Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs

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Abstract

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27Kip1/CDKN1B (p27) and p18Ink4C/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.

Highlights

► MEN1 is a autosomal dominantly inherited syndrome characterized by endocrine tumors. ► However, the mechanisms underlying the selectivity of tumors remain unclear. ► MEN1-related genes, MLL and p27, were predominantly expressed in the endocrine organs. ► Tissue-specific haploinsuffiency of MLL may lead to decreases in levels of p27 and p18.

Introduction

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Primary hyperparathyroidism is the most common clinical phenotype (>90%), while entero-pancreatic tumors are present in 30–75%, and pituitary adenomas, 10–60% of cases [1], [2]. The MEN1 gene, responsible for MEN1, is located on chromosome 11q13 and consists of 10 exons encoding a protein of 610 amino acids, referred to as menin [3]. A mutation of the MEN1 gene has been identified in approximately 90% of familial cases and 27% of sporadic cases of MEN1 [4]. As mentioned, although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, menin does not directly participate in the processes of hormone secretion. Furthermore, the Men1 gene has been reported to be broadly expressed, and so the specific expression of menin in endocrine organs does not explain the tissue-selectivity of MEN1.

Mixed lineage leukemia (MLL) was originally isolated as a common target of chromosomal translocations frequently found in human acute leukemias [5]. MLL is a functional ortholog of the Drosophila trithorax (Trx) gene and involved in the nuclear regulatory mechanism that establishes an epigenetic transcriptional memory system through SET domain-dependent histone methyltransferase activity. MLL specifically methylates the lysine 4 residue in the N-terminal tail on histone 3 (H3K4), a modification typically associated with transcriptionally active regions of chromatin [6]. In 2004, two groups simultaneously reported that MLL was associated with menin in a nuclear protein complex that activated the transcription of the Hox gene [7], [8]. More recently, MLL and menin have been found to cooperatively regulate cyclin-dependent kinase inhibitors (CDKI) such as p27Kip1/CDKN1B and p18Ink4C/CDKN2C, and some mutations of menin were shown to cause insufficient stimulation of these CDKI genes and deregulated cell growth [9], [10].

A mutation of the p27Kip1 gene was recently implicated in a family with what appeared to be MEN1-related tumors, which has now been proposed as MEN type 4 (MEN4). Pellegata et al. reported a family with a W76X mutation of the p27Kip1 gene and that the proband had an invasive GH-secreting adenoma (acromegaly) with high mitotic activity and cell atypia, and hyperparathyroidism. The proband’s father had acromegaly, and two other family members suffered from testicular carcinoma and renal angiomyolipoma, a MEN1-associated neuroendocrine tumor, respectively [11]. Furthermore, knockout of the p27Kip1 gene in mice resulted in hyperorganism and pituitary adenoma [12], [13], [14], and phenotypes similar to those of MEN1 have been reported in rats with a mutation of the p27Kip1 gene [11], [15]. In addition, Agarwal et al. reported a mutation of the p18Ink4C gene in a family with hyperparathyroidism [16] and the double knockout of p27Kip1 and p18Ink4C in mice caused combinations of all the major tumors of MEN syndromes [17], [18].

In the present study to gain insight into mechanism underlying the tissue-specific tumor phenotypes in MEN1, we examined expression of candidate genes related to MEN1 including Men1, MLL, p27Kip1 and p18Ink4C in several endocrine and non-endocrine organs in wild-type mice and mice with knockout of the Men1 or MLL gene.

Section snippets

Animals and Tissue extraction

Heterozygous MLL knockout (MLL+/−) mice carrying the anti-OVA-specific TCR-αβ (DO11.10) transgene were kindly provided by Prof. T. Nakayama (Chiba University, Japan: the MLL+/− mice were originally provided by Prof. T. Komori, Nagasaki University, Japan). Heterozygous Men1 knockout (Men1+/−) mice were generated as reported [19]. These mice were backcrossed at least 6 times with the C57BL/6J background (Clea Inc., Tokyo, Japan). All the mice were maintained under SPF conditions and used at the

Expression of MEN1-related genes in the wild-type mice

We first examined the expression of the Men1 gene in various organs of the wild-type mice using real-time PCR. As reported previously [21], Men1 mRNA was relatively broadly expressed in the tissues examined. However, a detailed analysis of Men1 mRNA levels by real-time PCR with the TaqMan probe revealed a difference in expression among organs. As compared to levels in the liver (set at 1.0), Men1 mRNA was expressed at significantly higher levels in all other organs examined. Interestingly, the

Discussion

The mechanism underlying the tissue-selectivity of the tumorigenesis in MEN1 is poorly understood, since menin, the product of Men1, is known to be ubiquitously expressed [21]. We therefore postulated that the selectivity of MEN1-associated tumorigenesis for endocrine tissue might be related to factors recently identified as involved in the menin/MLL-CDKI pathway. In this study, as previously reported, the Men1 gene was found to be widely expressed in both endocrine and non-endocrine organs. As

Disclosure summary

All authors have nothing to disclose.

Acknowledgments

We thank Dr. Stephan Marx, NIDDK, NIH for the critical discussion. This work was supported in part by a research grant from Health and Labor Sciences Research Grants, Research on Measures for Intractable Disease, the Hypothalamus-Pituitary Dysfunction Research Group (to M. M.) and the Grant from the Ministry of Health, Welfare and Labor [H21-Nanchi-Ippan-037] (to M.Y.).

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