Menin and JunD regulate gastrin gene expression through proximal DNA elements

Mutations in the MEN1 gene correlate with multiple endocrine neoplasia I (MEN1). Gastrinomas are the most malignant of the neuroendocrine tumors associated with MEN1. Because menin and JunD proteins interact, we examined whether JunD binds to and regulates the gastrin gene promoter. Both menin and JunD are ubiquitous nuclear proteins that we showed colocalize in the gastrin-expressing G cells of the mouse antrum. Transfection with a JunD expression vector alone induced endogenous gastrin mRNA in AGS human gastric cells, and the induction was blocked by menin overexpression. We mapped repression by menin to both a nonconsensus AP-1 site and proximal GC-rich elements within the human gastrin promoter. Chromatin immunoprecipitation assays, EMSAs, and DNA affinity precipitation assays documented that JunD and Sp1 proteins bind these two elements and are both targets for menin regulation. Consistent with menin forming a complex with histone deacetylases, we found that repression of gastrin gene expression by menin was reversed by trichostatin A. In conclusion, proximal DNA elements within the human gastrin gene promoter mediate interactions between JunD, which induces gastrin gene expression and menin, which suppresses JunD-mediated activation.     

Molecular and genetic mechanisms of tumorigenesis in multiple endocrine neoplasia type-1

Multiple endocrine neoplasia type 1 (MEN1) is a rare but informative syndrome for endocrine tumorigenesis. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. Mutations of menin have been reported in more than 400 families and tumors, most of which are truncating mutations, thus supporting the function of menin as a tumor suppressor. The exact function of menin is unknown, but overexpression of menin inhibits proliferation of Ras-transformed NIH3T3 cells. Since menin interacts with proteins from both the TGF beta and AP-1 signaling pathways, perhaps its tumor suppressor function is related to these key cell growth pathways. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN and sporadic endocrine tumors.     

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.     

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.     

Reduction of menin expression enhances cell proliferation and is tumorigenic in intestinal epithelial cells

Menin, the product of the tumor suppressor gene MEN1, is widely expressed in mammalian endocrine and non-endocrine tissues, including intestine. Its known abundant expression in several types of cells with high proliferative capacity led us to investigate the physiological function of the protein menin in intestinal epithelium, one of the most rapidly growing epithelia. Here we showed that the Men1 gene is mainly expressed in the crypt compartment of the proximal small intestine and that its expression was increased during fasting in vivo, both suggesting a role of menin in the control of cell growth. Indeed, specific reduction of menin expression by transfected antisense cDNA in the rat duodenal crypt-like cell line, IEC-17, increased cell proliferation. The latter is correlated to a loss of cell-cycle arrest in G(1) phase by resting cells and an overexpression of cyclin D1 and cyclin-dependent kinase (Cdk)-4. Furthermore, these cells lost the inhibition of proliferation induced by transforming growth factor-beta1, associated with a decrease of transforming growth factor-beta type II receptor expression. As a result of deregulated proliferation, antisense menin transfected IEC-17 cells became tumorigenic as shown in vitro as well as in vivo in immunosuppressed animals. These results indicate that menin contributes to proliferation control in intestinal epithelial cells. The present study reveals an unknown physiological function for menin in intestine that may be important in the regulation of epithelial homeostasis.     

Menin, a gene product responsible for multiple endocrine neoplasia type 1, interacts with the putative tumor metastasis suppressor nm23

Although the gene responsible for multiple endocrine neoplasia type 1 (MEN1) has been identified, the function of its gene product, menin, is unknown. To examine the biological role of the MEN1 gene, we searched for associated proteins with a yeast two-hybrid system using the MEN1 cDNA fragment as bait. On screening a rat fetal brain embryonic day 17 library, in which a high level of MEN1 expression was detected, we identified a putative tumor metastasis suppressor nm23/nucleoside diphosphate (NDP) kinase as an associated protein. This finding was confirmed by in vitro interaction assays based on glutathione S-transferase pull down experiments. The association required almost the entire menin protein, and several missense MEN1 mutations reported in MEN1 patients caused a loss of the binding activity for nm23. This result suggests that this interaction may play important roles in the biological functions of the menin protein, including tumor suppressor activity.     

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras^G12D-induced tumor formation in the Men1^f/f;K-Ras^G12D/+;Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.     

MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制

多发性内分泌肿瘤1-(multiple endocrine neoplasia type 1,MEN1)是一种常染色体显性遗传的肿瘤综合征,患者常表现出多发性的内分泌器官肿瘤,包括垂体瘤、甲状旁腺瘤和胰岛瘤.抑癌基因Men1的突变导致MENl的发生,其编码的蛋白为核蛋白menin.Menin可以抑制包括胰岛β细胞在内的...     

The 32-kilodalton subunit of replication protein A interacts with menin,the product of the MEN1 tumor suppressor gene

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.     

Menin represses malignant phenotypes of melanoma through regulating multiple pathways

Substantial genetic evidence suggests that chromosome 11q is involved in regulating initiation and progression of malignant melanomas. Mutations of the MEN1 gene, located in chromosome 11q13, predispose individuals to the multiple endocrine neoplasia type 1 (MEN1) familial syndrome. MEN1 patients develop primary malignant melanoma, suggesting a potential link between MEN1 syndrome and development of melanomas, but the precise molecular mechanism is poorly understood. Here we show that the MEN1 gene suppresses malignant phenotypes of melanoma cells through multiple signalling pathways. Ectopic expression of menin, the product of MEN1 gene, significantly inhibited melanoma cell proliferation and migration in vitro and in vivo. The inhibition was partly achieved through suppressing expression of growth factor pleiotrophin (PTN) and receptor protein tyrosine phosphatase (RPTP) β/ζ, accompanied with the reduced expression of phosphatidylinositol 3-kinase (pI3K) and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2). Interestingly, reduced expression of menin was associated with hypermethylation of the CpG islands of the MEN1 promoter in melanoma cells. Taken together, these findings suggest a previously unappreciated function for menin in suppressing malignant phenotypes of melanomas and unravel a novel mechanism involving in regulating PTN signalling by menin in development and progression of melanomas.