Journal of Reproduction and Development, Vol. 52, No. 2, 2006

—Full Paper—

High Level Expression of Prop-1 Gene in Gonadotropic Cell Lines

Satoko AIKAWA1), Takanobu SATO1), Tetsuo ONO1), Takako KATO1) and Yukio KATO1)

1)Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, School of Agriculture, Meiji University, 1–1–1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan

Abstract. Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH- producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHβ gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein α subunit (αGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in αT1- 1, TαT1, MtT/S, GH3, and TtT/GF cells, expression of the αGSU gene in αT1-1, αT3-1, LβT2, LβT4, TαT1, and GH3 cells, and expression of GnRH receptor gene in αT3-1, LβT2, LβT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LβT2 and LβT4 gonadotrope cell lines, which express the FSHβ gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production. Key words: Glycoprotein hormone, Transcription, Pig, Pituitary, Prop-1, Dwarfism (J. Reprod. Dev. 52: 195–201, 2006)

ituitary transcription factor Prop-1, an termination of development of GH, PRL, and TSH- upstream regulator of the Pit-1 gene, was producing cells, all of which are Pit-1-dependent originally cloned as a gene responsible for a lineage cells. In addition, the defect also reduces heritable form of murine pituitary-dependent the production of FSH and LH, causing dwarfism (Ames dwarf). The gene was confirmed hypogonadotropic hypogonadism (Combined as a novel, tissue-specific, paired-like Pituitary Hormone Deficiency; CPHD) [2]. The homeodomain transcription factor, termed Prophet molecular mechanism of CPHD is not yet fully of Pit-1 [1]. A defect in the Prop-1 gene causes understood. We reported that several pituitary failure of Pit-1 gene expression, resulting in nuclear proteins bind to the upstream region (– 852/–746 bp) of the porcine FSHβ gene [3] and Accepted for publication: November 9, 2005 Published online: December 28, 2005 demonstrated that Prop-1 is one of the binding Correspondence: Y. Kato (e-mail: [email protected]) proteins that directly regulates this gene [4]. This 196 AIKAWA et al. finding provides novel insight into better mammotropic pituitary tumors [9] and mouse understanding the roles of Prop-1 in pituitary thyrotropic pituitary tumors [10], respectively, by organogenesis, hormone gene regulation, and Dr. K. Inoue. AtT-20 [11] and GH3 [12] cell lines CPHD. derived from mouse and rat pituitary tumor, CHO It is noteworthy that the ontogeny of murine cells established from Chinese hamster [13], Prop-1 gene expression was first detected at around and mouse L929 fibrosarcoma cells [14] were embryonic day 10 (e10) after formation of the oral obtained from the RIKEN Cell Bank (Tsukuba, ectoderm on e8.5 and invagination leading to Ibaraki, JAPAN). The characteristics of the cell formation of a Rathke’s pouch on e9.5–e10 [1]. lines used are summarized in Table 1. These two events preceded expression of Pit-1 gene, which is a major target of Prop-1 and a crucial Preparation of total RNAs and cDNA synthesis stage in the progression of pituitary differentiation. Total RNAs were prepared from 60–70% Thus, the ontogeny of Prop-1 gene expression plays confluent cells of each cell line using ISOGEN an indispensable role in the early stages of pituitary (Nippon Gene Co., Ltd., Toyama, Japan) according organogenesis. Thereafter, Prop-1 gene expression to the instruction manual. The cDNA synthesis reaches its maximum level by e12, followed by a from the total RNAs was carried out using a decline to an extremely low level by e14.5 that SMART cDNA Synthesis Kit (BD Bioscience continues until birth [1]. Although postnatal Clontech, Palo Alto, CA) with ReverTra Ace expression has been confirmed in the porcine [4] reverse transcriptase (TOYOBO, Tokyo, Japan). and human [5] pituitary, information concerning mice, rats and other animals has yet to be PCR elucidated. The specific PCR primer sets for Pit-1, In this study, we attempted to examine the glycoprotein hormone α subunit (αGSU), GnRH presence of Prop-1 transcripts in various cell lines receptor (GnRH-R) and cyclophilin A shown in derived from pituitary tumors. Quantitative real- Table 2 were synthesized. PCR was essentially time PCR analyses revealed that the Prop-1 gene is performed as described previously [15]. Each expressed abundantly in gonadotrope cell lines. cDNA was amplified in a reaction mixture (5 µl) containing the two required primers (5 pmol each) and 0.125 U AmpliTaq Gold DNA polymerase (PE Materials and Methods Applied Biosystems, Foster City, CA), with 32 to 36 cycles of denaturation (94 C, 30 s), annealing (55 C, Cell lines 30 s), and extension reaction (72 C, 2 min). The Mouse pituitary tumor cell lines, αT1-1, αT3-1, resulting PCR products were analyzed on 2% LβT2, LβT4, and TαT1, were kindly supplied by Dr. agarose gels. P. L. Mellon [6–8]. MtT/S and TtT/GF cell lines were established from rat estrogen-induced

Table 1. Characteristics of the cell lines used

Cell line Origin Sex Cell lineage Reference αT1-1 mouse ? thyrotrope [8] αT3-1 mouse male gonadotrope [7] LβT2 mouse male gonadotrope [6] LβT4 mouse male gonadotrope [6] TαT1 mouse ? thyrotrope [8] MtT/S rat female somatotrope [9] GH3 rat female somatotrope/ [12] mammotrope AtT-20 mouse ? corticotrope [11] TtT/GF mouse male folliculostellate cell [10] CHO Chinese hamster female cell [13] L929 mouse male fibrosarcoma [14] PROP-1 GENE EXPRESSION IN PITUITARY-DERIVED CELL LINES 197

Table 2. Primers used for RT-PCR

Target Forward primer Reverse primer Product Accession size number*

Pit-1 (mouse) 5’-CAAACAACCATCTGTCGATTTG-3’ 5’-CTTGTTTTCACTCGTTTTTCTCTC-3' 325 NM_008849 Pit-1 (rat) 5’-CAAACAACCATCTGCCGATTTG-3’ 5’-CTCGTTTTCACCCGTTTTTCTCTC-3’ 325 NM_013008 αGSU** 5’-TCATGCTGTCCATGTTCCTGC-3’ 5’-CACTCTGGCATTTCCCATTACTG-3’ 275 M22992 GnRH-R** 5’-TCTACCTAGYMGACGGCTCTGG-3’ 5’-TCCAGCAGAYGACAAAGGAGG-3’ 297 NM_031038 Cyclophilin A** 5’-TGGTGACTTYACACGCCATAATG-3’ 5’-ATTCCTGGACCCRAAACGCTCC-3’ 256 NM_008907 * The nucleotide sequence is available from the DNA database using the accession number. ** The primers are common to rat and mouse targets.

Table 3. Primers used for real-time PCR

Target Species Forward primer Reverse primer MGB probe* Prop-1 rat** 5'-CCAGAACCGCAGGGCTAAG-3' 5'-ATGGGCTGTTGGCTGGAGTA-3' 5'-AACGGAAGCAAGAGC-3' mouse** same as above 5'-GGCTATCGGCTGGAGAAGTG-3' same as above FSHβ rat** 5'-GGGAGGAAAGGAAAGTGGAAA-3' 5'-ATGGCTCTGAAAAGCAGATGGT-3' 5'-TGTGAAAGAACTAAACCTAGC-3’ mouse** 5’-TCCCAGACCATGATGAAGTTGA-3’ 5’-CAGCTATGGCAGCAGATTGC-3’ 5’-CAGCTTTGCATCTTAT-3’ CyclophilinA rat and 5’-AGGATGAGAACTTCATCCTRAAGCA-3 5’-YTGGCAGTGCAGATAAAAAACTG-3’ 5’-TCCTGGCATCTTGTCCA-3’ mouse**

* The MGB probe is specific for real-time PCR by binding to the minor groove of the target sequence. ** Rat Prop-1: XM_153627. Mouse Prop-1: NM_008936. Rat FSHβ: XM_575192. Mouse FSHβ: NM_008045. Rat cyclophilin A: XM_345915. Mouse cyclo- philin A: NM_008907.

Comparative quantification by real-time PCR Reactions that exhibited a low amplification profile For real-time transcript quantification, cDNAs resulting in a low relative content against were subjected to a fluorogenic 5’–3’ exonuclease cyclophilin A of less than 0.02 were not included. assay utilizing the chemistry of the TaqMan system on an ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA). Gene-specific Results primers (forward and reverse) and TaqMan Minor Groove Binder (MGB) probe sets were created for Characterization of pituitary cell lines Prop-1, FSHβ, and cyclophilin A using the Primer RT-PCR was first performed for cyclophilin A, a Express Software v.1.5 (Applied Biosystems) as housekeeping gene, to determine the quantity of shown in Table 3. The TaqMan MGB probe for each cDNA sample needed to obtain the same cyclophilin A was labeled with VIC and used as a amount of PCR product (Fig. 1A). RT-PCRs for Pit- control for normalization. The TaqMan MGB 1, αGSU, and GnRH-R were then performed using probes for Prop-1 and FSHβ were labeled with 6- individual quantities of each cDNA sample having carboxyfluorescein (FAM). The experimental PCRs the same amount of cyclophilin A cDNA. The included 900 nM of each primer, 250 nM MGB major PCR products for each target were purified probe, 1 × TaqMan buffer A (50 mM KCl, 10 mM by separation on agarose gels, and were then used Tris-HCl, 0.01 mM EDTA), 5 mM MgCl2, dATP (200 for sequence analysis to confirm the exact product µM), dCTP (200 µM), dGTP (200 µM), dUTP (400 (data not shown). µM), 0.5 U AmpErase UNG, and 1.25 U AmpliTaq PCR product for Pit-1 was observed in αT1-1, Gold DNA Polymerase. The real-time PCR TαT1, MtT/S, and GH3, and a small amount was consisted of one cycle at 50 C for 2 min and 95 C for observed in TtT/GF cells (Fig. 1B). On the other 10 min, followed by 40 cycles at 95 C for 15 s and 60 hands, αT3-1, LβT2, LβT4, and AtT-20 did not yield C for 1 min. All data were calculated by the any Pit-1 product. The other non-pituitary cell comparative CT method (∆∆CT method) [16] lines, CHO and L929 cells, were also negative. according to the instruction manual to estimate the Figure 1C shows the results of PCR for αGSU. relative gene copy numbers using the same Products of αGSU were observed in αT1-1, αT3-1, threshold line for Prop-1, FSHβ, and cyclophilin A. LβT2, LβT4, TαT1, and GH3 cells. The other 198 AIKAWA et al.

Fig. 2. Real-time PCR analysis of Prop-1 and FSHβ transcripts. Real-time PCR was performed for Prop-1 (A) and FSHβ (B) with cyclophilin A as a control using specific primer sets as listed in Table 3. The results are indicated as relative amounts of Prop-1 and FSHβ compared with cyclophilin A.

known to be a specific receptor for gonadotrope lineage cells, RT-PCR for GnRH-R was performed. Products of GnRH-R were observed in αT3-1, LβT2, and LβT4 cells, and a small amount (invisible in the figure) was present in GH3 cells (Fig. 1D). It is worth noting that the product amount of GnRH-R was higher in αT3-1 cells than in LβT2 and LβT4 cells, which are thought to be part of a later stage of the development for gonadotrope lineage than αT3-1 because of acquisition of ability to express LHβ and FSHβ genes.

LβT2 and LβT4 cells express Prop-1 gene Real-time PCR was employed for quantitative estimation of Prop-1 gene expression. The results for Prop-1 were arranged in comparative values against those of cyclophilin A using the ∆∆CT Fig. 1. RT-PCR analysis of genes expressed in the cell lines. method (Fig. 2A). LβT4 cells showed the highest RT-PCR analyses of cyclophilin A (A), Pit-1 (B), αGSU content of Prop-1 (about 59% of that for cyclophilin (C), and GnRH-R (D) were performed using specific A), and another gonadotropic cell line, LβT2 cells, primer sets as listed in Table 2, followed by gel electrophoresis on a 2% agarose gel. The names of the contained about 57% of that for cyclophilin A. cell lines are indicated above each figure. Other cell lines had low contents of Prop-1 as follows: 3% (αT3-1), 2% (TαT1), 2% (AtT-20), and 3% (TtT/GF) of that for cyclophilin A. No products were found in αT1-1, MtT/S, and GH3 cells, or in pituitary cell lines, MtT/S, AtT-20, and TtT/GF the non-pituitary cell lines of CHO and L929 cells. cells, and non-pituitary cell lines, CHO and L929 Real-time PCR analysis of FSHβ was also carried cells, failed to yield any αGSU product. out (Fig. 2B). Expression of FSHβ gene was Therefore, the RT-PCR for αGSU and Pit-1 observed in LβT2 and LβT4 cells at a level 13 and confirmed that these genes express in a cell-type 15% of that for cyclophilin A, respectively, which specific manner as expected. Since GnRH-R is were lower amounts than for Prop-1 (Fig. 2A). PROP-1 GENE EXPRESSION IN PITUITARY-DERIVED CELL LINES 199

Table 4. Summary of RT-PCR and real-time PCR αT1-1 αT3-1 LβT2 LβT-4 TαT-1 MtT/S GH3 AtT20 TtT/GF CHO L929 Pit-1* + – – – ++ +++ +++ – + – – αGSU* ++ ++ ++ ++ ++ – + – – – – GnRH-R* – +++ ++ + – – ± –– –– FSHβ** ±±++ – – – – – – – Prop-1** – ± ++ ++ ± ––±± –– The mRNA quantity of each gene observed by RT-PCR (*) and real-time PCR (**) are arbitrarily indicated with an order of +++>++>+>±. Hyphens (–) indicate undetectable amounts.

Only a small amount of FSHβ gene expression was are classified into 7 cell lineages, including observed in αT1-1(4%) and αT3-1 cells (3%), folliculostellate cells, and covered all the cell respectively. lineages of the . The characteristics of the cell lines used are summarized in Table 1. The cell lineages reported are Discussion characterized by specific marker genes. The αT1-1 cell line, which expresses the αGSU gene, belongs Prop-1 is a crucial pituitary-specific transcription to a thyrotrope cell lineage. The αT3-1, LβT2, and factor for pituitary organogenesis. This factor LβT4 lines, which express the αGSU and GnRH-R appears at an early stage of pituitary development genes, belong to a gonadotrope cell lineage. The and disappears before completion of differentiation TαT1 cell line, which expresses the αGSU and of hormone producing cells [1]. Expression of TSHβ genes, belongs to a thyrotrope cell lineage. Prop-1 gene during the postnatal period was The MtT/S cell line, which expresses the GH gene, confirmed on P0 for the mouse in the report of belongs to a somatotrope cell lineage, and GH3, Sornson et al. [1], but Cushman et al. reported that which expresses both the GH and PRL genes, no Prop-1 expression was evident at or beyond belongs to a somato/mammotrope cell lineage. birth in the mouse [17]. However, by transient The AtT-20 cell line is known as a corticotrope cell transfection and immunohistochemical analysis, lines, and TtT/GF is a non-hormone producing we observed that Prop-1 directly regulates the pituitary cell line with the characteristics of porcine FSHβ subunit gene and is present in adult folliculostellate cells [10]. The CHO and L929 cell porcine gonadotrope cells [4]. Furthermore, by the lines are non-pituitary cell lines derived from RT-PCR analysis for porcine ontogeny, we Chinese hamster ovaries and mouse fibrosarcoma, observed expression of Prop-1 gene during the respectively. embryonic period and the expression increased The results of real-time PCR for Prop-1 and FSHβ after birth [18]. We hypothesize that Prop-1 might and RT-PCR for Pit-1, αGSU, and GnRH-R are play a role in regulation of specific hormone genes summarized in Table 4. It is noteworthy that the after accomplishment of its primary role in the cell lines expressing the Pit-1 gene, which is early stage of pituitary development. Our purpose regulated by Prop-1 in the early stages of pituitary in this study was to determine whether the Prop-1 development, did not express the Prop-1. The LβT2 gene is continues to express in hormone producing and LβT4 cells, which express αGSU and FSHβ cells during the course of the developmental stage, genes but not the Pit-1 gene, expressed the Prop-1 especially in Pit-1 dependent lineage cells and FSH- gene. Previously, Pernastetti et al. found expression producing cells. of FSHβ gene in LβT2 cells [19], and we confirmed Pituitary cell lines, which are immortalized at that a detectable amount of FSHβ transcripts are discrete stages of pituitary development, were used present in both LβT2 and LβT4 cells. This result in this study in addition to non-pituitary cell lines, corresponds to our previous finding that Prop-1 CHO cells derived from Chinese hamster ovary regulates the expression of FSHβ gene [4], and cells, and L929 cells derived from mouse supports our hypothesis described above. fibrosarcoma (Table 1). The pituitary cell lines used On the other hand, αT3-1 cells, which are 200 AIKAWA et al. classified as being of gonadotrope lineage by the the development of Pit-1 lineage cells failed to characteristics of producing αGSU, GnRH-R, and explain the hypogonadism symptoms involved in SF-1 [7], failed to express the Prop-1 gene. The CPHD caused by Prop-1 defect. However, two other αGSU-producing cell lines, αT1-1 and TαT1, possibilities may be considered. One is that Prop-1 which produce Pit-1 and are classified as being of participates in differentiation of gonadotropes in a thyrotrope lineage, also failed to produce Prop-1. manner specific to its role and similar to Pit-1 We recently found that Prop-1 also participates in lineage cells. The other is that it directly regulation of gene expression of αGSU (Sato et al., contributes to activation of subunit unpublished observation), and that the distal and gene expression. The experimental evidence proximal upstream regions of the porcine αGSU described above supports this later possibility. gene contain profound regulatory elements [20]. In this context, the regulation system for expression of αGSU gene in LβT2 and LβT4 cells is distinguished Acknowledgments from the systems in other αGSU producing cells, αT3-1, and thyrotrope lineage cells. This research was supported in part by Grant-in- The accumulation of these data probably Aid for Scientific Research (B) No. 12470217 and confirms our view that synthesis of FSH is involved No. 12557087 from the Ministry of Education, in Prop-1 function. It has already been established Culture, Sports, Science and Technology and by a that a Prop-1 gene defect causes CPHD, although Grant-in-Aid for Research Grant (A) from the its molecular mechanism is not yet fully Institute of Science and Technology at Meiji understood. Since Prop-1 definitely regulates University to Y. K. This study was also supported expression of the Pit-1 gene, a loss of Prop-1 by a matching fund subsidy (2001–2005) from the function by mutation does not result in the “High-Tech Research Center” Project for private differentiation of Pit-1 lineage cells, GH, PRL, and universities of the Ministry of Education, Culture, TSH producing cells. However, this disruption in Sports, Science and Technology of Japan.

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