Cell and Tissue Research

, Volume 322, Issue 2, pp 269–277

Induction of GH, PRL, and TSHβ mRNA by transfection of Pit-1 in a human pituitary adenoma-derived cell line

Authors

  • Shunsuke Miyai
    • Department of PathologyTokai University School of Medicine
  • Shinichi Yoshimura
    • Department of Molecular Life ScienceTokai University School of Medicine
  • Yasumasa Iwasaki
    • Department of Internal MedicineKochi University School of Medicine
  • Susumu Takekoshi
    • Department of PathologyTokai University School of Medicine
  • Ricardo V. Lloyd
    • Department of Laboratory Medicine and PathologyMayo Clinic and Mayo Foundation
    • Department of PathologyTokai University School of Medicine
Regular Article

DOI: 10.1007/s00441-005-0033-z

Cite this article as:
Miyai, S., Yoshimura, S., Iwasaki, Y. et al. Cell Tissue Res (2005) 322: 269. doi:10.1007/s00441-005-0033-z

Abstract

The functional development of pituitary cells depends on the expression of a combination of transcription factors and co-factors. Pituitary-specific transcription factor-1 (Pit-1) is required for the expression of growth hormone (GH), prolactin (PRL), and the thyroid-stimulating hormone β subunit (TSHβ) and acts synergistically with the estrogen receptor (ER) and GATA-binding protein 2 (GATA-2) to induce PRL and TSHβ expression, respectively. The glycoprotein hormone α subunit (αSU) is the first hormone to be expressed during pituitary development. In addition to being expressed in follicle-stimulating hormone, luteinizing hormone (LH), and TSH cells, αSU is reported to co-localize with GH in pituitary cells. These findings have led to the suggestion that the expression of Pit-1 in cells of the αSU-based gonadotropin cell lineage might also lead to the expression of GH. In this study, we transfected HP75 cells (derived from a human non-functioning pituitary adenoma that expressed αSU and LHβ) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSHβ mRNA. The HP75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSHβ mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active αSU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSHβ expression in the αSU-based gonadotroph lineage.

Keywords

Pituitary adenomasDifferentiationPit-1Glycoprotein α subunitTranscription factorHuman

Abbreviations

αSU

glycoprotein hormone alpha subunit

GH

growth hormone

PRL

prolactin

TSH

thyroid-stimulating hormone

POMC

pro-opiomelanocortin

ACTH

adrenocorticotropic hormone

FSH

follicle-stimulating hormone

LH

luteinizing hormone

Pit-1

pituitary-specific transcription factor-1

ER

estrogen receptor

Tpit

pituitary cell-restricted T-box factor

NeuroD1

neurogenic differentiation 1

SF-1

steroidogenic factor 1

GATA-2

GATA-binding protein 2

DAX1

dosage-sensitive sex-reversal, adrenal hypoplasia congenital, X chromosome

Ptx1

pituitary homeobox 1

LHX3

LIM homeobox protein 3

Prop-1

Prophet of Pit-1

GHRH-R

growth hormone-releasing hormone receptor

Introduction

The anterior pituitary consists of five distinct cell types, each of which produces distinct hormones (Simmons et al. 1990), arises from a single progenitor cell, and serves as a model for the investigation of the molecular mechanisms of cell differentiation (Dasen and Rosenfeld 2001). Several pituitary transcription factors and co-factors have been identified and are reported to be active in all or a subset of pituitary cell lineages during a specific period of development (Lefevre et al. 1987; Nelson et al. 1988; Lanctot et al. 1999; Gage et al. 1996; Barnhart and Mellon 1994; Poulin et al. 1997; Lamolet et al. 2001). For example, pituitary-specific transcription factor-1 (Pit-1) has been found to be required for the expression of growth hormone (GH), prolactin (PRL) and the thyroid-stimulating hormone β subunit (TSHβ) in somatotrope, lactotrope, and thyrotrope cells, respectively (Mangalam et al. 1989; Ingraham et al. 1990; Simmons et al. 1990). Pituitary cell types have been classified into the following three cell lineages based on the combination of transcription factors and co-factors that they possess: (1) GH-PRL-TSH cells, which contain Pit-1, (2) pro-opiomelanocortin (POMC) cells, which contain neurogenic differentiation 1 (NeuroD1)/pituitary cell-restricted T-box factor (Tpit), and (3) follicle-stimulating hormone (FSH)/lutenizing hormone (LH) cells, which contain steroidogenic factor 1 (SF-1)/GATA-binding protein 2 (GATA-2; Jong et al. 1994; Tremblay and Viger 2001; Chang et al. 1996; Tremblay et al. 1998).

Pituitary adenomas in humans are categorized based on clinical diagnosis and their ability to secrete hormones (Asa and Kovacs 1983; Sanno et al. 2001a). Human pituitary adenomas usually belong to one of the above three cell lineages (Suhardja et al. 2001), with some adenomas producing two or more hormones (Kovacs et al. 1996). These adenomas can be divided into monomorphous, bimorphous, or plurimorphous types. Monomorphous adenomas are defined as those that synthesize two or more hormones in the same cell (Thapar et al. 1995a,b). Multi-hormone production in monomorphous adenomas frequently includes combinations of GH, PRL, and TSHβ, or FSHβ and LHβ, which are derived from the same cell lineage that is dependent on Pit-1 and SF-1/GATA-2, respectively. In bimorphous and plurimorphous adenomas, each hormone is produced by a different cell population, and almost all possible combinations of hormone production involving GH and PRL, GH, PRL, glycoprotein hormone α subunit (αSU), TSHβ, and FSHβ have been observed (Felix et al. 1994; Furuhata et al. 1994; Gessl et al. 1994; Gil del Alamo et al. 1994; Bertholon Gregoire et al. 1999).

In normal human pituitary cells and in adenomas, some GH-producing cells have been reported as immunostaining positively for αSU and for FSHβ/LHβ (Osamura and Watanabe 1987). Furthermore, most clinically non-functioning adenomas that expressed Pit-1 protein are immunohistochemically positive for αSU (Sanno et al. 1996). As αSU is the first hormone that appears during fetal development in rodent pituitary glands (Horn et al. 1992; Tremblay et al. 1998), αSU-positive Pit-1-positive cells are thought to undergo differentiation within the GH cell lineage (Osamura et al. 1999; Scheme 1), and the expression of Pit-1 in αSU-based cells is postulated to play a key role in the development of GH/gonadotropin cells.
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Scheme 1

Proposed cell lineage for human pituitary cells (modified from Osamura et al. (1999). The appearance of Pit-1 in αSU-positive cells in clinically non-functioning human pituitary adenomas is speculated to account for the development of GH cells in the αSU-based gonadotroph lineage

This study has been designed to determine whether GH expression can be induced in αSU-positive gonadotrophs by transfection of Pit-1 transcripts. We have transfected the HP75 cell line established from a human non-functioning adenoma (Long Jin et al. 1998) and shown to express αSU, LHβ and various transcription factors including pituitary homeobox 1 (Ptx1), DAX1 (dosage-sensitive sex-reversal, adrenal hypoplasia congenital, X chromosome), GATA-2, and estrogen receptor β (ERβ) by immunocytochemistry and reverse transcription/polymerase chain reaction (RT-PCR). We have found that, in addition to GH mRNA, these transfected HP75 cells also express the mRNAs for PRL and TSHβ. These data lend credence to the previously proposed mechanism that Pit-1 plays a role in the development of GH-, PRL-, and TSH-producing cells within the αSU-based gonadotropin-producing cell lineage.

Materials and methods

Cell culture

HP75 cells were cultured in Dulbecco's modified essential medium (DMEM; 4.5 g/l glucose; GibcoBRL, Grand Island, N.Y., USA), supplemented with 15% horse serum (HS, Biowhittaker, Walkersville, Md., USA), 2.5% fetal bovine serum (FBS, GibcoBRL), and 100 U/l penicillin/streptomycin (GibcoBRL) in an atmosphere of 5% CO2-95% air at 37°C. COS-1 cells were cultured in DMEM supplemented with 10% FBS (GibcoBRL).

Adenoviral vector and infection

A chimera of the FLAG and Pit-1 genes was generated as follows. Human Pit-1 cDNA was amplified by RT-PCR from a human GH-secreting adenoma by using the hPit-1 primers, 5′- TTCGAATTCTATGAGTTGCCAAGCATTTACT-3′ and 5′- TTATCTAGATTTATCTGCACTCAAGATGTTCCT-3′. The RT-PCR product was subcloned into the HindIII/XbaI site of the pFLAG-CMV-4 plasmid (Sigma, St Louis, Mo., USA). The structure of the pFLAG-Pit-1 plasmid was verified by DNA sequencing with the pFLAG-CMV primers, 5′-AATGTCGTAATAACCCCGCCCCGTTGACGC-3′ and 5′-TATTAGGACAAG GCTGGTGGGCAC- 3′.

The adenoviral vector was prepared as follows. A replication-defective E1 and E3 recombinant adenoviral vector expressing the FLAG human Pit-1 gene was prepared using Adeno-X Expression System (Clontech, Palo Alto, Calif., USA). Briefly, the gene of interest was cloned into the multiple cloning site of the shuttle vector (pShuttle; Clontech) comprising a cytomegalovirus enhancer (pShuttle FLAG-hPit-1). The pShuttle FLAG-hPit-1 vector was then linearized by using two rare cutter enzymes (I-Ceu I and PI-Sce I) and ligated in vitro to the linearized Adeno-X Viral DNA (pre-cut with I-Ceu I and PI-Sce I). After additional digestion with the SwaI enzyme in order to remove nonrecombinant adenoviral DNA, the ligation mixture was transformed into competent Escherichia coli. Recombinant plasmid was purified, and HEK 293 cells were transfected with purified recombinant adenoviral DNA. The titer of the virus stock was assessed by using HEK 293 cells in a plaque-formation assay and was expressed as plaque-formation units (pfu). For the transfection, HP75 cells were infected for 2 h with FLAG-Pit-1 adenoviral vector at 90 pfu/cell in DMEM containing 15% HS and 2.5% FBS, with gentle agitation. The medium was then exchanged for growth medium (DMEM supplemented with 15% HS, 2.5% FBS), and the cells were incubated at 37°C in 5% CO2 until being assayed.

Luciferase assay

Transcription-promoting activity was assayed by using the Luciferase reporter system and pGH-Luc, which carries the rat GH promoter region fused to the Luciferase reporter gene (Niiori Onishi et al. 1999). The Luciferase assay was carried out as previously described (Kurotani et al. 2002). Briefly, COS-1 cells that were 70% confluent were transfected with pGH-Luc, adeno-Flag-Pit-1, or both vectors. Luciferase activity was then determined by using the Luciferase Reporter Assay System (Promega, Madison, Wis., USA) according to the manufacturer's instructions.

Immuno-blotting

Cells were collected by centrifugation (250g for 5 min), suspended in 0.1% NP40 in TRIS-EDTA (0.01 M TRIS-Hcl pH 7.2, 1 mM EDTA), and mixed gently. This cell lysate was centrifuged at 10,000g for 5 min, after which the supernatant (cytosol fraction) and precipitate (nuclear fraction) were separated. Each sample was solubilized in SDS sample buffer and boiled for 5 min. Equal amounts of total protein were separated on 12.5% polyacrylamide gels and transferred to a Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, Ill., USA). The membrane was treated with phosphate-buffered saline (PBS)-Tween 20 (PBST; 0.01 M PBS, 0.1% Tween 20) containing 5% non-fat dried milk (milk-PBST) overnight, followed by milk-PBST containing 2% normal HS for 30 min. The membrane was then incubated with either anti-FLAG M2 (Sigma) or anti-Pit-1 (Santa-Cruz, Heidelberg, Germany) antibody at 4°C overnight and washed in PBST. The membrane was incubated in milk-PBST with anti-rabbit IgG or anti-mouse IgG antibody for 30 min. Immunoreactive bands were developed by an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

RT-PCR protocol

Total RNA (5 μg) was isolated by using the TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and reverse-transcribed with Super Script II (Invitrogen) after treatment with DNaseI (Promega). PCR was carried out with AmpliTaq Gold (Applied Biosystems, Foster City, Calif., USA) according to the manufacturer's instructions. The primer sequences and the annealing temperatures for PCR amplification are listed in Table 1.
Table 1

Primers and annealing temperatures (LHX3 LIM homeobox protein 3; for other abbreviations, see text))

Gene

Sequence (5′ to 3′)

Annealing temp (°C)

Product size (bp)

Hormone

POMC

Forward

GAG GGC AAG CGC TCC TAC TCC

64

261

Reverse

GGG GCC CTC GTC CTT CTT CTC

αSU

Forward

AAG GAG CGC CAT GGA TTA C

60

394

Reverse

CAG CAG TCA TCA AGA CAG CAC

LHβ

Forward

GTC AAC ACC ACC ATC TGT GC

60

292

Reverse

GGG AGG GTC TTT AGA GGA AGA G

FSHβ

Forward

GCT GCA ATA GCT GTG AGC TG

60

284

Reverse

ATC AGT GCT GTC GCT GTC AC

TSHβ

Forward

GCA CAT CGA AAG GAG AGA GTG

60

238

Reverse

GCA CTT GCC ACA CTT ACA GC

PRL

Forward

CGT CCT GTC CCA CTA CAT CC

60

406

Reverse

CAT CTG CAG GGA TGG AAG TC

GH

Forward

ATC CAA CCT AGA GCT GCT CC

58

335

Reverse

ACT GCA CGA TGC GCA GGA AT

Transcription factor

Ptx1

Forward

ACC TCC ATG GAC GCC TTC AAG

58

290

Reverse

GCT GTT GTA CTG GCA CGC GT

LHX3α

Forward

ATG CTG CTG GAA ACG GGG CT

62

434

Reverse

TCA GAA CTG AGC GTG GTC TA

LHX3β

Forward

ATG GAG GCG CGC GGG GAG

62

370

Reverse

TCA GAA CTG AGC GTG GTC TA

Pit-1

Forward

ACA GCT GCT GAT TTC AAG CA

58

304

Reverse

ACA AAG CTC CTA CTT GCT CA

Prop-1

Forward

AAC CAG TAC CCC GAC ATC TG

62

175

Reverse

CTG GCA AGA AGC TGG AAA AG

GATA-2

Forward

CCC TAA GCA GCG CAG CAA GAC

58

163

Reverse

GAT GAG TGG TCG GTT CTG GCC

SF-1

Forward

GCA TCT TGG GCT GCC TGC AG

66

231

Reverse

CTT TGC CGT GCT GGA CCT GG

DAX1

Forward

AAG GAG TAC GCC TAC CTC AA

58

251

Reverse

TCC ATG CTG ACT GTG CCG AT

Tpit

Forward

CTT TTG CCA AAG CCT TCT TG

60

167

Reverse

AGC GGC ATA CTG GTA ATT GG

NeuroD1

Forward

AGT CCG CCT TAC GGT ACC ATG

58

448

Reverse

GAC AGT CAC TGT AAG CAC AG

ERα

Forward

TGG AGA TCT TCG ACA TGC TG

58

145

Reverse

TCC AGA GAC TTC AGG GTG CT

ERβ

Forward

GGA GTC TGG TCG TGT GAA G

58

167

Reverse

ACT TCA CCA TTC CCA CTT CG

Immunocytochemistry

Cells were cultured to approximately 70% confluency in 4-well Permanox slides, fixed in 4% paraformaldehyde in phosphate buffer for 20 min at room temperature, and pre-incubated in PBS containing 1% bovine serum albumin and 0.4% Triton X for 30 min. The cells were then incubated with antibodies specific for the following anterior pituitary hormones: anti-human GH (1:800), anti-human PRL (1:600), and anti-human adrenocorticotropic hormone (ACTH; 1:800; all DAKO, Carpinteria, Calif., USA); anti-human TSHβ (1:200), anti-human LHβ (1:200), and anti-human FSHβ (1:200; all Immunotech, Marseille, France); anti-human αSU antibody (1:100; Chemicon, Temecula, CA, USA). After being washed three times in PBS, the slides were incubated with horseradish-peroxidase-coupled second antibody (Dako Japan, Kyoto, Japan). Staining was visualized by using 0.05% 3, 3′-diaminobenzidine.

Results

Characterization of HP75 cell line

RT-PCR and immunocytochemical analysis were performed on wild-type HP75 cells to characterize them before they were transfected with the Flag-Pit-1 gene and showed that these cells expressed LHβ and αSU; FSHβ, GH, PRL and TSHβ were not detected (Fig. 1a,b). Furthermore, the transcription factors Ptx1, GATA-2, Dax1, and ERβ were detected by RT-PCR, whereas Pit-1, a key factor in the transcription of the GH, PRL, and TSHβ gene, was not (Fig. 1c). These results suggested that HP75 cells were not capable of producing GH, PRL, or TSHβ, and thus could serve as a valid model for determining whether a newly transfected Pit-1 gene could induce the expression of the genes regulated by this transcription factor.
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Fig. 1

Characterization of the HP75 cell line. a Immunocytochemical analysis of hormone production by HP75 cells revealed the presence of αSU and LHβ (brown). b RT-PCR analysis revealed the presence of LHβ (292 bp) and αSU (394 bp), whereas POMC (261 bp), FSHβ (384 bp), GH (335 bp), PRL (406 bp) and TSHβ (283 bp) were not detected (left 100-bp molecular marker ladder). c RT-PCR analysis of the following 12 pituitary-related transcription factor genes was carried out: lane 1 Ptx1 (290 bp), lane 2 LIM homeobox protein 3 α (LHX3α; 434 bp), lane 3 LHX3β (370 bp), lane 4 Pit-1 (304 bp),lane 5 Prop-1 (175 bp), lane 6 GATA-2 (163 bp), lane 7 SF-1 (231 bp), lane 8 DAX1 (251 bp), lane 9 Tpit (167 bp), lane 10 NeuroD1 (448 bp), lane 11 ERα (145 bp), lane 12 ERβ (167 bp). Ptx1 (lane 1), GATA-2 (lane 6), and Dax1 (lane 8) were expressed in these cells, whereas Pit-1, a key factor in the transcription for the GH, PRL, and TSHβ genes, was not (left 100-bp molecular marker ladder)

Construction and characterization of a Flag-Pit-1 fusion protein

A Flag-Pit-1 fusion gene was established in an adenoviral vector (Fig. 2a). Luciferase activity increased 20-fold when the adenoviral Flag-Pit-1 and pGH-Luc plasmids were co-transfected compared to when the pGH-Luc reporter gene was transfected (Fig. 2b).
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Fig. 2

Construction of the Flag-Pit-1 expression adenoviral vector and assessment of the transcription promoting activity of Flag-Pit-1. a The Flag and human Pit-1 cDNA fusion gene was ligated into Adeno-X Viral DNA comprising a cytomegalovirus (CMV) enhancer. b Effects of co-transfection of the adenoviral Flag-Pit-1 vector on pGH-Luc reporter gene expression in COS-1 cells. Luciferase activity was induced by approximately 20-fold when both plasmids (right bar) were transfected, compared with the case when only pGH-Luc (left bar) was transfected

Expression of Flag-Pit-1 in transfected HP75/Pit-1 cells

RT-PCR analysis revealed that HP75/Pit-1 cells expressed Flag/Pit-1 mRNA. The nucleotide sequence of the RT-PCR product was identical to that of Flag-Pit1 cDNA (Fig. 3a). Furthermore, immuno-blotting analysis of HP75/Pit-1 cell extracts also displayed immunoreactive bands for Flag and Pit-1 (Fig. 3b). Both anti-Flag and anti-Pit-1 antibodies reacted with the 33-kDa protein band in the nuclear fraction. This immunoreactive band was not observed in the non-transfected HP75 cell extract. These data suggested that transfected HP75/Pit-1 cells expressed the Flag-Pit-1 gene but not the gene for endogeneous Pit-1. Immunohistochemical analysis revealed labeling for Flag and Pit-1 in the nuclei of the HP75/Pit-1 cells (Fig. 3c); the morphology of these cells did not change as a result of transfection. Transfection efficiency, which was determined by counting the number of immunoreactive and non-immunoreactive cells, was estimated to be approximately 20%–30% in each experiment.
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Fig. 3

Expression of Flag-pit-1 in HP75/Pit-1 cells. a Flag-Pit-1 mRNA expression was analyzed in HP75/Pit-1 cells by RT-PCR (left 100-bp molecular marker ladder). b Immuno-blotting analysis for Flag-Pit-1 protein in HP75/Pit-1 cells. The Flag and Pit-1 33-kDa immunoreactive band (arrowhead) was only detected in nuclear fractions of HP75/Pit-1 cells (C cytoplasmic fraction, N nuclear fraction). c Immunocytochemical analysis of Flag-Pit-1 expression in HP75/Pit-1 cells (1–4 adeno Flag-Pit-1 infection group, 5–8 negative control group, 1, 5 immunostained for Flag, 2, 6 negative control for Flag staining, 3, 7 immunostained for Pit-1, 4, 8 negative control for Pit-1 staining). Immunoreactive signals for Flag (1) and Pit-1 (3) were localized in the nuclei of HP75 cells transfected with Flag-Pit-1 (×150 original magnification). Bars 100 μm

Characterization of HP75/Pit-1 cells

RT-PCR analysis revealed the presence of LHβ and αSU in the HP75/Pit-1 cells, suggesting that the ability of these cells to express their normal hormones was not affected by Flag-Pit-1 expression. This analysis also showed that these cells expressed mRNA for GH, PRL, and TSHβ (Fig. 4a). Transfections, which were carried out in four separate experiments, showed a frequency of GH induction of 75%, and a frequency of PRL and TSHβ induction of 25% (Fig. 4b).
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Fig. 4

RT-PCR detection of mRNA expression in the HP75/Pit-1 cell line. a mRNAs for LHβ and αSU were detected in both HP75 and HP75/Pit-1 cells. On the other hand, GH, PRL and TSHβ were only detected in HP75/Pit-1 cells (left 100-bp molecular marker ladder). b Hormone inductions for each transfection experiment (+ detected, not detected)

Discussion

Specific unique combinations of transcription factors participate in the differentiation of the individual pituitary cell lineages (Simmons et al. 1990; Lefevre et al. 1987; Nelson et al. 1988; Lanctot et al. 1999; Gage et al. 1996; Barnhart and Mellon 1994; Poulin et al. 1997; Lamolet et al. 2001; Dasen and Rosenfeld 2001). For example, Pit-1 is known to be a key factor in functional differentiation, promoting the production of GH, PRL, and TSHβ in the somatotrope, lactotrope, and thyrotrope lineages, respectively, in mice, rats, and humans (Mangalam et al. 1989; Ingraham et al. 1990; Li et al. 1990; Radovick et al. 1992). We have previously shown that human pituitary adenoma cells functionally differentiate into either the Pit-1 dependent cells, POMC (ACTH), or gonadotrope lineages (Sanno et al. 2001a; Umeoka et al. 2002; Oyama et al. 2001). Human pituitary adenomas are classified clinically as either functioning or non-functioning, the former including GH-producing adenomas (GHomas) and PRLomas, TSHomas, ACTHomas, and FSHomas. Many clinically non-functioning adenomas produce αSU, FSHβ, and LHβ (Kovacs 1991; Sanno et al. 1997; Sano and Yamada 1994). Multi-hormone production in pituitary adenoma cells has frequently been reported to include combinations of GH, PRL, and TSHβ, which are derived from the cell lineage that is dependent upon Pit-1 (Horvath et al. 1990; Sanno et al. 2001b).

In human pituitary adenomas, Pit-1 is reported to be highly expressed in GHoma and TSHoma, as anticipated (Sanno et al. 1996). Attention has also been paid to its expression in non-functioning adenomas, especially when the adenomas are positive for gonadotropin αSU and βSU. Nonfunctioning adenomas are currently thought to be composed of two groups, (1) glycoprotein-subunit-positive and multihormonal adenomas, and (2) adenomas with no hormone production (null cell adenomas). We previously reported that Pit-1 was expressed in 14 of 24 cases (58.3%) of nonfunctioning adenomas. Among these, all 10 cases of αSU-immunopositive adenomas expressed Pit-1 mRNA, whereas only 3 of 14 αSU-negative adenomas expressed Pit-1 mRNA (Sanno et al. 1996). Furthermore, the GH-producing cells in normal pituitary glands were immunohistochemically positive for αSU (Osamura et al. 1999; Childs et al. 2000), and FSHβ and LHβ co-localized with Pit-1 protein (Mukdsi et al. 2004). As αSU is the first hormone that appears during fetal development in the rodent pituitary gland (Horn et al. 1992; Tremblay et al. 1998), αSU-positive Pit-1-positive cells probably undergo differentiation within the GH cell lineage.

The present study was designed to determine whether hormone expression could be induced in αSU-positive human gonadotrophs by transfection of Pit-1 transcripts. Transfection was carried out in the HP75 cell line, which was established from a human non-functioning adenoma (Long Jin et al. 1998). Immunohistochemistry and RT-PCR showed that these cells expressed αSU and LHβ and various transcription factors including Ptx1, DAX1, GATA-2, and ERβ. HP75 cells transfected with Pit-1 expressed GH, PRL, and TSHβ mRNA, as detected by RT-PCR, although the expression of PRL and TSHβ was observed with a lower frequency than that of GH. These data suggest that Pit-1 lineage cells are derived from a subpopulation of gonadotrope cells. Furthermore, the normal expression of LHβ and αSU in HP75/Pit-1 cells is not affected by Flag-Pit-1 expression, suggesting that differentiated pituitary cells can redirect their fate and differentiate into alternative cell lineages.

Interestingly, GH mRNA was undetectable in a few transfections. The synergistic interaction of Pit-1 with thyroid hormone, retinoic acid (Palomino et al. 1998), GHRH-R signal, and CREB-binding protein (Cohen 2000) has been reported to play a role in GH gene transcription. Thus, other synergistic factors might similarly be involved in GH gene transcription. In our previous transfection studies of AtT-20 and αT3-1 cells, we found that only GH expression was induced by Pit-1 transfection (Kurotani et al. 2002). However, in HP75 cells, although infrequent, both PRL and TSHβ mRNA were induced by Pit-1 transfection. These latter mRNAs may have been inducible in HP75 cells because they contain ER and GATA-2, which are known to act synergistically with transfected Pit-1 to induce PRL (Shull and Gorski 1984) and TSHβ (Gordon et al. 1997) expression, respectively. These results support the hypothesis that pituitary cell function is determined by its combination of transcription factors and co-factors; the interactions of these transcription factors with their target genes remain to be determined.

The frequency of induction of GH (75%) and PRL and TSHβ (25%) varied in our study. We suspect that these differences in induction frequency were dependent on the number cells that expressed each hormone. Sometimes, we were unable to detect PRL and TSHβ expression following transfection, even though GH expression was observed. This may have been because only a few cells were induced to express these hormones or because the amount of hormone expression per cell was low. Another possibility is that the differences in hormone expression might have been attributable to transcriptional instability of the HP75 cell phenotype. When the HP75 cell line was initially established, it was reported to express FSHβ in addition to LHβ and αSU. Phenotypic instability of these cells could theoretically result in differences in the frequency of hormone production following Pit-1 transfection. However, this is unlikely to have had a significant impact on the induction of hormones in our experiments, since GH, PRL, and TSHβ were not observed in wild-type non-transfected HP75 cells; these hormones were only found to be expressed in the Pit-1-transfected cells.

In summary, this study has been designed to determine whether hormone expression can be induced in αSU-positive gonadotrophs by transfection of Pit-1 transcripts. Our data show that exogenous transfection of Pit-1 is sufficient to induce the transcription of the GH, PRL, and TSHβ genes in the HP75 cell line, supporting the hypothesis that these hormones can be induced in cells that possess an active αSU gene.

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© Springer-Verlag 2005