Biochemical and Biophysical Research Communications 324 (2004) 946–952 Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone b subunit gene expression Satoko Aikawaa, Takako Katoa, Takao Susaa, Kyoko Tomizawab, Satoshi Ogawab, Yukio Katoa,* a Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan b Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan Received 29 August 2004 Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone b subunit (FSHb) gene was performed by the yeast one-hybrid cloning system using the 852/746 upstream region (Fd2) as a bait sequence.
We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was inves-tigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-trans-fection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractivestimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of theProp-1 and FSHb subunit. This study is the first to report that Prop-1 participates in the regulation of FSHb gene. The presentfinding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD),since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.
 2004 Elsevier Inc. All rights reserved.
Keywords: Pituitary transcription factor; Prop-1; FSHb; FSH; Transcription; Gene regulation; CPHD; LH; Pit-1; Pig FSH is a pituitary glycoprotein hormone, including Several approaches have succeeded in providing an luteinizing hormone (LH) and thyroid-stimulating hor- understanding of the molecular mechanisms and the mone (TSH). Each of these hormones shares a common transcription factors governing basal and cell-specific a-glycoprotein subunit (a-GSU) and contains a unique expression of the a-GSU and LHb subunit genes .
b-subunit that confers physiological specificity on the However, for the FSHb subunit gene, only a few lines respective hormone. The synthesis and secretion of of evidence for the basal expression have accumulated.
FSH as well as LH are restricted to pituitary gonado- We previously observed that GnRH significantly stimu- troph cells. The regulatory mechanism of gene expres- lates the expression of both c-Jun and c-Fos genes that sion of three subunits forming gonadotropin hormones form transcription factor, AP1 Later, it was revealed is an interesting issue.
that the GnRH stimulation of FSH is mediated by theprotein kinase C signaling cascade through AP-1 sitesin the region of 120/83 bp and the distal re-gion between 4152/2878 and 2550/1089 bp of the ovine FSHb subunit gene Several steroid hor- Corresponding author. Fax: +81 44 934 7035.
E-mail address: (Y. Kato).
mones, such as progesterone and estrogen, are known 0006-291X/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.09.124 S. Aikawa et al. / Biochemical and Biophysical Research Communications 324 (2004) 946–952 to suppress the expression of FSHb subunit gene a Whatman No. 50 filter paper (Whatman International, Maidstone, but our previous analysis of porcine FSHb subunit gene England), followed by visualization as described previously .
Sequence analysis. DNA samples were prepared from Escherichia failed to identify typical responsive sequences for pro- coli by the alkaline mini-preparation method and were employed in the gesterone (PRE) and estrogen (ERE) Thereafter, fluorescence-labeled dye-terminator reaction using the Big Dye ter- multiple PREs are identified in the proximal region of minator system (Applied Biosystems, Foster City, CA), followed by the ovine and rat FSHb subunit gene Similarly, analyzing on the ABI PRISM 310 (Perkin–Elmer Cetus, Norwalk, ERE was identified in 105/72 of the ovine FSHb Production of recombinant protein. Porcine Prop-1 cDNA was subunit gene . Interestingly, it was reported that cloned in-frame into the pET32a vector (Novagen, Darmstadt, Ger- the 270/248 region of the rat FSHb subunit gene is many) and introduced into E. coli BL21 (DE3) Codon Plus RP responsible for activin-dependent activation mediated (Stratagene). TrxÆTag and HisÆTag-fused porcine Prop-1 protein was by the transcription factors, Smad and Pitx2 . A sim- isolated after induction by 1 mM isopropyl thiogalactoside and puri- ilar region is also responsible for Pitx1, the homologue fied by the His-Tag Mag beads (Toyobo, Tokyo, Japan).
Electrophoretic gel mobility shift assay. FAM-labeled DNA frag- of Pitx2 . Nevertheless, the mechanism of cell/tis- ment was produced by PCR using FAM-labeled oligonucleotide 50- sue-specific expression of FSHb subunit gene remains primer. The binding reaction mixture included 10 fmol FAM-labeled probe DNA (1 ng) and 100 ng of porcine recombinant Prop-1 with In our previous study we found that multiple 250 ng poly(dI–dC) in 10 ll of 10 mM Hepes buffer, pH 7.9, containing porcine nuclear proteins bind to the region (Fd2) of 0.4 mM MgCl2, 0.4 mM DTT, and 50 mM NaCl, and 4% glycerol wasincubated at 30 C for 30 min. Then samples were subjected to elec- around 100 bp in length located between 852 and trophoresis on a 4% polyacrylamide gel as described in the previous 746 bp upstream of the porcine FSHb subunit gene.
paper TrxÆTag and HisÆTag-fused porcine Msx-1 protein Hence, we have attempted to find the binding proteins (unpublished data) was used for the negative binding control.
by the yeast one-hybrid cloning system. The transcrip- DNase I footprinting assay. The 50-labeled DNA was incubated tion factor eventually cloned was a paired-like homeo- with recombinant porcine Prop-1 protein in binding buffer under thesame conditions used for electrophoretic gel mobility shift assay box transcription factor, Prop-1, which had been (EMSA). After a 20-min incubation at 30 C, 0.1 or 0.2 U DNase I identified as a pituitary transcription factor essential (RQ1 RNase-Free DNase, Promega, Madison, WI) was added, and for the expansion of the pituitary primordia and for the mixture was incubated for 15 min at 25 C. The reaction was the differentiation and/or function of the hormone-pro- stopped by the addition of EDTA to a final concentration of 65 mM, ducing cells of the anterior pituitary gland Our and then proteins were removed by phenol–chloroform extraction.
DNA fragments were precipitated, dissolved in 10 ll formamide con- finding may provide novel clues to understand the taining 1 ll ROX-labeled GS-500 (Applied Biosystems) as a molecular development of gonadotroph cells and why a Prop-1 size marker, and resolved in a Gene Scan analyzer equipped in ABI defect ablates gonadotroph cells as well as the Pit-1-de- pendent cell-lineage of GH/PRL/TSH-producing pitui- Expression vectors and secreted alkaline phosphatase (SEAP) re- porter gene constructs. Porcine Prop-1 cDNA was excised by digestionwith EcoRI and XhoI from cloned Prop-1-pADGAL4 plasmid andligated into the EcoRI and XhoI site of mammalian expression vector,pcDNA3.1Zeo+ (Clontech). The reporter gene constructs were gener- Materials and methods ated by ligation of an upstream region of porcine FSHb subunit geneinto pSEAP2-Basic (Clontech) (Fb3-basic: 985/+10, DFb3-basic: Construction of reporter vectors and integration into chromosome deletion mutant of Fb3-basic from 745 to 104 bp, and Fb5-basic: of yeast YM4271. The reporter vectors, pHISi-1 and pLacZi 238/+10) and pSEAP2-Promoter vectors (Clontech) (Fd2-promoter: (Clontech, Palo Alto, CA), were digested with EcoRI and XbaI, and Fd2, 852/746).
SmaI, respectively. The 50 upstream region of the porcine FSHb Cell culture, DNA transfections, and reporter gene assays. Chinese subunit gene, Fd2 (852 to 746 bp), was ligated to each vector, hamster ovary (CHO) cells were cultured in F12 medium supplemented resulting in Fd2ÆpHISi-1 and Fd2ÆpLacZi, respectively. Fd2ÆpHISi-1 with 10% fetal bovine serum (CELLect Gold FBS, ICN, Aurora, OH).
was linearized by XhoI and integrated into the chromosome of Transfections of CHO cells were performed after 24 h from seeding YM4271 (Clontech) generating YM4271::[Fd2pHISi-1]. Fd2ÆpLacZi approximately 1 · 104 cells/100 ll/well in 96-well plates using Fu- was then linearized with StuI and integrated into YM4271::[Fd2- GENE 6 (Roche Diagnostic, Indianapolis, IN) with 20 ng DNA/0.3 ll FuGENE 6/well. Then cells were incubated in 5% CO2–95% air for genomic integrants were selected according to the manufacturers 24 h at 37 C. Each 5 ll of cultured medium was assayed for secreted alkaline phosphatase activity using the Phospha-Light Reporter Gene One-hybrid screening of pituitary transcription factors binding to Fd2 Assay System (Applied Biosystems).
region. The adult porcine pituitary cDNA library was constructed in Double immunocytochemical staining of porcine anterior pituitary pADGAL4 using the HybriZAP Two-Hybrid cDNA Gigapack cells. Fresh porcine pituitaries (7 months, male and female) were kindly Cloning Kit (Stratagene, La Jolla, CA) according to the manufac- supplied by Dr. M. Kikuchi of Kitasato University. Fixation, prepa- turers instructions, as described previously The plasmid library ration of section, and staining were essentially performed as described was introduced into the yeast reporter cells, YM4271::[Fd2pHISi- previously Anti-FSH monoclonal antibody (Nichirei, Tokyo, 1][Fd2pLacZi], by LiAcetate method, and yeast transformants were Japan) diluted with 4 volume of PBS and anti-Prop-1 polyclonal selected on synthetic medium lacking leucine, uracil, and histidine, but antibody (5 lg/ll in PBS) originally raised by synthetic peptide were containing 3-amino-1,2,4-triazole (3-AT, to inhibit leaky expression of used. FSH was visualized by goat Cy3-labeled anti-mouse IgG (1:500), HIS3 integrated in pHISi-1).
and Prop-1 was reacted with goat biotin-labeled anti-rabbit IgG b-Galactosidase assay. The colony-lift b-galactosidase filter assay (1:1000) followed by visualization with the TSA system (Perkin–Elmer was performed for yeast cells which were grown at 30 C for 2 days on Life Science, Boston, MA).

S. Aikawa et al. / Biochemical and Biophysical Research Communications 324 (2004) 946–952 In vitro binding assay by EMSA and DNase I footprinting Screening of porcine pituitary cDNA library In the Fd2 region, there is one putative Prop-1-binding site, TAATtaATTA between 836 and 827. To We carried out one-hybrid screening, in which trans- confirm the binding site of Prop-1 in Fd2, in vitro binding formants of the yeast YM4271::[Fd2pHISi-1][Fd2pLac- assay was performed by EMSA as well as DNase I Zi] harboring a porcine pituitary cDNA library in pAD-GAL4 (pAD-GAL4/cDNA library) were selected EMSA demonstrated that the shift band generated by on SD-Ura-Leu-His agar plates containing 3-AT. Screening the addition of TrxÆTag and HisÆTag-fused Prop-1 ( of 4.1 · 106 transformants at the efficiency of 2 · 105 col- lane 2) migrated slightly faster by removal of the non- ony forming units per 1 lg DNA yielded several pre- specific binding (lane 3) under the excessive poly(dI–dC) sumptive positive clones. Eleven clones exhibited both conditions (250-fold excess amount). Then, the binding specific amino acid requirement and b-galactosidase was decomposed by the addition of an excess amount activity. DNAs of the clones were amplified in E. coli of unlabeled Fd2 (50-fold molar excess), forming multi- cells and re-transformed into YM4271::[Fd2pHISi-1] ple fast migrating bands (lane 4). On the other hand, [Fd2pLacZi] cells. The transformants exhibited the abil- the addition of TrxÆTag and HisÆTag-fused Msx1 showed ity of growth on SD-agar plates lacking histidine, leu- no shift band (lanes 6 and 7), indicating that not only cine, and uracil (as well as the b-galactosidase Msx1 but also the Tag peptide has no binding affinity activity B), indicating an interaction between to Fd2 sequence.
the products selected by one-hybrid cloning and the DNase I footprinting demonstrated that many frag- Fd2 sequence of [Fd2pHISi-1] and [Fd2pLacZi] inte- ments covering the entire region of the Fd2 were gener- grated into the yeast gene.
ated by DNase I without recombinant Prop-1 upper panel). In contrast, the digestion in the presence Nucleotide sequence of Prop-1 of Prop-1 produced a marked change in the profile ofthe fragment. Apparently the signals of fragments be- Three of the selected clones were almost completely tween 846 and 778 had disappeared or decreased, ex- identical to the porcine Prop-1 The nucleotide se- cept for the position at 797 bp. Downstream of the quence of the cloned Prop-1 revealed the entire sequence binding region, the signals of the sequence AGTT be- of 967 bp with a poly(A) tract (DDBJ Accession No.
tween 777 and 774 increased, but the other 30 part AB187272). Open reading frame of 678 bp codes 226 did not change, presumably providing DNA-bending amino acids with 50 and 30 untranslated regions of 43 at this site to increase the nuclease sensitivity. Thus, and 246 bp, respectively. There is no typical polyadenyl- Prop-1 protects the Fd2 fragment over the 60-bp region ation signal sequence, AATAAA. As described previ- of 844/780 which has 80% AT-content including a ously , the amino acid sequence of the central putative Prop-1-binding site.
DNA-binding homeo-domain and the major trans-acti-vation domain at the carboxyl terminus are well con-served, although the amino terminus is considerablychanged among porcine, human, rat, and mouse Prop-1 as described in Sloop et al.
Fig. 1. Amino acid requirements and b-galactosidase activity of yeasttransformants. The amino acid requirements using SD-Ura-Leu-His agar Fig. 2. Electrophoretic gel mobility shift assay of Prop-1. Complex plates (A), and b-galactosidase activity of transformants grown on with recombinant Prop-1 and FAM-labeled Fd2 was analyzed on 4% SD-Ura-Leu agar plates (B) were examined. The transformants con- polyacrylamide gel followed by visualization with a fluorescence tained cDNA/pADGAL4 in YM4271::[Fd2pHISi-1][Fd2pLacZi] (a), viewer. The composition of each binding mixture is indicated under the pADGAL4 in YM4271::[Fd2pHISi-1][Fd2pLacZi] (b), cDNA /pAD- electrogram. Recombinant Msx-1, which does not bind to Fd2, was GAL4 in YM4271 (c), and YM4271::[Fd2pHISi-1][Fd2pLacZi] (d).
used as a negative control.

S. Aikawa et al. / Biochemical and Biophysical Research Communications 324 (2004) 946–952 Fig. 3. Footprinting assay of Prop-1-binding site in Fd2 region. Labeling of Fd2 was performed by PCR using a FAM-labeled forwardoligonucleotide. The DNase I digests were prepared with or without recombinant porcine Prop-1 (lower panel and upper panel, respectively) asdescribed in Materials and methods. The nucleotide sequence of Fd2 corresponding to each signal is indicated below.
Transcriptional activation of the porcine FSHb subunit tory elements present in the vector construct. By gene by Prop-1 in CHO cells transfection of the reporter vector fused with the985/+10 region (Fb3-basic), the expression level of To ascertain whether Prop-1 serves as a transcription the SEAP gene increased significantly by about 2.5-fold factor for the FSHb subunit gene, we investigated the in the presence of the Prop-1 expression vector. The transcriptional activity by co-transfection of Prop-1 deletion of 745/104 leaving Fd2 and the endogenous expression vector and reporter vector fused with the proximal promoter region (103/+10 bp, DFb3-basic) upstream region of the FSHb subunit gene into CHO did not alter the activation by Prop-1. In addition, the 238/+10 region (Fb5-basic) does not have any effect.
Transfection of pSEAP2-Basic and pSEAP2-Promot- In contrast, when the reporter vector fused with er (SV40 promoter) with or without expression vector pSEAP2-Promoter with Fd2 was co-transfected with of porcine Prop-1 gave almost the same expression level Prop-1 expression vector, transcriptional activity of of SEAP gene in CHO cells indicating the ab- Prop-1 was not observed, indicating the promoter-spe- sence of endogenous activation by unexpected regula- cific activation of Prop-1.
Fig. 4. Transcriptional activation of FSHb gene in CHO cells. Porcine Prop-1 cDNA ligated in pcDNA3.1Zeo+ vector or pcDNA3.1Zeo+ vectoralone was cotransfected into CHO cells with pSEAP2-Basic or reporter vector fused with region of 985/+10 (Fb3-basic), 745/104 bp deletedfrom Fb3-basic (DFb3-basic), and 238/+10 (Fb5-basic). pSEAP2-Promoter fused with Fd2 (Fd2-promoter) was also co-transfected. After 48 hincubation, an aliquot of cultured medium was assayed for the alkaline phosphatase activity. Each activity was normalized using that of transfectionwith pcDNA3.1+ vector alone.

S. Aikawa et al. / Biochemical and Biophysical Research Communications 324 (2004) 946–952 gene expression to result in the terminal differentiationof the Pit1-lineage of the thyrotroph, somatotroph,and lactotroph cell types Both Ames df mice andhuman CPHD patients exhibit a reduction of Pit1-line-age cells and reduced gonadotropin levels Thisloss of Pit1-lineage cells is explained by a failure of ini-tial proliferation of three Pit1-dependent cell types Fig. 5. Double immunohistochemical staining. The 5 lm-thick section caused by the mutation of Prop-1 gene, but the reason of porcine adult pituitary was immunohistochemically stained by anti- for the ablation of the gonadotroph cells remains ob- Prop-1 antisera (left) and anti-FSH antibody (right), respectively.
scure The present finding that Prop-1 participates Arrows indicate positive cells reacted with both antibodies.
directly in the FSHb subunit gene expression may wellprovide a breakthrough for the understanding of the Double immunohistochemical staining CPHD mechanism.
The ontogeny of the murine Prop-1 gene expression Double immunohistochemical staining using a por- could first be detected at about embryonic day 10 cine adult pituitary was performed for Prop-1 ( (e10) preceding the formation of Rathkes pouch. The left) and FSH (right). More cells were positive Prop-1 gene expression reached the maximum level by for anti-Prop-1 antibody than those for anti-FSH anti- e12, followed by a decrease to an extremely low level body. The cells positive for both Prop-1 and FSH stain- by e14.5 with the distribution of the dorsal/ventral gra- ing are indicated by arrows. The cells positive only for dient During anterior pituitary development, the anti-Prop-1 antibody are indicated by arrowheads.
differentiation of hormone-producing cell types takesplace with a temporal order in distinct regions. In themurine anterior pituitary, POMC is first detected at e12.5 on the dorsal side of the lobe towards the rostraltip when and where the expression level of the Prop-1 The present study demonstrated that a binding factor is high. TSH is detected at e14.5 in the central region, cloned by the yeast one-hybrid system using the 50-up- GH and PRL at e15.5 in the dorsal region, and finally stream region of the porcine FSHb gene (Fd2 region: LH and FSH at e16.5 and e17.5, respectively, in the ven- 852/746 bp) as a bait sequence is the pituitary-specific tral region where the expression level of Prop-1 is low transcription factor, Prop-1, and that porcine Prop-1 cer- Thus, Prop-1 plays its role for the determination tainly participates in the regulation of the FSHb gene.
and differentiation of pituitary hormone-producing cells Recombinant porcine Prop-1 binds to Fd2 fragment at by a spatially and temporally unique expression.
multiple binding sites presented in the AT-rich sequence In this study we demonstrated for the first time that extensively expanded from 846 to 778 bp, including a Prop-1 directly regulates the FSHb subunit gene expres- consensus Prop-1-binding sequence of TAATtaATTA sion. However, the above observation that LH and FSH (). In CHO cells, the expression of re- are detected in the region of low Prop-1 expression may porter gene fused with 50-upstream region of the porcine indicate the importance for the differentiation of gonad- FSHb gene (985/+10 bp) was significantly activated otroph. Actually, the transgenic mouse that overex- (2.5-fold) with Prop-1 (). This transcriptional presses Prop-1 gene shows the interference of anterior activation caused by Prop-1 is specific to the set of Fd2 pituitary cell differentiation and the susceptibility for sequence and endogenous FSHb-promoter without rela- pituitary tumors, followed by a delay in the terminal dif- tion to the intervening sequences ). Immunohisto- ferentiation of pituitary gonadotroph and transient chemical analysis using a porcine adult pituitary shows hypogonadism On the other hand, it would be con- the co-localization of Prop-1 and FSH, suggesting that tradictory to hold that the mutation of Prop-1 gene Prop-1 participates in the regulation of FSHb gene found in Ames df mice and human CPHD patients expression in adult gonadotroph cells.
triggers hypogonadism as well as the deficiency Here we have indicated for the first time that Prop-1 of GH, PRL, and TSH. These observations indicate that directly participates in the regulation of the FSHb sub- Prop-1 participates in the development of gonadotroph unit gene expression. Originally, Prop-1 had been posi- cells and gene expression of gonadotropins by altering tionally cloned as a gene apparently responsible for a the expression level spatially and temporally. At least heritable form of murine pituitary-dependent dwarfism in part the mechanism of hypogonadism in CPHD (Ames dwarf, df). The gene was confirmed as a novel, may be accounted for by the loss of Prop-1 function that tissue-specific, paired-like homeodomain transcription regulates the FSHb subunit gene expression as demon- factor, termed Prophet of Pit-1 (Prop-1) Prop-1 strated by the present study. Interestingly, Sornson heterodimerizes with Rpx/Hesx1, which expresses in et al. found that Prop-1 forms a dimer with the early pituitary development, and activates the Pit1 Hesx1/Rpx, which inhibits the expression of Pit-1 gene.
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