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Original Studies |
First Department of Internal Medicine (M.Y., K.H., T.S., N.S, M.M.) and Department of Neurosurgery (H.K.), Gunma University School of Medicine, Maebashi, Gunma; and the Departments of Endocrinology and Metabolism (Y.O.) and Neurosurgery (S.Y.), Toranomon Hospital, Tokyo, Japan
Address all correspondence and requests for reprints to: Masanobu Yamada, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-15 Showa-machi, Maebashi, Gunma 371, Japan. E-mail: myamada{at}sb.gunma-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits (3, 4, 5, 6, 7, 8). Furthermore, the oncogenic potential of
constitutively active forms of G protein-coupled receptors that were
induced by mutations has been demonstrated in endocrine diseases in man
(9, 10). We and others have reported that both TRH binding and TRH receptor (TRHR) messenger ribonucleic acid (mRNA) were identified in pituitary tumors, including not only PRL-, TSH-, and GH-secreting tumors but also nonfunctioning tumors (11, 12, 13). A paradoxical response of GH to TRH and a blunted response of TSH and PRL to TRH have been observed in patients with pituitary tumors (14, 15, 16). Based on these observations, it is speculated that TRHR may be involved in the pathogenesis of pituitary tumors.
We have recently cloned and characterized a human pituitary TRHR complementary DNA (cDNA) and its entire genomic DNA and found that the TRHR belongs to the family of G protein-coupled receptors containing putative seven transmembrane domains (11, 17). Using this cDNA and genes, we and others have attempted to find mutations in the TRHR gene in pituitary adenomas. However, no such mutations have yet been identified (11, 18).
In the present study, we measured TRHR mRNA levels in normal pituitary and pituitary tumors and found a new TRHR gene transcript that showed significant differences in expression among pituitary tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary adenoma tissues and normal pituitaries were obtained
from patients at the time of transsphenoidal surgery or autopsy.
Informed consent was obtained from each patient or their relatives. As
shown in Table 1
, samples included eight cases of GH-secreting
adenomas, seven prolactinomas, five TSH-producing tumors, four
nonfunctioning tumors, and five normal pituitaries.
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Total RNA was prepared from each adenoma and normal pituitary using the modified acid-phenol method as described previously (11). Aliquots of 3 µg total RNA were subjected to reverse transcription for 2 h at 37 C using Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN) in a total volume of 20 µL.
Quantitative reverse transcription-PCR (RT-PCR)
To overcome the problem of variability in efficiency of
amplification, TRHR mRNA was coamplified with a standard dilution
series of a plasmid differing from the target sequence by a single base
pair prepared by site-directed mutagenesis. This minor modification
would not be expected to affect amplification efficiency, but it
allowed us to distinguish the amplified product of the competitor
template from unknown samples after restriction enzyme digestion
(EcoRI; Fig. 1). Aliquots of 1
µL cDNA and standard templates were incubated with specific antisense
and sense primers, deoxynucleoside triphosphates, ExTaq, and PCR buffer
(Boehringer Mannheim). Thirty cycles of amplification were carried out
using a step program (94 C for 1 min, 60 C for 2 min, 72 C for 2 min),
followed by a 15-min final extension at 72 C. Primers were designed to
span the exon 2/exon 3 boundary (CompS, 5'-tgctattgtgatatcctgtggc-3';
CompAS, 5'-gtaaatcaccgggttgatgg-3') as shown in Fig. 1a. After
digestion with EcoRI, PCR products were subjected to
electrophoresis on a 2% agarose gel and stained with ethidium bromide.
To quantify TRHR mRNA, the density of amplified TRHR mRNA was compared
to that of the competitor (Fig. 1b).
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To identify HTRHR2 and HTRHR1 mRNAs simultaneously, PCR primers
NTR1 (5'-agatgtttctgcagcacagtatcttca-3') and NTR2
(5'-gttctcccttttctagatgatgactgcac-3') were used (Figs. 2a and 3).
To compare their expression levels, Southern blot analysis was
performed. Briefly, amplification was performed as described above,
except with 40 cycles of amplification. PCR products were subjected to
electrophoresis on a 2% agarose gel (Seaplaque), transferred onto a
nylon membrane (GeneScreen Plus), then hybridized with an
32P-labeled TRHR cDNA fragment that encompassed the
region between 547 and 1207 bp from the translational start site. The
relative amount of each fragment was determined by densitometric
scanning.
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In all PCR experiments, the presence of possible contaminants was checked by control reactions in which amplification was carried out without template.
Stable expression in Chinese hamster ovary (CHO) cells
To establish a cell line expressing human wild-type (HTRHR1) and
the short form (HTRHR2) of TRHR, cloned cDNAs were subcloned into an
expression vector pSVL (Pharmacia, Piscataway, NJ) and cotransfected
with plasmid pSD(M) that expresses dihydrofolate reductase in
dihydrofolate reductase-deficient CHO cells by the calcium phosphate
method (20). Twelve days after transfection, well isolated colonies
were selected to obtain stable cell lines. The expression of the TRHR
gene was amplified by incubation of these cell lines in complete
-medium containing increasing amounts of methotrexate.
Competitive binding assay
Cells in replicate wells, 30 mm in diameter, were incubated at 4 C for 2 h in a buffer containing 4 nmol/L [3H]MeTRH (87 Ci/mmol), a high affinity analog of TRH. Cells were incubated in the presence or absence of an excess of unlabeled TRH. At the end of incubation, free ligand was removed by washing cells three times with 1 mL cold buffer. Cells were then solublized with 1 mL 0.4 N NaOH, and radioactivity was counted with scintillation liquid. Specific binding was calculated by subtracting nonspecific binding from [3H]MeTRH binding in the absence of unlabeled TRH.
Measurement of intracellular Ca2+concentrations
Cells were gently scraped into 1 mL Hanks’ Balanced Salt Solution and 10 mmol/L HEPES (pH 7.4; HBSS), centrifuged, and incubated for 30 min at 37 C in 1.5 mL of buffer containing 4 µmol/L Fura-2/AM. Cells were diluted into 5 mL HBSS, and the pellets were resuspended at a density of 1 x 107 cells/mL in HBSS buffer. Cells were placed in a thermostatted cuvette maintained at 37 C and kept in suspension with a magnetic stir bar, and intracellular calcium concentrations were measured.
Statistical analysis
All data were analyzed by ANOVA and Duncan’s multiple range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patients examined are summarized in Table 1
. Thirteen patients were female, and the
mean average age of the patients was 48 yr.
Employing competitive PCR with primers, CompS and CompAS, HTRHR1 mRNA was specifically and quantitatively amplified. The detectable range was from 103–107 ag of the amplified product. All measurements were performed within this range, and results are expressed as the amount of amplified cDNA from 1 µL cDNA sample.
TRHR mRNA was identified in all samples, including nonfunctioning
tumors, but its expression levels were variable from less than
102 to 105 ag in each type of adenoma. The
responsiveness of pituitary hormones to TRH administration (500 µg,
iv) is also summarized in Table 1.
cDNA isolation and sequence analysis of a novel HTRHR transcript (HTRHR2)
When the primers NTR1 and NTR2 were used to amplify the region
between the sixth transmembrane domain and the carboxyl-terminus, an
additional 0.4-kilobase product was observed (Fig. 2a). Cloning and
sequence analysis of this short fragment revealed it to be 128
nucleotides shorter than the wild-type TRHR mRNA (HTRHR1), and the
deleted sequence corresponded exactly to the 5'-sequence of exon 3
(Fig. 2b). Therefore, alternative splicing occurs in the internal
acceptor site in exon 3, and this alternative splicing results in a
frame shift that yields a C-terminal truncated protein on translation.
The predicted HTRHR2 is 303 amino acids in length, 95 amino acids
shorter than the wild-type HTRHR1, and hydropathy analysis revealed
that HTRHR2 lacks the sixth and seventh transmembrane domains, as shown
in Fig. 2c.
Expression level of HTRHR2 mRNA in various pituitary tumors
The expression levels of HTRHR1 mRNA were higher than those of
HTRHR2 in all samples examined. The percentages of HTRHR2 mRNA relative
to HTRHR1 mRNA in the TSH-, PRL-, and GH-producing tumors;
nonfunctioning tumors; and normal pituitary were 10.4 ± 2.6%,
27.7 ± 3.3%, 1.6 ± 1.4%, 18.3 ± 1.7%, and
16.2 ± 4.1%, respectively (Fig. 3C). Interestingly, in six of eight
GH-secreting tumors, HTRHR2 mRNA was undetectable, and there was no
detectable signal even after long exposure. Therefore, this ratio in
the GH-secreting tumors was significantly lower than those in other
tumors and the normal pituitary (P < 0.01).
Functional analysis of HTRHR2 in CHO cells
To study the functions of HTRHR2, HTRHR2 was expressed in CHO cells. Although the wild-type (HTRHR1) expressed in CHO cells bound [3H]MeTRH significantly (>3000 cpm/dish) and showed significant elevation of intracellular calcium in response to TRH, there was no significant binding or elevation of intracellular calcium concentration even with 100 µmol/L TRH in the HTRHR2-expressing CHO cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We next demonstrated the existence of a novel short transcript of the TRHR gene, designated HTRHR2, in human pituitary and adenomas, which is generated by alternative splicing at the internal acceptor site in exon 3. An isoform of the murine TRHR has been reported recently that is produced by alternative splicing of a retained intron in the 3'-untranslated region, and its function is not distinguishable from that of the wild-type receptor (22). However, in the case of the human TRHR gene, there is no acceptor site in the corresponding region, suggesting that HTRHR may have no similar isoform. Therefore, the HTRHR2 mRNA is the first isoform of the human TRHR mRNA identified to date.
Recently, we cloned the entire human TRHR gene and reported that the
gene contains 3 exons and 2 introns (17). Intron 2 is more than 25
kilobases in length and interrupts the region between the fifth and
sixth transmembrane domains, which is located in the region of the
third cytoplasmic loop. Several G protein-coupled receptors have an
intron in the third cytoplasmic loop in a location similar to that of
the human TRHR gene (23, 24, 25, 26). Among these genes, those encoding
receptors including the dopamine D2 receptor and neuropeptide Y-Y1
receptor can produce individual isoforms by alternative splicing at the
region around this intron. An isoform of the neuropeptide Y-Y1ß
receptor, which is a truncated form of the Y1- receptor and lacks
the seventh transmembrane domain and C-terminal cytoplasmic tail, can
bind to neuropeptide Y-1 to the same extent as the wild-type receptor,
but has no signal transduction activity. On the other hand, an isoform
of the D2 receptor with an insert of 21 amino acids in the cytoplasmic
loop generated by alternative splicing of a cassette exon, has the same
properties as the wild-type receptor. It was, therefore, anticipated
that the new isoform, HTRHR2, may exhibit functional differences from
the wild-type HTRHR1. Functional analysis of the HTRHR2 showed neither
significant binding to [3H]MeTRH nor significant
elevation of intracellular calcium level in response to TRH. HTRHR2
shows conservation of most of the third cytoplasmic loop, but lacks the
sixth and seventh transmembrane domains, as shown in Fig. 2c. The loss
of its function can be explained by recent observations showing the
importance of certain residues in the sixth and seventh transmembrane
domains of the receptor for both TRH binding and signal transduction
(27, 28).
The question remains regarding the functional properties of the HTRHR2 mRNA. It is of interest to note that the expression ratio of HTRHR2 vs. HTRHR1 mRNA was consistent in individual pituitary tumors, but differed significantly among different tumors. The precise mechanism by which the splicing of the HTRHR mRNA was regulated in these tumors remains unclear. However, considering the strong expression of the HTRHR2 mRNA in certain tumors, HTRHR2 mRNA may alter the cellular responsiveness to TRH by affecting the degradation rate of HTRHR1 mRNA, its transcription rate, or translation itself. Furthermore, as the HTRHR2 mRNA is just 128 nucleotides shorter than HTRHR1 mRNA, the existence of high levels of HTRHR2 mRNA, particularly in prolactinomas, must be taken into account when measuring levels of TRHR mRNA by conventional Northern blot analysis or PCR methods.
In conclusion, although the expression levels of the HTRHR1 mRNA were variable among pituitary tumors, splicing of TRHR mRNA to generate HTRHR1 and HTRHR2 was regulated in a tumor-specific manner. Thus, the specific ratio of HTRHR2/HTRHR1 mRNA may be a useful marker for evaluation of the characteristics of pituitary tumors.
Received May 19, 1997.
Revised July 16, 1997.
Accepted August 14, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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