene is expressed throughout the fetal (from f40 through f110)
and postnatal periods. We have also observed that Prop-1
mRNAs are present at high levels in the pituitary tumorderived cell lines LβT2 and LβT4 (unpublished observation). Our data suggested that Prop-1 might participate in
the development of hormone-producing cells up to thebirth and in the control of gene regulation of pituitary hormones during the postnatal period.
In this study, we have demonstrated that Prop-1 is atranscription factor for FSHβ gene. It is known that combined pituitary hormone deficiency (CPHD) caused by adefective Prop-1 gene is accompanied by hypogonadism aswell as by a defect in Pit-1-dependent lineage cells. Therefore, the present finding provides a novel insight into ourunderstanding of CPHD defects.
In summary, we have confirmed the function of Prop-1 inregulation of the FSHβ gene, its marked presence in postnatal pituitaries, and its role in developing porcine fetal pituitaries, suggesting that Prop-1 is involved in the pituitary
function in addition to the determination of cell lineages
that produce pituitary hormones. Finally, the present results also provide new information for an improved understanding of CPHD.

Acknowledgements
This study was partly supported by grants-in-aid (Nos.
02640578 and 06454019) to Y. K., and by the High-Tech Research Center Project for Private Universities: matching fundsubsidy, 2001-2005, from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan).

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