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First Department of Internal Medicine, Kagawa Medical University, 1750–1, Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan
Address all correspondence and requests for reprints to: Makoto Sato, M.D., First Department of Internal Medicine, Kagawa Medical University, 1750–1, Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan. E-mail: makoto{at}kms.ac.jp
Abstract
The multiple endocrine neoplasia type 1 (MEN1) gene seems to be a tumor suppressor that encodes a 610-amino acid protein termed menin and that plays an important role in the development of MEN1 syndrome. Recent reports indicate that heterozygous germline mutations of this gene are responsible for the disease onset of MEN1. In this study we examined the expression of menin in parathyroid tumors from primary hyperparathyroidism (PHP), secondary hyperparathyroidism (SHP), and MEN1 and thyroid tumors including Basedow’s disease, thyroid cancer, and adrenocortical tumors. Both ribonucleic acid and protein from these tumors were applied to RT-PCR and Western blotting, respectively. Primers for RT-PCR were designed to amplify the sequence between exons 2 and 3 of the MEN1 gene. Specific antibody against menin was generated in guinea pigs immunized with the recombinant peptide from amino acid residues 443–535 of menin made by using glutathione-S-transferase (GST) gene fusion. Menin messenger ribonucleic acid was strongly expressed on RT-PCR analysis in the parathyroid tumors from both PHP and SHP. Western blotting revealed a specific band of approximately 67 kDa in parathyroid tumors from PHP and SHP, with a much weaker such band detected in thyroid tumors. Menin expression was down-regulated in MEN1 samples, including nonsense mutation and deletion mutant. These findings suggest that menin is predominantly synthesized and stored in parathyroid tumors resulting from PHP and SHP.
MULTIPLE ENDOCRINE neoplasia type 1 (MEN1) is an autosomal dominant familial tumor syndrome characterized by the combined occurrence of tumors in parathyroids, enteropancreatic endocrine tissues, and the anterior pituitary (1). Parathyroid tumors are the most common manifestation of this disorder (2). In addition, adrenal cortical tumors, foregut carcinoids, and lipomas have been observed in association with MEN1 (1). The genetic basis for MEN1 is the homozygous inactivation of the putative tumor suppressor gene on chromosome 11q13 (3). Larsson and co-workers (4) were instrumental in initially identifying chromosome 11q13 as the location of the gene responsible for MEN1, and such patients are frequently found to have somatically lost the wild-type allele of markers in the vicinity of the gene (4, 5). MEN1 patients inherit a mutation in one copy of the gene, and susceptible cells in the target organs are transformed through inactivation of the wild-type copy of the gene via point mutations or deletions (4, 6). Moreover, allelic deletions on chromosome 11q13 have been reported in 63–100% of MEN1-associated parathyroid tumors and in 25–35% of sporadic parathyroid tumors, suggesting a role for the MEN1 gene in the pathogenesis of such tumors (6, 7, 8).
The MEN1 gene contains 10 exons and expresses a 2.8-kb transcript that encodes a 610-amino acid protein termed menin. Although analysis of the predicted menin amino acid sequence exhibits no apparent similarities to any known proteins, nuclear localization signals were identified in the C-terminals of menin (9, 10). It has been reported that the wild-type allele is consistently lost in MEN1 tumors, and both alleles of the MEN1 gene are often inactivated in sporadic tumors, indicating that tumorigenesis is very likely due to loss of function of the protein product as a tumor suppressor (7, 8, 11). Thus, the MEN1 gene seems to be an excellent example of a classic tumor suppressor. However, no reports have demonstrated the exact tissue distribution and cellular localization of menin. Therefore, we investigated the tissue-specific expression of the MEN1 gene and menin in parathyroid, thyroid, and adrenal tumors by highly sensitive RT-PCR and Western blot analysis.
Subjects and Methods
Subjects
Freshly frozen tumor samples from surgical specimens of parathyroid, thyroid, adrenal gland, breast, and muscle tissues were used in this study. Informed consent was obtained from every patient, and the study was approved by the ethical committee of Kagawa Medical University Hospital. Total ribonucleic acid (RNA) from each tumor tissue was extracted using a QIAGEN RNA/DNA minikit (QIAGEN, Hilden, Germany) and was used for PCR analysis. Total proteins extracted from the corresponding samples with 1% Nonidet P-40 were used for Western blot analysis.
RT-PCR
Total RNA (2 µg) was reverse transcribed using avian
myeloblastosis virus reverse transcriptase (Life Science Co.,
Petersburg, FL) and random primer (Takara Shuzo Co., Osaka, Japan), and
the synthesized complementary DNA (cDNA) was amplified by PCR as
previously described (12). The primers used for RT-PCR were 5'-GAG CTG
TCC CTC TAT CCT CG-3' (sense) and 5'-TGA CCT CAG CTG TCT GCT CC-3'
(antisense), designed to amplify the sequence between exons 2 and 3 of
the MEN1 gene (Fig. 1
). The
primers for amplification of the ß-actin sequence in the
corresponding tissues were designed as previously described (12). The
initial denaturation was carried out for 7 min at 94 C followed by 30
cycles of PCR for both the MEN1 gene and ß-actin using a
thermal cycler (Sanko Junyaku, Tokyo, Japan) according to a step
program of 94 C for 80 s, 55 C for 80 s, and 72 C for 80
s, followed by a 15-min extension at 72 C. Eight microliters of each
PCR product were electrophoretically separated on a 1.5% agarose gel
containing 0.5 µg/mL ethidium bromide and photographed.
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A fragment of menin cDNA corresponding to the amino acid
residues 443–535 of menin (Fig. 1
) from parathyroid tissue containing
restriction sites for HindIII and XbaI was
amplified by PCR using an appropriate set of primers matching the
published sequence of the MEN1 gene (10). The purified PCR
product was then digested with HindIII and XbaI
and ligated to the HindIII and XbaI restriction
sites of pGEX2T plasmid vector (Pharmacia Biotech,
Uppsala, Sweden) by T4 DNA ligase (Takara Shuzo Co. Ltd., Osaka,
Japan), forming a recombinant plasmid DNA. Competent Escherichia
coli were then transformed with the recombinant plasmid DNA and
cultured for GST fusion protein expression. The protein expression was
induced by 0.1 mmol/L isopropyl B-D-thiogalactoside. Stable
transformation of the cells was maintained by their resistance to
ampicillin. Cells were harvested and lysed by mild sonication. The GST
fusion protein was isolated with glutathione-Sepharose 4B beads
(Pharmacia Biotech) from the bacterial lysate and used to
generate a specific antibody in guinea pigs directed against the amino
acid residues 443–535 of the reported sequence of the menin containing
610 amino acid residues (10). The IgG fraction was purified using an
ImmunoPure Plus immobilized protein A IgG purification kit
(Pierce Chemical Co., Rockford, IL) and used for Western
blot analysis to detect menin in various tumor tissues, where a
horseradish peroxidase-conjugated goat antiguinea pig IgG
(Sigma, St. Louis, MO) was used as the second
antibody.
Western blot analysis of menin
The plasmid pCMV-sport menin clone (A11), containing a
full-length menin cDNA (9) from the National Human Genome Research
Institute of NIH, was used for transfection of HEK-293T cells.
Forty-eight hours after transfection, cells were harvested by washing
with phosphate-buffered saline (PBS) and mechanical scraping from the
flask, and total cellular proteins were extracted with 1% Nonidet P-40
for Western blot analysis. Cellular proteins from tissue samples stored
at -80 C were prepared (13) and resuspended under reducing conditions;
40 µg were fractionated by size on a 7.5% SDS-polyacrylamide gel for
menin, and only 10 µg were fractionated on a 15% gel for cyclophilin
A (cyp), and transferred to a polyvinylidene difluoride membrane
(Millipore Corp., Bedford, MA) using Trans-Blot
(Bio-Rad Laboratories, Inc., Richmond, CA) for
immunoblotting (14). The membranes were blocked overnight at 4 C with
PBS containing 0.2% Tween-20 (PBS-T) and 5% skimmed milk followed by
incubation for 2 h at room temperature with the antimenin antibody
(1:2000) or the anticyclophilin A antibody (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA; 1:1000). The membranes
were washed with PBS-T, incubated with the secondary antibody
conjugated to peroxidase for 2 h at room temperature, and then
washed with PBS-T. The staining signal was detected using the enhanced
chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) followed by exposure to
Kodak x-ray film (Eastman Kodak, Rochester,
NY). The blots of menin and cyp were scanned by an image scanner, and
the intensities of the blots were quantified using NIH Image (version
1.59). The ratio of menin to cyp is shown in Fig. 4.
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RT-PCR analysis of MEN1 gene
Menin messenger RNAs (mRNAs) were successfully amplified to an
expected size (286 bp) by RT-PCR using exon-specific primers in various
tumor tissues. Representative data regarding menin and ß-actin mRNAs
from each tissue are shown in Fig. 2.
Menin mRNA was expressed strongly and in approximately equivalent
amounts in parathyroid tumors of primary hyperparathyroidism (PHP) and
secondary hyperparathyroidism (SHP; Fig. 2A). Somatic mutation of the
MEN1 gene was not found in any samples of PHP tested. In
thyroid tumors of Basedow’s disease, expression of menin mRNA was
relatively low compared with that in the parathyroid tumors of PHP and
SHP (data not shown). Figure 2B shows substantial variations in the
levels of expression of menin mRNA in various parathyroid tumors from
MEN1 patients. The expression of menin mRNA was very low in the MEN1
cases with a deletion mutant and nonsense mutation, whereas it did not
differ among PHP and MEN1 cases with missense mutations. The expression
level of ß-actin mRNA was constant in all samples examined. In
addition, we examined menin mRNA in several other tissues, including
adrenal gland, breast, and skeletal muscle. Menin mRNA was clearly
detected in adrenal cortical tumors; however, very little or almost no
expression of menin mRNA was seen in breast and muscle tissues (data
not shown).
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Our antibody detected menin with an estimated molecular size of
approximately 67 kDa in HEK-293T cells transfected with the plasmid A11
DNA containing a full-length menin cDNA (9) (Fig. 3). In the untransfected cells, menin
expression was much weaker, although it was still detectable,
suggesting that these cells expressed endogenous menin. In parathyroid
tumors from PHP and SHP, menin was expressed almost equally with a
molecular size of about 67 kDa. In contrast, the expression of menin
was down-regulated or almost not detected in thyroid tumor from
Basedow’s disease (Fig. 3). Menin was expressed in various types of
cell lines and several samples of adrenal cortical tumors (data not
shown). Figure 4 shows the ratio of menin
to cyp in various parathyroid tumors from PHP, SHP, and MEN1 with
different mutations (missense, three deletions, nonsense) and in
thyroid tumors from patients with Basedow’s disease and thyroid
cancer. Although the expression of menin depends on each tumor sample,
there is no significant difference in menin expression between PHP and
SHP (Fig. 4). In contrast, the expression of menin was decreased in two
samples of MEN1 with same missense mutation (Fig. 4). However, in the
samples of MEN1 with three deletions and the nonsense mutation, the
expression of menin was dramatically decreased compared with that in
PHP and SHP (Fig. 4). In thyroid tumors from Basedow’s disease and
thyroid cancer, however, the expression of menin was also greatly
down-regulated compared with that in PHP and SHP (Fig. 4). Western blot
analysis of cyp as an internal control revealed a specific band of
about 18 kDa, which was expressed almost equally in all corresponding
tissues examined.
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As the prevalence of hyperparathyroidism approaches 90–100% in MEN1 patients (2, 8), our screening particularly focussed on the detection and analysis of menin in parathyroid tumors resulting from PHP, SHP, and MEN1. Our study showed that menin was expressed equally at mRNA and protein levels in parathyroid tumors from both PHP and SHP. The pathogenesis is quite different in these two diseases, and only a single parathyroid gland is involved in most cases of PHP, whereas two or more glands are involved in SHP. Histological findings have shown adenoma in the parathyroid tumors resulting from PHP and hyperplasia in SHP. Despite these dissimilarities, menin expression was constant between the two types of parathyroid tumors. Menin might therefore play an essential role in the cellular function of parathyroids, although menin expression remains to be studied in the normal parathyroid gland. Recent reports have shed light on the cellular function of menin. Guru et al. reported that menin is a nuclear protein (9) and possibly plays a role in transcriptional regulation, DNA replication, or cell cycle regulation. Agarwal et al. (15) indicated that menin protein directly interacts with the AP1 transcription factor JunD and represses transcriptional activation mediated by JunD. It has also been reported that naturally occurring and clustered MEN1 missense mutations disrupt menin interaction with JunD (15). Other observations have indicated that many of the mutations detected to date in the MEN1 gene most likely result in loss of function of its protein product, which is consistent with a tumor suppressor mechanism (4, 10, 16, 17, 18). In our study expression of menin was down-regulated in thyroid tumors, including Basedow’s disease and thyroid cancer. As the prevalence of hyperparathyroidism reaches 90–100% in MEN1 patients (2, 8) and as our study interestingly demonstrates, parathyroid tumor harbors an abundance of menin; therefore, one might speculate that the MEN1 gene in parathyroid tumors might play an important role in the onset of the disease process.
The incidences of the less frequently observed lesions, such as foregut carcinoid, adrenocortical tumor, lipoma, and thyroid tumor (1, 19) are 4%, 5%, 1%, and 5%, respectively, in MEN1 (1). It is unclear whether such lesions appear incidentally or in association with MEN1 in each case. The adrenal and thyroid tumors in association with MEN1 particularly cause confusion about their pathogenesis. In this study menin expression was very weak in the thyroid tumors and was abundant in the adrenal cortical tumors as well as in the parathyroid tumors, suggesting that menin is less important in thyroid than in adrenal gland. Therefore, it might play an important role as a nuclear protein (9, 15) in some specific tissues, including parathyroid and adrenal glands. Germline and somatic mutations of menin possibly disturb the tumor suppressor function of menin, resulting in the development of adrenal tumors in association with parathyroid tumor in MEN1 syndrome. On the other hand, thyroid tumor appears to be incidental in MEN1, where most likely menin is not distributed in the thyroid tissue by such mutations.
In the present study we detected menin in parathyroid tumors obtained from three MEN1 patients who had germline mutations such as missense mutation (E45G), nonsense mutation (Y312X), and deletion (L414del) (20). If a germline mutation of the MEN1 gene had altered the structure of menin, our antibody against the synthetic peptide of menin would have failed to detect the mutant menin. In addition, a somatic mutation of the MEN1 gene, such as loss of heterozygosity (LOH) could cause a loss or decrease in the immunoreactivity of menin. In both cases, menin expression should have been decreased. In two samples of the same missense mutation at the amino acid position 244, distant from the recognition sites (443–535) of our antibody, expression of menin protein was decreased compared with that in PHP. It is unlikely that such a mutation can change the binding affinity of our antibody. Therefore, the decrease in menin expression in these two samples might reflect LOH in the parathyroid tumors. Although RT-PCR showed no distinct difference in the expression of menin mRNA in PHP and in the samples of MEN1 with missense mutation, it might be difficult to quantify the difference between them by this method. In the sample of deletion, the position of the deletion mutant is close to the antibody recognition sites, suggesting that the antibody might loose its binding capacity with such mutant menin, possibly due to conformational changes. This is supported by the observation of decreased expression of menin in this sample of deletion compared with that in missense mutation. The nonsense mutation in the MEN1 gene resulted in the expression of a truncated menin protein lacking the antibody recognition sites. In combination with LOH, the immunoreactivity of menin protein was decreased in the sample of nonsense mutation compared with that in missense mutation, although it was still detectable in Western blotting, possibly due to the normal admixture.
In addition to the highly sensitive RT-PCR used for screening the expression of menin mRNA, our Western blot analysis revealed that menin was predominantly found in parathyroid tumors resulting from PHP and SHP. Our results, although not entirely conclusive, strongly suggest that the menin is synthesized and located mainly in the parathyroid tumors. Moreover, these findings are consistent with a strong possibility that mutations of the MEN1 gene would most likely result in loss of function of the protein product as a tumor suppressor and might play an important role in the development of the MEN1 syndrome.
Acknowledgments
We are grateful to Settara C. Chandrasekharappa (National Human Genome Research Institute of NIH, Bethesda, MD) for providing the generous gift of plasmid A11 DNA containing a full-length menin cDNA.
Received September 10, 1999.
Revised January 18, 2000.
Revised March 14, 2000.
Accepted March 21, 2000.
References
This article has been cited by other articles:
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  S Corbetta, L Vicentini, S Ferrero, A Lania, G Mantovani, D Cordella, P Beck-Peccoz, and A Spada Activity and function of the nuclear factor kappaB pathway in human parathyroid tumors Endocr. Relat. Cancer, December 1, 2005; 12(4): 929 - 937. [Abstract] [Full Text] [PDF]
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 I. Cavallari, D. M. D'Agostino, T. Ferro, A. Rosato, L. Barzon, C. Pasquali, P. Fogar, M. Theodoropoulou, G. Esposito, M. Boscaro, et al. In Situ Analysis of Human Menin in Normal and Neoplastic Pancreatic Tissues: Evidence for Differential Expression in Exocrine and Endocrine Cells J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3893 - 3901. [Abstract] [Full Text] [PDF]
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X174 DNA.