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Original Studies |
Department of Medicine and Clinical Science (Y.N., T.U., H.Mi., H.Mu., S.M., M.S., K.T., I.T., K.N.), and Department of Laboratory Medicine (A.S.), Kyoto University Graduate School of Medicine, Kyoto 606-8397, Japan; and International Institute for Advanced Studies (T.U.), Kizugawa-dai Kizu-cho Soraku-gun Kyoto 619-0225, Japan
Address all correspondence and requests for reprints to: Dr. Takeshi Usui, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: tusui{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that some transcription factors are
related to human endocrine disorders, such as combined pituitary
hormone deficiency, which results from Pit-1 gene mutations (6).
Recently, Wei Wu et al have cloned and sequenced human
Prop-1 and found that its mutations cause familial combined pituitary
hormone deficiency (7). Subsequently, two other groups reported the
Prop-1 mutations in familial combined pituitary hormone deficiency (8, 9). According to their reports, 2-bp deletions (7, 8, 9) leading to
frameshifts with premature terminations, which result in the loss of
the DNA-binding HD and the C-terminal transactivation domain of Prop-1,
and amino acid substitutions (7) in the third helix of the HD leading
to the reductions of DNA binding affinity and transactivation
abilities, were found in these patients. All these disorders result
from inactivating mutations of these transcription factors, leading to
diminished target gene expression. On the other hand, several genes
were reported to be responsible for functioning endocrine tumors, such
as Gs 
gene for a subset of GH-secreting tumors (10, 11), TSH
receptor gene for hyperfunctioning thyroid adenomas (12), and RET
protooncogene for MEN type 2 (13). Although no constitutively
activating mutations of transcription factors have been reported in
hyperfunctioning endocrine disorders, it is conceivable that an
activating mutation of pituitary transcription factors might result in
hypersecretion of pituitary hormone and be responsible for the
tumorigenesis of pituitary adenomas. Pit-1 (14) and Prop-1 are thought
to be required for both the determination of cell phenotypes and
proliferation. Although several researchers have demonstrated human
Pit-1 gene expression in pituitary adenomas and investigated its
contribution to pituitary tumorigenesis (15), Prop-1 gene expression in
normal pituitary and pituitary adenomas has not been studied. In this
study, we cloned human Prop-1 complementary DNA (cDNA) and analyzed the
transcriptional activity of human Prop-1 protein. We also investigated
human Prop-1 expression in normal pituitary and pituitary adenoma
cells. Finally, we sequenced the Prop-1 cDNAs derived from pituitary
tumors to determine the mutations that might have led or contributed to
their tumorigenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human pituitary cDNA (CLONTECH Laboratories, Inc., Palo Alto, CA) was amplified by PCR using degenerate primers designed based on the amino acid sequence of mouse Prop-1 (4). The amplified fragments were subcloned into plasmid pCR 2.1 using TA cloning kit (Invitrogen, San Diego, CA), and several clones were sequenced. One of the clones, which showed strong sequence homology to mouse Prop-1, was shown to be human homolog of Prop-1. Based on this partial nucleotide sequence of the human Prop-1, 5' and 3' rapid amplification of cDNA ends (RACE) was used to obtain the entire open reading frame of human Prop-1 cDNA.
Northern blot analysis
One microgram of human pituitary poly A (+) RNA (CLONTECH Laboratories, Inc.) was separated on a 1% agarose/formaldehyde gel, transferred by capillarity to a nylon membrane (GeneScreen Plus, DUPONT, Boston, MA), UV-cross-linked, and baked. This membrane and a commercial membrane, containing 2 µg poly A (+) RNA per lane from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Human Multiple Tissue Northern Blot, from CLONTECH Laboratories, Inc.), were hybridized with 32P-labeled human Prop-1 cDNA and human G3PDH probes using ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.). Prehybridization was performed at 68 C for 30 min and hybridization at 68 C for 1 h. The blots were washed at room temperature for 30 min in 2 x saline-sodium citrate (SSC) and 0.05% SDS, and then at 50 C for 30 min in 0.1 x SSC and 0.1% SDS.
Plasmid construction
Various fragments of human Prop-1 were generated by PCR. The
primers used were as follows: S1 and A3 for N terminus (N), S2 and A2
for HD, and S3 and A1 for C terminus (C). The sense and antisense
primers contained BamHI and SalI recognition
sites, respectively (Fig. 1b
). Either
human pituitary cDNA or pituitary adenoma cDNA (case 13, Table 1) were used as templates for PCR in a
50-µL reaction mixture containing 20 mmol/L Tris-HCl (pH 8.0), 2
mmol/L MgCl2, 10 mmol/L KCl, 6 mmol/L
(NH4)2SO4, 0.1% Triton X-100, 10
µg/mL BSA, 1.25 U Pfu DNA polymerase
(Stratagene, La Jolla, CA), and 0.2 µmol/L
oligonucleotide primers. The PCR mixtures were denatured for 1 min at
94 C and followed by 35 cycles of PCR (94 C for 30 sec; 50 C for 1 min;
and 72 C for 3 min), with a final 7-min extension at 72 C. The PCR
products were collected from 1.5% low-melting agarose (Gibco BRL, Grand Island, NY) and purified by QIAquick Gel Extraction
Kit (Qiagen, Hilden, Germany). The purified products were
digested by BamHI and SalI and then ligated into
pM plasmid (CLONTECH Laboratories, Inc.) in frame, so as
to fuse to the GAL4 DNA binding domain. In pM-hProp-C, two kinds
of plasmids were obtained; one had Thr at codon 142 and the other had
Ala at codon 142. These plasmids were designated as pM-hProp-N,
pM-hProp-HD, and pM-hProp-C(142T or 142A) each
of which generated a fusion protein of the N-terminal domain (amino
acids 1–68), HD (amino acids 69–128), and C-terminal domain (amino
acids 129–226) of human Prop-1 and GAL4 DNA binding domain in
transfected mammalian cells, respectively. pGAL-Luc was generated by
fusing five consensus GAL4 binding sites and an adenovirus E1b minimal
promoter to the multicloning site of PicaGene basic vector (Toyo
Ink Product, Tokyo, Japan).
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CV-1 cells, derived from the kidney of African green monkey, were grown in MEM medium supplemented with 10% FBS, 50 U/mL penicillin G, 50 µg/mL streptomycin, in a humidified atmosphere of 95% air-5% CO2 at 37 C. CV-1 cells were plated onto 12-well microplates (105 cells/well) and cultured for 24 h. The mixture, containing 0.5 µg of various pM-derived plasmids, 0.25 µg pGAL-Luc, and 0.25 µg pßgal-control plasmid with 5 µL of PLUS Reagent (Gibco BRL) in 250 µL serum-free MEM, was incubated at room temperature for 15 min; then 250 µL of the same media, containing 4 µL LipofectAMINE Reagent (Gibco BRL), was added. After incubation at room temperature for 15 min, the mixture was applied to cell cultures previously washed with serum-free MEM. At 24 h after the start of transfection, the medium containing LipofectAMINE was replaced with fresh, complete MEM medium and was further cultured for 24 h. After being washed with PBS, the cells were lysed using PicaGene ReporterLysis Buffer (Toyo Ink Product). The lysates were centrifuged at 14,000 rpm, and the supernatants were used for luciferase and ß-galactosidase assay with PicaGene luminescent substrate (Toyo Ink Product) and Luminescent ß-gal Detection Kit II (CLONTECH Laboratories, Inc.). Luminescence was measured with a luminometer (LUMICOUNTER 700).
RT-PCR
Fourteen human pituitary adenoma tissues (5 somatotroph, 3 lactotroph, 2 thyrotroph, 1 corticotroph, and 3 nonfunctioning adenomas) were obtained by transsphenoidal surgery. All these tumors were classified by immunohistochemical staining using polyclonal antibodies specific for GH, PRL, TSH, ACTH, LH, and FSH (Dako Corp. Japan, Tokyo). Total RNAs of human pituitary adenoma cells were isolated by acid guanidinium thiocyanate-phenol-chloroform extraction, using ISOGEN (Nippon Gene Co, Tokyo, Japan), and were reverse-transcribed with SuperScript II reverse transcriptase in the presence of oligo dT as primer (SuperScript Preamplification System for First Strand cDNA Synthesis; Gibco BRL). RT-PCR was performed using sets of primers under the same conditions as in plasmid construction, except with 30 cycles instead of 35 cycles for human cyclophilin. To control for extraneous contaminating genomic DNA or cDNA in experiment reagents, a tube containing the PCR reaction mixture with no template was included in all the amplifications and was negative for all experiments. In addition, the primers for Prop-1 were designed to span intron. To ensure semiquantitive results, the number of PCR cycles for each set of primers was determined to be in the linear range of amplification, and all cDNA samples were adjusted to yield equal amplification of a fragment of cyclophilin cDNA as internal standard. The forward and reverse primers were as follows; S1 and A1 for human Prop-1, 5'-GGAATGAGGATCCAAGCTTTTACTTCGGCT-3' and 5'-GGTTCTGCAGATGACAGGA-AGGAACAG-3' for human Pit-1, and 5'-CCGCGTCTCCTTTGAGCTGTTTGCAG-3' and 5'-ACCCAAAGGGAACTGCAGCGAGAGC-3' for human cyclophilin (16). PCR products were separated by electrophoresis on 1.5% agarose gels and visualized by staining with ethidium bromide.
In situ hybridization for human Prop-1
One of the somatotroph adenoma tissues obtained by transsphenoidal surgery (case 5) was fixed in 4% paraformaldehyde/PBS (pH 7.2) for 24 h, dehydrated in graded concentrations of ethanol, and embedded in paraffin. Five-micron sections were placed on aminopropyltriethoxysilane (APS)-coated glass slides (Matsunami Glass Industries, Inc., Osaka, Japan). The sense and antisense probes for Prop-1 and GH were labeled with digoxigenin-deoxyuridine 5-triphosphate, which was obtained from an RNA labeling kit (Boehringer Mannheim, Mannheim Germany). Briefly, after deparaffinization, sections were treated with 0.2 N HCl and 50 µg/mL proteinase K for 15 min. After postfixation with 4% paraformaldehyde/PBS, sections were immersed in 2 mg/mL glycin PBS and kept in 50% formamide/5 x SSC. Hybridization was carried out at 50 C, for 16 h, in the hybridization medium (50% formamide, 10 mmol/L Tris/HCl, 0.25% SDS, 1 mmol/L EDTA, 250 µg/mL transfer RNA, 250 µg/mL salmon sperm DNA) with digoxigenin-labeled RNA probes (17). After the hybridization, the sections were washed with 5 x SSC for 10 min, 50% formamide in 2 x SSC for 30 min, and PBS containing 20 µg/mL ribonuclease A (Wako Pure Chemicals Industries, Inc., Tokyo, Japan) for 30 min at 37 C. After further washing by 2 x SSC and 0.2 x SSC at 50 C for 15 min, the sections were incubated with antidigoxigenin horseradish peroxidase (HRP) (Boehringer Mannheim), washed, and treated with biotinyl tryamide and streptavidin-HRP for amplification, according to the Tyramide Signal Amplification system using the Tyramide Signal Amplification-Indirect kit (NEN Life Science Products, Boston, MA) (18). The localization of HRP was visualized using 3,3'-diaminobenzidine (Dojindo, Kumamoto, Japan) with 0.01% H2O2. The sections were not counterstained.
Sequencing of human Prop-1 expressed in pituitary adenomas
To examine whether the Prop-1 gene could be an oncogene in human
pituitary tumors, we amplified and directly sequenced the entire coding
region of Prop-1. cDNAs derived from pituitary adenomas were amplified
by PCR using Pfu DNA polymerase, as previously described
with S1 and Am, and with Sm and A1 primers. Sm and Am primers contained
-21M13 primer sequences on their 3' ends (Fig. 1b
). The PCR products
were collected from 1.5% low-melting agarose gel and purified by
QIAquick Gel Extraction Kit. The purified products were used for direct
sequencing with Dye Primer Cycle Sequence FS Ready Reaction Kit
(-21M13; PE Applied Biosystems, Warrington, UK).
Sequencing was performed using a 373A DNA Sequencer (PE Applied Biosystems).
Statistical analysis
All data are expressed as means ± SEM. Comparison was performed with ANOVA followed by Scheffe’s test. P < 0.0001 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1a shows the nucleotide sequence of human Prop-1 cDNA that we
cloned. The amino acid sequence of the human Prop-1 cloned was
identical to that which had been reported previously (4), except for an
Ala-to-Thr substitution at amino acid 142 (Table 1). To investigate the
responsible domain of transcriptional activity of human Prop-1,
pM-derived plasmids, which contained N-terminal, HD, and
C(142T or 142A)-terminal domains of human
Prop-1, were transfected into CV-1 cells with reporter plasmids. As
shown in Fig. 2, both
pM-hProp-C(142T) and C(142A) significantly
increased reporter gene expression to the same degree. On the other
hand, the other two plasmids (pM-hProp-N and pM-hProp-HD) did not
exhibit any increase in reporter gene expression, compared with
transfection with the empty vector. This result indicates that only the
C-terminal domain, not the HD or N-terminal domain, of human Prop-1 is
responsible for its transcriptional activity. Furthermore, no
difference in the transcriptional activity of Prop-1 was observed
between Thr and Ala at codon 142. Similar results were also found using
GH3 cells (data not shown).
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Northern blot analysis showed the existence of human Prop-1
transcript, approximately 5 kb in size, only in the pituitary but not
in any other tissues (Fig. 3). Using
RT-PCR analysis, it was demonstrated that human Prop-1 mRNA was
expressed in all of the 14 human pituitary adenomas examined. On the
other hand, human Pit-1 mRNA was expressed in only somatotroph,
lactotroph, and thyrotroph adenomas, but not in corticotroph and
nonfunctioning adenomas (Fig. 4).
In situ hybridization analysis in one of somatotroph
adenomas was performed to ascertain the existence of human Prop-1 mRNA
in the pituitary adenoma cells but not in fibroblasts, endothelia, or
contaminating surrounding nontumorous adenohypophysis (Fig. 5a). Human Prop-1 mRNA was colocalized
with GH mRNA in the nuclei of pituitary adenoma cells (Fig. 5, a and
b).
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The nucleotide sequences of PCR products were identical to those
of human Prop-1 cDNA, except for five silent mutations (amino acid 9,
Ala, GCC for GCT; amino acid 56, Phe, TTT for TTC; amino acid 111, Ala,
GCG for GCC; amino acid 112, Arg, AGA for CGA; amino acid 203, Ala, GCG
for GCC), which were all heterozygotes. Amino acid 142 was a homozygote
of Thr in eight cases and Ala in one case, and a heterozygote of Thr
and Ala in five cases (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization of mouse Prop-1 during ontogeny revealed that its expression was detected by e10-e10.5, maximal by e12.0, and markedly decreased after e14.5. With Southern analysis, the amount of mouse Prop-1 was only detected in trace amounts in the adult pituitary (4). In this study, we demonstrated that detectable amounts of mRNA are expressed in the pituitary, but not in any other tissues, by Northern blot analysis, producing a transcript of approximately 5 kb in size. The coding region of human Prop-1 is reported to consist of three exons and spans, approximately 3.5 kb on the genomic DNA (7). The discrepancy between the size of the mRNA in Northern blots and the size of the genomic DNA spanning the coding region might be explained by the existence of noncoding exons and/or a long 5' or 3' noncoding region. Because a discrete band was observed when probed with G3PDH in blots of the pituitary, the fuzzy band in the pituitary when probed with Prop-1 might reflect the existence of multiple transcripts of different sizes.
We examined human Prop-1 mRNA expression in pituitary adenomas using
RT-PCR analysis, because it is usually difficult to obtain sufficient
amounts of tumor tissues by transsphenoidal surgery to perform Northern
blot or ribonuclease protection assay, and the amounts of tissues we
obtained were not sufficient to do such experiments. As shown in Fig. 4, Prop-1 mRNA was expressed in all the pituitary adenomas we examined,
whereas Pit-1 mRNA expression was restricted to somatotroph,
lactotroph, and thyrotroph adenomas. Previous reports on Pit-1 mRNA
expression have been controversial. Some investigators have reported
that Pit-1 mRNA is expressed in pituitary adenomas other than
somatotroph, lactotroph, and thyrotroph adenomas (19, 20, 21, 22); and others
have reported that, in most cases, it is expressed only in somatotroph,
lactotroph, and thyrotroph adenomas (16, 23, 24, 25, 26). The expression of
Pit-1 in corticotroph and nonfunctioning adenomas might be explained by
the existence of tumor cells that are focally differentiated into Pit-1
lineage cells or of trapped, intermingled normal pituitary cells in the
adenomas (27). No evidence of Pit-1 mRNA in ACTH-producing and
clinically nonfunctioning adenoma cells in this study indicates that
these tumor tissues were not contaminated with normal pituitary cells
or tumor cells that have characteristics of Pit-1 lineage cells.
Recently, it has been reported that some patients who have human Prop-1
gene mutations have not only GH, PRL, and TSH deficiencies but also
have lower LH, FSH, and ACTH levels in serum than normal subjects (7, 28, 29), which are not observed in patients who have Pit-1 gene
mutations. Therefore, the expression of Prop-1 (but not Pit-1) gene in
corticotroph and nonfunctioning adenomas, many of which are reported to
secrete gonadotropin (30, 31), suggests that human Prop-1 might have
functions other than as a Pit-1 gene enhancer, such as regulating cell
division, which is independent of Pit-1 lineage directly or regulating
pituitary-specific transcription factor(s) other than Pit-1. In
addition, these results suggest that the additional factor(s), other
than Prop-1, might be necessary for Pit-1 gene expression.
To date, several genes have been reported to play important roles in the pathogenesis of pituitary adenomas, such as gsp-activating mutations in GH-secreting adenomas (10, 11) and menin gene mutation in sporadic pituitary adenomas (32). Because Pit-1 is required for both determination of cell phenotype and proliferation, some investigators have tried to reveal its contribution to abnormal pituitary cell proliferation and/or development of pituitary adenomas (16, 19, 20, 21, 22, 23, 24, 25, 26, 27). However, no significant difference had been found in the Pit-1 mRNA expression level between functioning and nonfunctioning tumors. In addition, possible involvement of Pit-1 gene mutation in pituitary adenomas was studied by two groups (18, 28), but no mutations with functional significance were found in these studies. These studies suggest that pituitary tumorigenesis might not be associated with a gross alteration of Pit-1 gene expression in humans. Although no constitutively activating mutation in the Prop-1 gene has been found, it is important to identify such mutations in human pituitary adenomas that might lead to alterations in DNA binding, protein interactions, or transcriptional activities of human Prop-1. Therefore, to investigate the possible involvement of Prop-1 gene mutation in pituitary adenomas, we carried out direct sequencing of the entire coding region of Prop-1 cDNA in pituitary adenomas. Although five nucleic acid substitutions were observed, they did not alter amino acids. These substitutions were considered to be polymorphisms. We also observed the amino acid at codon 142 as Thr/Thr in eight cases, Ala/Ala in one case, and Thr/Ala in five cases, suggesting that Ala-to-Thr substitutions should be polymorphisms, which is also supported by the fact that this amino acid substitution did not affect the transcriptional activity of human Prop-1 in transfected cells.
In conclusion, we cloned human Prop-1 cDNA and showed its gene expression in normal pituitary and pituitary adenoma cells. The transcriptional domain of human Prop-1 was located at the C-terminal as mouse Prop-1. No mutations, except five silent nucleic acid changes, were found in human Prop-1 cDNA in somatotroph, lactotroph, thyrotroph, and corticotroph adenomas, suggesting that abnormalities of the Prop-1 gene might not be frequent mechanisms of human pituitary tumorigenesis. Further studies are required to elucidate its relation to the pathogenesis of pituitary adenomas.
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Acknowledgments |
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Footnotes |
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Received November 10, 1998.
Revised January 4, 1999.
Accepted January 12, 1999.
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