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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

(Requests for offprints should be addressed to T Sato; Email: yukato{at}isc.meiji.ac.jp)

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone ß subunit (FSHß) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone subunit (GSU), and luteinizing hormone ß subunit (LHß). A series of deletion mutants of the porcine GSU (up to –1059 bp) and LHß (up to –1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine GSU and LHß promoters by Prop-1, which was found to activate the GSU promoter of –1059/+12 bp up to 11.7-fold but not the LHß promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, –1038/–1026, –942/–928, –495/–479, –338/–326, –153/–146, and –131/–124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of GSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for GSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of GSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHß gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of GSU and FSHß gene expressions.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
The pituitary glycoprotein hormones, LH, FSH, and thyroid-stimulating hormone (TSH), are a family of heterodimeric proteins that consist of a common subunit (GSU) noncovalently associated with a hormone-specific subunit (Pierce & Parsons 1981). In the pituitary gland, LH and FSH are synthesized within gonadotropes, while TSH is synthesized in thyrotropes. In addition, this GSU is also expressed in the primate and equine placenta to produce chorionic gonadotropins (Fiddes & Goodman 1981). Thus, GSU gene is expressed within two different cell types in the pituitary and in different tissue in a cell-type specific as well as tissue-specific manner. Meanwhile, the expression of specific ß-subunit genes, LHß and FSHß, are differently regulated (Papavasiliou et al. 1986, Kato et al. 1989). To clarify the specific regulatory mechanism of GSU and specific ß-subunit genes, several investigations have been conducted and several regulatory elements and transcription factors were reported (for review, see Brown & McNeilly 1999, Savage et al. 2003, Jorgensen et al. 2004).

We recently demonstrated that one of the DNA-binding proteins for the Fd2 region (–852/–746 bp) of the porcine FSHß gene (Kato et al. 1999) is a pituitary-specific transcription factor, Prophet of Pit-1 (Prop-1; Aikawa et al. 2004). Our finding may throw light on a novel aspect that may be helpful in clarifying the regulatory mechanism of the FSHß gene and also serve to pose the question of whether Prop-1 regulates other subunit genes of gonadotropins, GSU and LHß, which are expressed in gonadotropes as well as FSHß gene.

The Prop-1 gene was originally identified as the gene responsible for a heritable form of murine Ames dwarfism (df; Sornson et al. 1996). Similarly, recessive mutations in the human Prop-1 gene result in combined pituitary hormone deficiency (CPHD) with hypogonadism (Wu et al. 1998). Sornson et al. demonstrated that Prop-1 is essential as an upstream transcription factor of Pit-1 that determines the development of Pit-1 lineage hormone-producing cells, somatotrope, lactotrope, and thyrotrope that produce growth hormone (GH), prolactin (PRL), and TSH. It is notable that the defect of murine Prop-1 causes Ames dwarfism, which reduces levels of FSH and LH (Tang et al. 1993) in addition to inducing a deficiency in GH, PRL, and TSH as shown in human CPHD. Accordingly, such evidence encouraged us to undertake this investigation into whether Prop-1 participates in the regulation of the genes encoding gonadotropin subunits.

In this study, we have examined the stimulation of the promoter activity of both GSU and LHß genes by Prop-1 using transient transfection assay, followed by analyzing the Prop-1-binding site by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. Our results show that the promoter of GSU gene was activated by Prop-1, whereas that of LHß gene was not. EMSA and DNase I footprinting demonstrated that the GSU promoter has at least six Prop-1-binding sites.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Construction of reporter vectors and expression vector

To obtain serial truncated upstream regions of the porcine GSU gene (accession number: D00768 [GenBank] ; Kato et al. 1991) and porcine LHßgene (accession number: D00579 [GenBank] ; Ezashi et al. 1990; Fig. 1B and E), specific primer sets for PCR were designed and synthesized (Table 1). PCR was carried out in a reaction mixture (5 µl) containing two required primers (5 pmol each) and 0.125 U AmpliTaqGold DNA polymerase (PE Applied Biosystems, Foster City, CA, USA) with 32–36 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension reaction (72 °C, 2 min) steps. The resulting amplified products were ligated to the secreted alkaline phosphatase (SEAP) plasmid vector, pSEAP2-Basic (Clontech Laboratories, Inc., Mountain View, CA, USA), which has no eukaryotic promoter, resulting in reporter vectors GSU(–1059/+12), GSU(–798/+12), GSU(–540/+12), GSU(– 239/+12), GSU(–100/+12), and GSU(–53/+12) for GSU promoter, and LHß(–1277/+7), LHß(– 950/+7), LHß(–676/+7), LHß(–331/+7), and LHß(–57/+7) for LHß promoter. Using oligonucleotides listed in Table 2, we have constructed expression vectors containing putative Prop-1-binding sites and their mutants as follows. The –1059/–1015, –946/–909, –503/–474, –359/–319, –163/–142, and –147/– 114 bp regions of GSU, and their mutants were directly connected to the minimum GSU promoter vector, GSU(–53/+12), in forward orientation resulting in GSU(–1059/–1015), GSU(–1059/–1015)-m, GSU(–946/–909), GSU(–946/–909)-m, GSU (–503/–474), GSU(–503/–474)-m, GSU(– 359/–319), GSU(–359/–319)-m, GSU(–163/– 142), GSU(–163/–142)-m, GSU(–147/–114), and GSU(–147/–114)-m. The Prop-1 consensus binding element (PRDQ9; Sornson et al. 1996) and its mutant were also connected to GSU(–53/+12) resulting in GSU(PRDQ9) and GSU(PRDQ9)-m. The integrity of all DNA fragments inserted to vectors was confirmed by DNA sequencing on the ABI PRISM 310 (PE Applied Biosystems).

View larger version (12K): [in this window] [in a new window]   Figure 1 Diagram of porcine GSU, LHß promoters, and DNA fragments. (A) and (D) Positions of regulatory elements and binding sites of transcription factors identified in other mammals are shown by nucleotide numbers corresponding to the relevant porcine sequence. (B) and (E) Truncated promoters were ligated to reporter vector, pSEAP2-Basic, and used for transient transfection assay. (C) FAM-labeled DNA fragments of GSU promoter were used for EMSA and DNase I footprinting analysis. LIM-HD, LIM homeodomain transcription factor binding site (Roberson et al. 1994); GSE/SF-1, gonadotrope-specific element/SF-1-binding site (Barnhart & Mellon 1994); GATA, GATA-binding element (Steger et al. 1994); CRE, cyclic AMP-responsive element (Delegeane et al. 1987); JRE, junctional response element (Andersen et al. 1990); TRE, thyroid hormone responsive element (Attardi et al. 1995); CArG, CArG box (Weck et al. 2000); NF-Y (Keri et al. 2000); EGR-1 (Tremblay & Drouin 1999); Ptx-1 (Tremblay et al. 1998); SF-1 (LHß) (Brown & McNeilly 1997); TATA: TATA-box.

View this table: [in this window] [in a new window]   Table 1 Primers used to generate porcine GSU and LHß vectors and FAM-labeled GSU fragment

View this table: [in this window] [in a new window]   Table 2 Synthetic oligonucleotides used to generate porcine GSU vectors containing putative Prop-1-binding elements and mutations in these elements

Cell culture, transfection, and reporter gene assay

LßT2 and GH3 cells were used for transient transfection assay. LßT2 cells, which endogenously express gonadotropin genes of GSU, LHß, and FSHß (Pernasetti et al. 2001), are the pituitary gonadotrope cell line established by targeted oncogenesis in transgenic mice (Alarid et al. 1996) and were kindly provided by Dr P Mellon. GH3 cells are the clonal somatomammotrope cell line of the rat pituitary (Bancroft et al. 1969), which endogenously express several pituitary specific transcription factors, including Pit-1 and pituitary hormones of GH and PRL, but not GSU, FSHß, or LHß. GH3 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). The maintenance of cells was performed in monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (Sigma-Aldrich Co.) for LßT2 cells or in DMEM/F-12 medium (GIBCO-BRL) supplemented with 10% (v/v) horse serum (JRH Biosciences, Lenexa, KS, USA), 2.5% (v/ v) FBS (JRH Biosciences), and antibiotics (Sigma-Aldrich Co.) for GH3 cells in humidified 5% CO₂–95% air at 37 °C. LßT2 cells were plated at 1.0 x 10⁴ cells/ 100 µl per well in a 96-well plate (Corning Inc., Corning, NY, USA). Then, 24 h after seeding, cells were transfected with the complex of 0.2 µl Lipofectamine2000 (Invitrogen) and 20 ng reporter vector or pSEAP2-Basic DNA with or without Prop-1/pcDNA3.1 per well in quadruplicate (n = 4) in two independent experiments according to the manufacturer’s instructions. In GH3 cells, the complex of 0.3 µl FuGENE6 (Roche Diagnostics GmbH) and 10 ng reporter vectors or pSEAP2-Basic DNA was used. After incubation for more than 24 h, an aliquot (5 µl) of cultured medium was assayed for SEAP activity using the Phospha-Light Reporter Gene Assay System (PE Applied Biosystems) according to the manufacturer’s instructions with a MiniLumat LB 9506 luminometer (Berthold, Wildbad, Germany). All values were expressed in mean ± S.D. of quadruplicate transfections in two independent experiments. Statistical significance was calculated by Student’s t-test.

Electrophoretic mobility shift assay

Production and purification of the Trx/His-tag fused recombinant porcine Prop-1 and Trx/His-tag protein (Tag protein) were carried out as previously described (Aikawa et al. 2004). Purified recombinant porcine Prop-1 and Tag protein were analyzed on 10% SDS-PAGE followed by staining with Bio-Safe Coomassie Blue G-250 (Bio-Rad Laboratories). To accomplish EMSA, FAM-labeled DNA fragments (Fig. 1C) were produced by PCR using 5' FAM-labeled oligonucleotide primer (Table 1). The binding reaction mixture included 100 ng recombinant porcine Prop-1 or Tag protein, 100 fmol FAM-labeled DNA and 2 µg poly(dI-dC) in 10 µl of 10 mM HEPES buffer (pH 7.9), containing 0.4 mM MgCl₂, 0.4 mM DTT, 50 mM NaCl, and 4% glycerol, and was incubated at 30 °C for 30 min. Samples were then subjected to electrophoresis on a 4% polyacrylamide gel as described in our previous paper (Kato et al. 1999).

DNase I footprinting assay

The 5' FAM-labeled DNA fragments were incubated with 200 or 400 ng recombinant porcine Prop-1 in binding buffer under the same conditions used for EMSA. After a 30-min incubation at 30 °C, 0.2 or 0.4 U RQ1 RNase-Free DNase (Promega) was added, and the mixture was incubated for 5 min at 25 °C. The reaction was stopped by the addition of EDTA to a final concentration of 100 mM, and proteins were then removed by phenol–chloroform extraction. DNA fragments were precipitated, dissolved in 10 µl formamide containing 0.5 µl ROX-labeled GS-500 (PE Applied Biosystems) as a molecular size marker, and analyzed on a GeneScan analyzer equipped with an ABI PRISM 310 (PE Applied Biosystems).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Prop-1 activates porcine GSU promoter, but not porcine LHß promoter

Several regulatory elements of the porcine GSU promoter (Kato et al. 1991) and the porcine LHß promoter (Ezashi et al. 1990) have been identified in the proximal region (Fig. 1A and D). To investigate whether Prop-1 regulates the transcription of the GSU and LHß genes, we constructed each series of reporter vectors by fusing sequential deletion mutants of the promoter regions, –1059/+12 bp for GSU (Fig. 1B) and –1277/+7 bp for LHß (Fig. 1E), to pSEAP2-Basic and assayed the promoter activities by transfection in two pituitary cell lines, LßT2 cells and GH3 cells, with or without Prop-1/pcDNA3.1. The real-time PCR analysis using total RNA isolated from cultured cells revealed that Prop-1 gene expresses in LßT2 cells, but not in GH3 cells (Aikawa et al. 2006).

In LßT2 cells, as shown in Fig. 2, the basal promoter activity of porcine GSU was 47-, 89-, 58-, and 7-fold higher than that of pSEAP2-Basic, for GSU(–1059/+ 12), GSU(–798/+12), GSU(–540/+12), and GSU(–239/+12) respectively. GSU(–100/+12) and GSU(–53/+12) showed the same basal level as that of pSEAP2-Basic. In the presence of Prop-1/ pcDNA3.1, the promoter activity in reporter vectors of GSU promoter up to –239 bp was not enhanced. The promoter activity of GSU(–540/+12), in spite of the high basal level, was enhanced significantly to 1.4-fold (P < 0.01) by Prop-1 and the further distal promoter was also likely to be enhanced. However, the basal promoter activity of porcine LHß gene in LßT2 cells was lower than or equal to that of pSEAP2-Basic without obvious enhancement by Prop-1.

View larger version (15K): [in this window] [in a new window]   Figure 2 Transient transfection assay of porcine GSU and LHß promoters in LßT2 cells. Truncated promoters of (A) GSU and (B) LHß fused with SEAP gene in pSEAP2-Basic vector (shown in left panel) were transfected in LßT2 cells with/without Prop-1 expression vector. An aliquot of cultured medium was used for SEAP assay. Reporter gene activities are indicated relative to pSEAP2-Basic vector. Shaded and solid bars indicate values with pcDNA3.1 and Prop-1/pcDNA3.1 respectively. Data (mean ± S.D.) are means of quadruplicate transfections in two independent experiments. Asterisk (*) indicates statistical significance by Student’s t-test (P < 0.01).

View larger version (17K): [in this window] [in a new window]   Figure 3 Transient transfection assay of porcine GSU and LHß promoters in GH3 cells. Truncated promoters of (A) GSU and (B) LHß (shown in left panel) fused with SEAP gene in pSEAP2-Basic vector were transfected in GH3 cells with/without Prop-1 expression vector, followed by assaying reporter gene activities and processing data as described in Fig. 2.

Prop-1 binds to GSU promoter

Recombinant porcine Prop-1 and Tag protein were isolated from E. coli, and the purities were confirmed by 10% SDS-PAGE (Fig. 4A). The binding activity of recombinant porcine Prop-1 was assayed in comparison with Tag protein as a negative control using FAM-labeled GSU fragments (Fig. 1C) by EMSA in the presence of about a 200-fold excess amount of poly(dI-dC) as a competitor to prevent nonspecific binding (Fig. 4B). Prop-1 certainly binds to –1059/– 740, –540/–190, and –239/+12 bp fragments, giving multiple shift bands. Only small amounts of shift bands were observed in –798/–501 bp fragment. Since Tag protein did not give any shift band, these shift bands were created by the binding with Prop-1 itself. These binding features are consistent with the results of the transfection experiment described earlier.

View larger version (46K): [in this window] [in a new window]   Figure 4 EMSA of Prop-1 binding of GSU gene promoter. (A) Protein used for EMSA was analyzed on 10% SDS-PAGE. Lane 1, protein molecular marker (in kilo Daltons, kDa); lane 2, Trx/His-tag protein (20 kDa); lane 3, Trx/His-tag fused Prop-1 (43 kDa). (B) FAM-labeled fragments of porcine GSU gene were analyzed on 4% PAGE after binding with recombinant Prop-1 or Tag protein under the condition described in Materials and methods. Arrow () indicates the shift band.

Prop-1 binds to A/T rich sequences in GSU promoter

Transfection assay and EMSA demonstrated the presence of responsive elements for Prop-1 in the GSU promoter between –1059 and –101 bp. However, there was no consensus Prop-1-binding site, so-called PRDQ9 TAATtgaATTA (Sornson et al. 1996), in this region. DNase I footprinting analysis was performed to determine the nucleotide sequence of Prop-1-binding sites using 5' FAM-labeled fragments of the GSU promoter. FAM-labeled DNA fragments with or without the binding of Prop-1 were digested by DNase I, and the digested fragments were recovered, followed by separation on capillary electrophoresis and analysis on the GeneScan Analyzer using ABI PRISM 310 (Fig. 5A–E). In the distal region over –800 bp, a loss of signals caused by the protection against DNase I digestion by Prop-1 binding was observed in the region –1038/–1026 bp (5'-ACTAATTCATATC-3') (Fig. 5A), and an increase in signals caused by conformational change against DNase I digestion by Prop-1 binding was found in the region –1020/–1015 bp. The region –942/–928 bp (5'-AAGAAATCAACTTAT-3'; Fig. 5B) also lost signals, and an increase in signals was found in the adjacent region at –927/–921 bp. In the region –540/–190 bp, which showed maximal binding to Prop-1, decreased signals were observed in the regions –495/–479 bp(5'-CATCCTTATTAAATCCA-3'; Fig. 5C), and –338/–326 bp (5'-AGCTAATTAAATG-3'; Fig. 5D). It is noteworthy that the latter sequence overlaps with that of Lhx2-binding site (5'-TACTTAGC-TAATTA-3', –343/–330; Roberson et al. 1994). In addition, the increased signals by Prop-1 binding appeared in the regions –476/–474 and –325/– 314 bp. Finally, in the proximal region, decreased signals were observed in the regions –153/–146 bp (5'-AGATAAGA-3') and –131/–124 bp (5'-TGGTAATT-3'; Fig. 5E). Adjacent to these regions, the increased signals were also observed in –119/–113 bp. The DNase I footprinting analysis for –798/–501 bp did not show any marked change of signals (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 5 DNase I footprinting analyses. DNase I digests were prepared with or without recombinant porcine Prop-1 (lower panel and upper panel respectively), followed by analysis using a capillary sequencer with GeneScan system. Solid bar indicates Prop-1-binding region. Nucleotide sequence and number are shown below and summarized in Table 3. Gray bar indicates signal increased by Prop-1 binding. Nucleotide sequence and number are shown in box.

View this table: [in this window] [in a new window]   Table 3 Summary of Prop-1-binding sequences in porcine GSU promoter

DNase I footprinting demonstrated that Prop-1 strongly binds to the region –338/–326 bp located in PGBE (–345/–301 bp; Schoderbek et al. 1993) with marked sensitivity to DNase I (Fig. 5D). In addition, five other Prop-1-binding sites were identified as described earlier. Hence, each cis-acting activity regardless of its position was examined using the consensus PRDQ9 as a positive control. We generated the reporter vectors constructed by directly ligating the wild type and mutated sequences of Prop-1-binding site and PRDQ9 to the minimum GSU promoter vector, GSU(–53/+12), followed by a transfection assay of GH3 cells with/without Prop-1/ pcDNA3.1 (Fig. 6). Synthetic oligonucleotides used to generate wild type and mutated-binding sequences were listed in Table 2. With PRDQ9, Prop-1 activated the SEAP gene expression up to 2.3-fold, and the mutation decreased its activation to the level of GSU(–53/+12; Fig. 6A). The expression levels of SEAP gene in the reporter vector constructed with wild-type sequence of other six Prop-1-binding sites were increased to 1.8- to 3.4-fold by Prop-1 (Fig. 6B). The mutation generated in five binding sites significantly decreased the Prop-1 activation level to almost the same level of GSU(–53/+12), though GSU(–359/– 319)-m still preserved substantial activity. However, GSU(–503/–474) showed a decrease from 1.8- to 1.6-fold, which was not significant. The mutation introduced to –1059/–1015, –946/–909, –163/–142, and –147/–114 bp led to a loss of Prop-1 activation, indicating that TAAT in –1059/– 1015, AAAT and TTAT in –946/–909, ATAA in –163/–142, and TAATT in –147/–114 bp are the core sequences of Prop-1 activation. On the other hand, the mutation introduced to –359/–319 bp, in which the maximum induction by Prop-1 was observed remained the Prop-1 activation to show only a 20% decrease in expression level, indicating that not only TAAT but also other A/T-rich sequences contribute to Prop-1 activation in the –359/–319 bp region. These data revealed that at least five of six Prop-1-binding sites play the role of a cis-acting element, regardless of their position.

View larger version (29K): [in this window] [in a new window]   Figure 6 Cis-acting activity of Prop-1-binding sites. Transient transfection assays of reporter vectors constructed by connecting synthesized Prop-1-binding sites to minimum GSU promoter vector, GSU(–53/+12), were performed using GH3 cells with/without Prop-1 expression vector. An aliquot of cultured medium was used for SEAP assay. Reporter gene activities are indicated relative to pSEAP2-Basic vector. Shaded and solid bars indicate values with pcDNA3.1 and Prop-1/pcDNA3.1 respectively. Data (mean ± S.D.) are means of quadruplicate transfections in two independent experiments. (A) PRDQ9 and its mutant. (B) Prop-1-binding sites in comparison with mutants (nucleotide sequences are described in Table 2). Asterisk (*) indicates statistical significance by Student’s t-test (P < 0.01). NS means nonsignificance.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The GSU gene expresses in a cell type or tissue-specific manner by several trans-acting factors interacting with their respective cis-acting elements (Maurer et al. 1999). The major regulatory elements identified are shown in Fig. 1A. This study revealed that Prop-1 binds to several positions and promotes GSU promoter activity. Our comprehensive investigations using transfection assay, EMSA, DNase I footprinting, and estimations of cis-acting activities demonstrated that Prop-1 binds to six cis-acting elements, i.e., –1038/–1026, –942/–928, –495/–479, –338/– 326, –153/–146, and –131/–124 bp.

In the distal region, no information on typical regulatory elements has yet been provided, while we recently demonstrated the importance of the distal region of porcine GSU promoter for the high level and cell-type-specific expression of GSU gene (Aikawa et al. 2005a). Indeed, the study of cis-acting activity revealed two cis-acting elements of Prop-1 located at –1038/–1026 and –942/–928 bp. The mutation of the region at –503/–474 showed a nonsignificant decrease of Prop-1-dependent activation (1.8-fold) to 1.6-fold, indicating that its activity in this region, if any, is weak. This finding may provide new knowledge that demonstrates the regulatory factor and its target sequence in the distal region of GSU promoter. In comparison with the nucleotide sequences of the corresponding positions among other mammalian GSU promoters, this porcine sequence is not well conserved, indicating a possibility of species specificity.

The proximal region is well conserved among mammalian species, containing a number of regulatory elements important for the basal transcription of GSU gene (Delegeane et al. 1987, Silver et al. 1987, Jameson et al. 1988, Heckert et al. 1995). The present study revealed that three cis-acting elements of Prop-1 located in the proximal region at –338/–326, –153/–146, and –131/–124 bp. These three elements were compared with the elements already identified in the proximal region of human GSU (Fig. 7). The element –338/–326 bp mostly overlapped the LIM homeodomain transcription factor-binding element (Roberson et al. 1994), which is characterized as an important regulatory element for the basal transcription of GSU gene in the pituitary. The element –153/–146 bp completely overlapped with GATA (element for GATA-binding proteins; Steger et al. 1991). Another Prop-1-binding site, –131/–124 bp, overlaid half of the junctional response element (JRE), which is originally characterized as an important element for placenta-specific expression (Andersen et al. 1990) and recognized by the homeobox factor, Distal-less 3 (Dlx 3; Roberson et al. 2001). Our study further reveals an interesting feature of the regulatory mechanism of GSU gene expression, i.e., that three of the Prop-1-binding sites are shared with other transcription factors, LIM homeodomain transcription factors, GATA-binding proteins, and JRE-binding proteins. The interaction or synergy with other regulatory factors is of interest for future investigation.

View larger version (9K): [in this window] [in a new window]   Figure 7 Nucleotide sequence of proximal region of porcine and human GSU promoter. Prop-1-binding sites identified in porcine sequence are shown in reversed type. Nucleotide numbers from transcription start sites are shown above (pig) and under (human) sequences. Transcription factor-binding sites and regulatory elements are boxed. Arrow-indicated regions are regulatory regions. LIM-HD, LIM homeodomain transcription factor-binding site (Roberson et al. 1994); PGBE, pituitary glycoprotein hormone basal element (Schoderbek et al. 1993); GSE/SF-1, gonadotrope-specific element/SF-1-binding site (Barnhart & Mellon 1994); TSE, trophoblast-specific element (Steger et al. 1991); GATA, GATA-binding element (Steger et al. 1991); URE, upstream regulatory element (Jameson et al. 1988); CRE, cyclic AMP-responsive element (Delegeane et al. 1987); JRE, junctional response element (Andersen et al. 1990); CCAAT, CCAAT box (Jameson et al. 1988); DRE, distal regulatory element (Budworth et al. 1997).

Generally, most homeodomain factors recognize nucleotide sequences containing a TAAT core motif (Catron et al. 1993, Damante et al. 1994, Jagla et al. 1994, Pomerantz & Sharp 1994). Prop-1 is a homeodomain transcription factor and known to bind to the consensus sequence, PRDQ9 (5'-TAATtgaATTA-3', a palindromic sequence of a TAAT core motif; Sornson et al. 1996). However, the binding regions of Prop-1 found in the porcine GSU promoter show a variety of sequences and lengths (Table 3). Four of the six sequences contain a TAAT motif, and the others contain A/T clusters of 5'-AAAT-3' or 5'-TTAT-3'/5'-ATAA-3'. Prop-1 certainly recognizes these sequences, and stimulates the promoter activity of GSU except for –495/–479 (Fig. 6). Taken together, Prop-1 may have broader binding properties. In fact, the systematic evolution of ligands by exponential enrichment (SELEX) analysis for porcine Prop-1 demonstrated that Prop-1 can bind firmly to a variety of AT-rich sequences, though the strongest binding sequence is 5'-TAATnnnATTA-3' (personal communication). Mutation analysis of sites –946/–909, –163/–142, and –147/–114 also confirmed that Prop-1 binds to AAAT/ATTT, ATAA/TTAT, and TAAT/ATTA.

In the development of the pituitary gland, Prop-1 expression is first detected on e10–10.5 at the dorsal portion of the gland and reaches a maximum by e12, after which its expression expands to the full caudomedial area, where Pit-1 is first expressed on e13.5 (Sornson et al. 1996). On the other hand, GSU expression is first detected on e11.5 in the anteroventral aspect of Rathke’s pouch and expands to the pars tuberalis by e12.5. After e13.5, dorsal and lateral gradients of its expression appear in cells of the anterior lobe with increasing age, although GSU-expressing cells remain in the pars tuberalis (Japon et al. 1994, Lanctot et al. 1999). This expression pattern of GSU coincides with that of TSHß expression that first appears transiently in the pars tuberalis on e12.5 prior to preceding Pit-1 gene activation, and then appears in another region of the anterior lobe on e15.5. This transient TSHß expression in the pars tuberalis is Pit-1-independent and disappears by the day of birth (Japon et al. 1994). Thyrotrope embryonic factor is a candidate for potential activator of TSHß expression in the pars tuberalis, and Pit-1 is required to activate TSHß gene in the caudomedial area (Lin et al. 1994). Although many transcription factors for activating GSU expression have been reported as reviewed in Savage et al.(2003), factors which serve as an activator of GSU expression limited to the pars tuberalis have not been clarified. GSU expression in the anterior lobe appears on e13.5 when and where Pit-1 expression is introduced by Prop-1. This expression pattern of GSU in the developmental pituitary together with our data in this report indicates that Prop-1 might be one of the transcription factors that activates the limited expression of GSU in the anterior lobe of the pituitary.

Several findings support the Prop-1 function in an adult pituitary. We histochemically demonstrated the presence of Prop-1 in the adult porcine gonadotrope and other cells (Aikawa et al. 2004). Furthermore, Prop-1 mRNAs are detected in the fetal as well as the postnatal pituitaries (Aikawa et al. 2005b), and in the pituitary tumor-derived cell lines LßT2 and LßT4 (Aikawa et al. 2006). In the adult human pituitary, Prop-1 mRNAs have also been detected (Nakamura et al. 1999). Taken together, Prop-1 might play multiple roles in the early stage of pituitary development and in the control of GSU and FSHß gene expressions in postnatal pituitaries.

In summary, this study demonstrated for the first time that the paired-like homeodomain transcription factor Prop-1 participates in the regulation of GSU gene but not that of LHß gene. Together, with our preceding study, showing that Prop-1 directly regulates FSHß gene (Aikawa et al. 2004), we have now demonstrated that Prop-1 modulates the synthesis of FSH at the transcriptional level.

	Acknowledgements

 
This research was partially supported by the Ministry of Education, Culture Sports, Science and Technology, Grant-in-Aid for Scientific Research (B) Nos 12470217 and 12557087, and by the Institute of Science and Technology, Meiji University, Grant-in-Aid for Research Grant (A) to Y K. Our study was also supported by the ‘High-Tech Research Center’ Project for Private Universities: matching fund subsidy, 2001–2005, from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Aikawa S, Kato T, Susa T, Tomizawa K, Ogawa S & Kato Y 2004 Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone ß subunit gene expression. Biochemical and Biophysical Research Communications 324 946–952.[CrossRef][Web of Science][Medline]

Aikawa S, Susa T, Sato T, Kitahara K, Kato T & Kato Y 2005a Transcriptional activity of the 5' upstream region of the porcine glycoprotein hormone alpha subunit gene. Journal of Reproduction and Development 51 117–121.[CrossRef][Web of Science]

Aikawa S, Susa T, Sato T, Kitahara K, Ono T, Suzuki K, Nakayama M, Kato T & Kato Y 2005b Prop-1 activation of porcine FSHß subunit gene requires AT-rich recognition sites. Japanese Journal of Reproductive Endocrinology 10 43–48.

Aikawa S, Sato T, Ono T, Kato T & Kato Y 2006 High level expression of Prop-1 gene in gonadotropic cell lines. Journal of Reproduction and Development 52 195–201.[CrossRef][Web of Science]

Alarid ET, Windle JJ, Whyte DB & Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122 3319–3329.[Abstract]

Andersen B, Kennedy G & Nilson JH 1990 A cis-acting element located between the cAMP responsive elements and CCAAT box augments cell-specific expression of the glycoprotein hormone subunit gene. Journal of Biological Chemistry 265 21874–21880.[Abstract/Free Full Text]

Attardi B, Klatt B & Little G 1995 Repression of glycoprotein hormone alpha-subunit gene expression and secretion by activin in alpha T3-1 cells. Molecular Endocrinology 9 1737–1749.[Abstract/Free Full Text]

Bancroft FC, Levine L & Tashjian AH Jr 1969 Control of growth hormone production by a clonal strain of rat pituitary cells. Stimulation by hydrocortisone. Journal of Cell Biology 43 432–441.[Abstract/Free Full Text]

Barnhart KM & Mellon PL 1994 The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone -subunit gene in pituitary gonadotropes. Molecular Endocrinology 8 878–885.[Abstract/Free Full Text]

Brown P & McNeilly AS 1997 Steroidogenic factor-1 (SF-1) and the regulation of expression of luteinising hormone and follicle stimulating hormone ß-subunits in the sheep anterior pituitary in vivo. International Journal of Biochemistry and Cell Biology 29 1513–1524.[CrossRef][Web of Science][Medline]

Brown P & McNeilly AS 1999 Transcriptional regulation of pituitary gonadotrophin subunit genes. Reviews of Reproduction 4 117–124.[Abstract]

Budworth PR, Quinn PG & Nilson JH 1997 Multiple characteristics of a pentameric regulatory array endow the human alpha-subunit glycoprotein hormone promoter with trophoblast specificity and maximal activity. Molecular Endocrinology 11 1669–1680.[Abstract/Free Full Text]

Catron KM, Iler N & Abate C 1993 Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins. Molecular and Cellular Biology 13 2354–2365.[Abstract/Free Full Text]

Damante G, Fabbro D, Pellizzari L, Civitareale D, Guazzi S, Polycarpou-Schwartz M, Cauci S, Quadrifoglio F, Formisano S & Di Lauro R 1994 Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain. Nucleic Acids Research 22 3075–3083.[Abstract/Free Full Text]

Delegeane AM, Ferland LH & Mellon PL 1987 Tissue-specific enhancer of the human glycoprotein hormone -subunit gene: dependence on cyclic AMP-inducible elements. Molecular and Cellular Biology 7 3994–4002.[Abstract/Free Full Text]

Ezashi T, Hirai T, Kato T, Wakabayashi K & Kato Y 1990 The gene for the ß subunit of porcine LH: clusters of GC-boxes and CACCC elements. Journal of Molecular Endocrinology 5 137–146.[Abstract/Free Full Text]

Fiddes JC & Goodman HM 1981 The gene encoding the common alpha subunit of the four human glycoprotein hormones. Journal of Molecular and Applied Genetics 1 3–18.[Medline]

Heckert LL, Schultz K & Nilson JH 1995 Different composite regulatory elements direct expression of the human alpha subunit gene to pituitary and placenta. Journal of Biological Chemistry 270 26497–26504.[Abstract/Free Full Text]

Jagla K, Stanceva I, Dretzen G, Bellard F & Bellard M 1994 A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster. Nucleic Acids Research 22 1202–1207.[Abstract/Free Full Text]

Jameson JL, Jaffe RC, Deutsch PD, Alanese C & Habener JF 1988 The gonadotropin -gene contains multiple protein binding domains that interact to modulate basal and cAMP-responsive transcription. Journal of Biological Chemistry 263 9879–9886.[Abstract/Free Full Text]

Japon MA, Rubinstein M & Low MJ 1994 In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development. Journal of Histochemistry and Cytochemistry 42 1117–1125.[Abstract]

Jorgensen JS, Quirk CC & Nilson JH 2004 Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone. Endocrine Reviews 25 521–542.[Abstract/Free Full Text]

Kato Y, Imai K, Sakai T & Inoue K 1989 Simultaneous effect of gonadotropin-releasing hormone (GnRH) on the expression of two gonadotropin ß genes by passive immunization to GnRH. Molecular and Cellular Endocrinology 62 47–53.[CrossRef][Web of Science][Medline]

Kato Y, Ezashi T & Kato T 1991 The gene for the common subunit of porcine glycoprotein hormone. Journal of Molecular Endocrinology 7 27–34.[Abstract/Free Full Text]

Kato Y, Tomizawa K & Kato T 1999 Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5'-flanking region of the porcine follicle-stimulating hormone (FSH) beta-subunit gene. Molecular and Cellular Endocrinology 158 69–78.[CrossRef][Web of Science][Medline]

Keri RA, Bachmann DJ, Behrooz A, Herr BD, Ameduri RK, Quirk CC & Nilson JH 2000 An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone beta subunit gene. Journal of Biological Chemistry 275 13082–13088.[Abstract/Free Full Text]

Lanctot C, Gauthier Y & Drouin J 1999 Pituitary homeobox 1 (Ptx1) is differentially expressed during pituitary development. Endocrinology 140 1416–1422.[Abstract/Free Full Text]

Lin SC, Li S, Drolet DW & Rosenfeld MG 1994 Pituitary ontogeny of the Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of the thyrotrope. Development 120 515–522.[Abstract]

Maurer RA, Kim KE, Schoderbek WE, Roberson MS & Glenn DJ 1999 Regulation of glycoprotein hormone alpha-subunit gene expression. Recent Progress in Hormone Research 54 455–484.[Medline]

Nakamura Y, Usui T, Mizuta H, Murabe H, Muro S, Suda M, Tanaka K, Tanaka I, Shimatsu A & Nakao K 1999 Characterization of prophet of Pit-1 gene expression in normal pituitary and pituitary adenomas in humans. Journal of Clinical Endocrinology and Metabolism 84 1414–1419.[Abstract/Free Full Text]

Papavasiliou SS, Zmeili S, Khoury S, Landefeld TD, Chin WW & Marshall JC 1986 Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone and ß subunits in male rats. PNAS 83 4026–4029.[Abstract/Free Full Text]

Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang HJ, Miller WL & Mellon PL 2001 Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LßT2 pituitary gonadotrope cell model. Endocrinology 142 62284–62295.

Pierce JG & Parsons TF 1981 Glycoprotein hormones: structure and function. Annual Review of Biochemistry 50 465–495.[CrossRef][Web of Science][Medline]

Pomerantz JL & Sharp PA 1994 Homeodomain determinants of major groove recognition. Biochemistry 33 10851–10858.[CrossRef][Medline]

Roberson MS, Schoderbek WE, Tremml G & Maurer RA 1994 Activation of the glycoprotein hormone -subunit promoter by a LIM-homeodomain transcription factor. Molecular and Cellular Biology 14 2985–2993.[Abstract/Free Full Text]

Roberson MS, Meermann S, Morasso MI, Mulvaney-Musa JM & Zhang T 2001 A role for the homeobox protein distal-less 3 in the activation of the glycoprotein hormone alpha subunit gene in choriocarcinoma cells. Journal of Biological Chemistry 276 10016–10024.[Abstract/Free Full Text]

Savage JJ, Yaden BC, Kiratipranon P & Rhodes SJ 2003 Transcriptional control during mammalian anterior pituitary development. Gene 319 1–19.[CrossRef][Web of Science][Medline]

Schoderbek WE, Roberson MS & Maurer RA 1993 Two different DNA elements mediate gonadotropin releasing hormone effects on expression of the glycoprotein hormone -subunit gene. Journal of Biological Chemistry 268 3903–3910.[Abstract/Free Full Text]

Silver BJ, Bokar JA, Virgin JB, Vallen EA, Milsted A & Nilson JH 1987 Cyclic AMP regulation of the human glycoprotein hormone subunit gene is mediated by an 18-base-pair element. PNAS 84 2198–2202.[Abstract/Free Full Text]

Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carriere C, Ryan AK, Miller AP et al. 1996 Pituitary lineage determination by the prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 384 327–333.[CrossRef][Medline]

Steger DJ, Altschmied J, Buscher M & Mellon PL 1991 Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin -subunit genes. Molecular Endocrinology 5 243–255.[Abstract/Free Full Text]

Steger DJ, Hecht JH & Mellon PL 1994 GATA-binding proteins regulate the human gonadotropin -subunit gene in the placenta and pituitary gland. Molecular and Cellular Biology 14 5592–5602.[Abstract/Free Full Text]

Tang K, Bartke A, Gardiner CS, Wagner TE & Yun JS 1993 Gonadotropin secretion, synthesis, and gene expression in human growth hormone transgenic mice and in Ames dwarf mice. Endocrinology 132 2518–2524.[Abstract/Free Full Text]

Tremblay JJ & Drouin J 1999 Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance luteinizing hormone beta gene transcription. Molecular and Cellular Biology 19 2567–2576.[Abstract/Free Full Text]

Tremblay JJ, Lanctot C & Drouin J 1998 The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit-1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Molecular Endocrinology 12 428–441.[Abstract/Free Full Text]

Weck J, Anderson AC, Jenkins S, Fallest PC & Shupnik MA 2000 Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes. Molecular Endocrinology 14 472–485.[Abstract/Free Full Text]

Wu W, Cogan JD, Pfaffle RW, Dasen JS, Frisch H, O’Connell SM, Flynn SE, Brown MR, Mullis PE, Parks JS et al. 1998 Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genetics 18 147–149.[CrossRef][Web of Science][Medline]

Received 22 June 2006
Accepted 28 June 2006

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