Induction of GH, PRL, and TSHβ mRNA by transfection of Pit-1 in a human pituitary adenoma-derived cell line
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.
KeywordsPituitary adenomasDifferentiationPit-1Glycoprotein α subunitTranscription factorHuman
glycoprotein hormone alpha subunit
pituitary-specific transcription factor-1
pituitary cell-restricted T-box factor
neurogenic differentiation 1
steroidogenic factor 1
GATA-binding protein 2
dosage-sensitive sex-reversal, adrenal hypoplasia congenital, X chromosome
pituitary homeobox 1
LIM homeobox protein 3
Prophet of Pit-1
growth hormone-releasing hormone receptor
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).
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
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.
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.
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).
Primers and annealing temperatures (LHX3 LIM homeobox protein 3; for other abbreviations, see text))
Sequence (5′ to 3′)
Annealing temp (°C)
Product size (bp)
GAG GGC AAG CGC TCC TAC TCC
GGG GCC CTC GTC CTT CTT CTC
AAG GAG CGC CAT GGA TTA C
CAG CAG TCA TCA AGA CAG CAC
GTC AAC ACC ACC ATC TGT GC
GGG AGG GTC TTT AGA GGA AGA G
GCT GCA ATA GCT GTG AGC TG
ATC AGT GCT GTC GCT GTC AC
GCA CAT CGA AAG GAG AGA GTG
GCA CTT GCC ACA CTT ACA GC
CGT CCT GTC CCA CTA CAT CC
CAT CTG CAG GGA TGG AAG TC
ATC CAA CCT AGA GCT GCT CC
ACT GCA CGA TGC GCA GGA AT
ACC TCC ATG GAC GCC TTC AAG
GCT GTT GTA CTG GCA CGC GT
ATG CTG CTG GAA ACG GGG CT
TCA GAA CTG AGC GTG GTC TA
ATG GAG GCG CGC GGG GAG
TCA GAA CTG AGC GTG GTC TA
ACA GCT GCT GAT TTC AAG CA
ACA AAG CTC CTA CTT GCT CA
AAC CAG TAC CCC GAC ATC TG
CTG GCA AGA AGC TGG AAA AG
CCC TAA GCA GCG CAG CAA GAC
GAT GAG TGG TCG GTT CTG GCC
GCA TCT TGG GCT GCC TGC AG
CTT TGC CGT GCT GGA CCT GG
AAG GAG TAC GCC TAC CTC AA
TCC ATG CTG ACT GTG CCG AT
CTT TTG CCA AAG CCT TCT TG
AGC GGC ATA CTG GTA ATT GG
AGT CCG CCT TAC GGT ACC ATG
GAC AGT CAC TGT AAG CAC AG
TGG AGA TCT TCG ACA TGC TG
TCC AGA GAC TTC AGG GTG CT
GGA GTC TGG TCG TGT GAA G
ACT TCA CCA TTC CCA CTT CG
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.
Characterization of HP75 cell line
Construction and characterization of a Flag-Pit-1 fusion protein
Expression of Flag-Pit-1 in transfected HP75/Pit-1 cells
Characterization of HP75/Pit-1 cells
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.