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Endocrinology Vol. 145, No. 7 3443-3450
Copyright © 2004 by The Endocrine Society

Tumor Suppressor Menin Regulates Expression of Insulin-Like Growth Factor Binding Protein 2

Ping La, Robert W. Schnepp, Clark D. Petersen, Albert C. Silva and Xianxin Hua

Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160

Address all correspondence and requests for reprints to: Dr. Xianxin Hua, Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160. E-mail: huax{at}mail.med.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that menin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild-type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild-type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the deoxyribonuclease I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MULTIPLE ENDOCRINE NEOPLASIA type I (MEN1) is a hereditary tumor syndrome characterized by the development of tumors in multiple endocrine organs, such as the parathyroids, the pituitary, and pancreatic ß islet cells (1, 2). Recently, nonendocrine tumors such as collagenomas have also been described in MEN1 patients (3). The gene mutated in MEN1 patients, Men1, which encodes a nuclear protein of 610 amino acid residues, menin, was identified by positional cloning (4). However, there are no structural motifs in menin that suggest any biochemical function for menin. Targeted disruption of Men1 in mouse leads to a tumor syndrome that closely mimics MEN1 in humans, demonstrating a critical role for menin in suppressing the development of MEN1 (5).

Menin interacts with a variety of proteins including the transcription factors JunD, nuclear factor (NF)-{kappa}b, Smad3, and Pem (6, 7, 8, 9), and represses the transcriptional activity of JunD and NF-{kappa}b. Ectopic expression of menin in Chinese hamster ovary cells inhibits insulin-induced transcription of c-Fos (10). Because regulation of these transcriptional factors by menin was observed using either reporter gene assays or the overexpression of menin, it is unclear whether menin is essential for repressing the expression of endogenous genes. Identification of endogenous genes that are repressed by menin will provide a desired system to further understand how menin regulates gene transcription and suppresses tumorigenesis.

Using DNA microarray analysis of menin-null cells, we show that targeted disruption of Men1 leads to the enhanced expression of IGFBP-2 (IGF binding protein 2), a secreted protein that binds IGFs (11). IGFBP-2 is a member of the IGFBP family and plays a crucial role in regulating cell proliferation. It can stimulate cell proliferation in an IGF-independent manner in certain types of cells but can also inhibit cell proliferation by suppressing the activities of IGFs (11). For example, IGFBP-2 inhibits proliferation of normal epithelial cells but stimulates proliferation of cancer cells (12, 13). However, it has been reported that IGFBP-2 partly mediates TGF-ß-induced inhibition of proliferation of mink lung epithelial cells (14). It is well known that TGF-ß inhibits proliferation of normal epithelial cells but stimulates growth of many cancer cells (11, 15). This positive and negative regulation of cell proliferation may be analogous to what is observed for the role of IGFBP-2.

We show that complementation of the menin-null cells with wild-type menin, but not a common MEN1-related K119{Delta} mutant, restores the cells’ ability to repress the expression of IGFBP-2. Additionally, wild-type menin, but not the K119{Delta} mutant, inhibits the promoter of IGFBP-2. This is the first time that menin is found to be essential for repression of an endogenous gene via targeted gene disruption, providing a foundation for further understanding the mechanism of menin-mediated regulation of gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
To generate recombinant retroviruses, pMX-menin and pMX-2xFlag-menin were constructed by inserting PCR-amplified menin cDNA into the BamHI/NotI site of the retroviral vector pMX-puro or pMX-2xFlag. pMX-K119{Delta} and pMX-D418N were generated from pMX-menin using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). To generate menin NLS mutants, pMX-2xFlag-menin was used as a template for site-directed mutagenesis to delete the nuclear localization sequence 1 (NLS1) and/or NLS2. To generate pIGFBP2-Luc, a 1.0-kb promoter of IGFBP-2 from –76 to –1096 bp was isolated from mouse genomic DNA by PCR amplification, and cloned into pGL3-Basic (Promega, Madison, WI). To generate the IGFBP-2 retroviral construct, murine IGFBP-2 cDNA was released from an expressed sequence tag (EST) clone (8909261) and inserted into the EcoRI/XhoI site of pMX-puro.

Cell lines and cell culture
Mouse embryonic fibroblast (MEF) cell lines were isolated from Men1{Delta}N3–8/+ mice heterozygous for the Men1 locus (5), and cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, penicillin (100 U/ml), streptomycin (100 U/ml), 1% MEM nonessential amino acids, and 1% L-glutamine, as previously described (16). To generate a variety of retrovirus-infected MEF cell lines, retroviruses expressing vector, menin, menin mutant, or IGFBP-2 were packaged in 293T cells by cotransfection with a defective helper retroviral DNA as previously described (16). The Men1–/– MEFs were infected with either one type of retrovirus or with two types of retroviruses. The resulting cells were selected with puromycin as previously described (16).

Transient transfection and luciferase assays
293T cells were transfected by the calcium phosphate precipitation method as previously described (17). For luciferase assays, Men1–/– MEFs were cotransfected with pSV40-ß encoding the lacZ gene as an internal control to normalize the luciferase activity. To transfect Men1–/– MEFs, 5 x 104 cells per well were plated in 12-well plates on d 0. On d 1, cells were transfected using the GenePORTER method as instructed by the manufacturer (GTS, Inc., San Diego, CA). After a 36-h incubation (5% CO2, 37 C), cells were harvested for luciferase and ßgalactosidase assays as previously described (17). All luciferase activities were normalized to the ß-galactosidase activities and presented as the average of duplicate samples.

DNA microarray and Northern blotting analysis
Vector or menin-complemented MEFs (2.5 x 105) were seeded in a 100-mm dish. The cells were cultured for 48 h before the total RNA was isolated using the cesium chloride centrifugation method (18). To reduce variation in gene expression profiles, two independent preparations of RNA from vector and menin-complemented MEFs were isolated and processed to generate biotin-labeled RNA probes (19). The probe was hybridized to the Affymetrix Murine GeneChip U74A array at The Penn Microarray Core Facility. For Northern blotting analysis, cells (4 x 106) were seeded per 150-mm dish. After a 24-h incubation, cells were harvested and the total RNA was isolated. Total RNA (30 µg) was used for Northern blotting assays as previously described (17). The probe for IGFBP-2 was derived from an EST clone (8909261).

Western blotting and Immunofluorescent staining
The whole cell lysates, nuclear or cytoplasmic fractions were separated on SDS-PAGE gels, transferred to Hybond-C+ membranes, and blotted with primary and secondary antibodies. For immunofluorescent staining, cells were seeded on coverslips, processed for incubation with an anti-Flag antibody (M2), followed by incubation with the fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 4',6-diamidino-2phenylindole before being photographed under a fluorescence microscope (Nikon E800, Tokyo, Japan), as previously described (20).

Deoxyribonuclease (DNase) I hypersensitivity analysis
DNase I hypersensitivity analysis was performed as previously described (21). Briefly, 5 x 106 MEFs were used to isolate the nuclei, which were then digested with increasing concentrations of DNase I (Sigma, St. Louis, MO), followed by proteinase K digestion at 55 C overnight. DNA was isolated from the digested nuclei by phenol/ chloroform extraction and ethanol precipitation and then digested with restriction enzyme BstXI. The resulting DNA was subjected to Southern blotting with a P32-labeled probe corresponding the 5' end of the BstXI fragment (~200-bp fragment downstream from the 5' BstXI site). The membrane was exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) for quantitation of the intensity of the BstXI fragment and the DNase I-sensitive fragment. The intensity of the DNase I-sensitive fragment was divided by the intensity of the BstXI fragment to obtain the normalized sensitivity of the promoter DNA to DNase I.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Targeted disruption of Men1 leads to up-regulation of IGFBP-2, and complementation of menin-null cells with wild-type menin reduces expression of IGFBP-2
Menin interacts with several transcription factors including JunD and inhibits their activities (6, 22). However, it is not known whether menin is essential for repressing any endogenous genes via their promoter. To investigate the role of menin in repressing gene expression, we isolated and then immortalized a pair of menin-expressing or menin-null mouse MEFs, from 9.5-d murine embryos. Loss of expression of menin in the menin-null cell line was confirmed by Western blotting analysis (Fig. 1AGo, top panel, lane 2). To identify genes that are repressed by menin expression, RNA was isolated from a pair of menin-null and menin-expressing cells, and DNA microarray analysis was performed on RNA isolated from these cells to identify such genes. Genes whose expression was down-regulated more than 2-fold in the menin-expressing cells are listed in Table 1Go. IGFBP-2 was one of the genes whose expression was repressed by menin and confirmed by Northern blot analysis. In the menin-expressing cells, the expression of IGFBP-2 was reduced approximately 14-fold in two independent DNA microarray analyses, compared with that in the menin-null cells (Table 1Go). IGFBP-2 was originally identified for its ability to bind IGFs (11). It can antagonize the IGF-induced cell proliferation, but it can also promote cell growth in an IGF-independent manner (12, 23). Additionally, IGFBP-2 is a marker for both prostate and colon cancer (11, 23).



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  		FIG. 1. Ablation of Men1 leads to up-regulation of IGFBP-2, and complementation of menin-null cells with menin down-regulates expression of IGFBP-2. A, IGFBP-2 expression is lowered in Men1+/+ cells (no. 53), but not in Men1–/– cells (no. 55). Whole cell lysates (152 µg) were used for Western blotting analysis with an antimenin antibody (#80) (top panel). The asterisk (*) denotes a nonspecific cross-reactive band. Total RNA from the indicated cells was separated on an agarose gel, transferred to the membrane, and hybridized to the probe corresponding to the first exon-1 of murine IGFBP-2 (middle panel). As a control for loading, the membrane was also hybridized with the probe of GAPDH (bottom panel). B, IGFBP-2 expression is reduced in menin-complemented cells. Men1–/– cells (no. 21) were infected with control retroviruses or menin-expressing retroviruses. The cell lysates (60 µg) from the infected cells were subjected to Western blotting analysis (top panel) to confirm menin expression. Total RNA isolated from these cells was subjected to Northern blotting analysis using the IGFBP-2 probe or a control GAPDH probe (middle and bottom panels) as described in Fig. 1AGo. The asterisk (*) denotes a nonspecific cross-reactive band. C, IGFBP-2 protein expression is repressed by menin. Menin-null or menin-expressing cells as noted in panel B (0.5 x 106 per 100-mm dish) were seeded, and 48 h later, the whole cell lysates were isolated from these cells. The cell lysates (200 µg) were subjected to Western blotting using an anti-IGFBP-2 (sc-6002 Santa Cruz Biotechnology, Santa Cruz, CA; top panel). As a control for equal loading, the same membrane was reprobed with an antiactin antibody (sc-1615, Santa Cruz Biotechnology; bottom panel).

 

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  		TABLE 1. Genes whose expression was down-regulated more than 2-fold in menin-expressing cells

 
Further examination of IGFBP-2 expression in a pair of Men1+/+ and Men1–/– cells by Northern blotting analysis confirms that menin reduces the level of the IGFBP-2 mRNA (Fig. 1AGo, middle panel, lanes 1 and 2). As a control, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each cell line is similar (Fig. 1AGo, bottom panel). If ablation of Men1 up-regulates IGFBP-2, complementation of menin-null cells with menin should reduce the expression of IGFBP-2. Thus, menin-null cells were infected with control retroviruses or retroviruses expressing menin, and the expression of menin in the infected cells was confirmed by Western blotting analysis (Fig. 1BGo, top panel). The infected cells were subjected to Northern blotting analysis to detect the expression level of the IGFBP-2 mRNA. Figure 1BGo shows that, indeed, expression of menin reduces the steady level of the IGFBP-2 mRNA (middle panel, lane 1), whereas the level of the control GAPDH in each cell line is similar (Fig. 1BGo, bottom panel). We further examined the protein levels of intracellular IGFBP-2 in both menin-null and menin-expressing cells that were in an exponential growth phase. The results indicate that menin reduces expression of IGFBP-2 (Fig. 1CGo, top panel), whereas the level of control actin is the same between the menin-null and menin-expressing cells (Fig. 1CGo, bottom panel). Collectively, these results demonstrate a critical role for menin in repressing the expression of IGFBP-2.

The menin mutant, K119{Delta}, fails to repress expression of IGFBP-2
Numerous mutations in Men1 have been reported in MEN1 patients (1, 3). We chose to examine the effects of two common mutations in the Men1 gene, K119{Delta} and D418N, on the expression of IGFBP-2. The menin-null cells were infected with either control viruses, or viruses expressing wild-type menin, K119{Delta}, or D418N. Western blotting analysis shows that wild-type menin and its mutants (K119{Delta} and D418N) were expressed as expected (Fig. 2AGo, top panel, lanes 2–4), with a higher level of expression for the K119{Delta} mutant (lane 3). Northern blotting analysis shows that, as expected, wild-type menin reduces the expression of IGFBP-2, but the K119{Delta} mutant fails to repress IGFBP-2 expression (Fig. 2AGo, middle panel, lane 3). However, the point mutant D418N is still capable of repressing the expression of menin (Fig. 2AGo, middle panel, lane 4). The expression of GAPDH in each of the cell lines is similar (Fig. 2AGo, bottom panel).



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  		FIG. 2. The K119{Delta} point mutant loses its ability to repress the expression of IGFBP-2. A, Menin mutant K119{Delta} fails to suppress the expression of IGFBP-2 on mRNA level. Men1–/– cells were infected with control viruses, or viruses expressing wild-type menin, K119{Delta} or D418N. Whole cell lysates were isolated from each of the above cell lines, and analyzed by Western blotting using an antimenin antibody (no. 80) (top panel). The asterisk (*) denotes a nonspecific crossreactive band. Total RNA (30 µg) from the above various cell lines were separated and hybridized to a probe generated from either IGFBP-2 (middle panel) or GAPDH cDNAs (bottom panel). B, Menin mutant K119{Delta} fails to suppress the expression of IGFBP-2 on protein level. The MEFs described in Fig. 2AGo were seeded at a density of 3.2 x 105 per 100-mm dish, and harvested after 48 h culture for Western blot analysis. The whole cell lysates (150 µg for each cell line) were subjected to Western blotting analysis using an anti-IGFBP-2 antibody (sc-6002 Santa Cruz Biotechnology; top panel). As a control for equal loading, the same membrane was reprobed with an antiactin antibody (sc-1615, Santa Cruz Biotechnology; bottom panel).

 
We further examined the IGBP-2 protein level in the menin-null cells and the cells expressing either wild-type or mutant menin. Consistent with the mRNA level of IGFBP-2, the protein level of IGFBP-2 is also decreased in cells expressing wild-type menin (Fig. 2BGo, top panel, lane 2). Notably, cells expressing mutant K119{Delta} fail to suppress the IGFBP-2 expression (Fig. 2BGo, top panel, lane 3). However, menin mutant D418N still represses the expression of IGFBP-2 protein (Fig 2BGo, top panel, lane 4). An equal loading of the total proteins for each lane is confirmed by Western blotting using an antiactin antibody (Fig. 2BGo, bottom panel). Collectively, these results indicate that a common disease-related mutant, K119{Delta}, compromises menin’s ability to repress IGFBP-2 expression, suggesting that repression of gene expression may related to the tumor-suppressing function of menin. However, because another mutation, D418N, does not affect menin’s role in regulating IGFBP-2, regulation of IGFBP-2 may not be the only or even major means by which menin suppresses MEN1 development.

Wild-type menin, but not the K119{Delta} mutant, inhibits the promoter of IGFBP-2
Because menin represses the expression of IGFBP-2, we tested whether menin affects the activity of the promoter of IGFBP-2. A 1.0-kb promoter of IGFBP-2 was isolated from the mouse genomic DNA and cloned upstream of a luciferase reporter gene. To determine whether menin inhibits the promoter of IGFBP-2, the IGFBP-2 promoter construct was cotransfected into a menin-null cell line along with increasing amounts of a menin-expressing construct. Figure 3AGo shows that menin inhibits the expression of the luciferase reporter gene more than 2-fold (at 500 ng). To verify that the small amount of menin cDNA used in Fig. 3AGo was indeed expressed, increasing amounts of the epitope-tagged menin construct were transfected into 293T cells, followed by Western blotting analysis to determine the expression of the transfected menin cDNA. Indeed, transfection of the exogenous menin construct leads to expression of the corresponding protein (Fig. 3BGo, lanes 3 and 4). The K119{Delta} mutant, but not the D418N mutant, fails to significantly repress the expression of the reporter gene (P = 0.017, menin vs. K119{Delta} and P = 0.08, menin vs. D418N) (Fig. 3CGo). This is consistent with the data in Fig. 2Go that the K119{Delta} mutant fails to repress the expression of IGFBP-2. Because there is no evidence that menin binds DNA directly, it is unclear how menin regulates the promoter. Menin interacts with a variety of transcription factors, such as JunD and NF-{kappa}b (6, 7), and it is possible that menin regulates the promoter by associating with other transcription factors.



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  		FIG. 3. The K119{Delta} mutant fails to suppress the IGFBP-2 promoter activity. A, Inhibition of the IGFBP-2 promoter by menin is dose dependent. The menin-null cells in 12-well plates were cotransfected with the reporter gene driven by the IGFBP-2 promoter (0.6 µg per well), pSV-ß gal (0.1 µg) and the indicated amounts of pMX-menin, followed by luciferase assays. The total amount of DNA per well was 1.2 µg per well, using pMX-puro vector as control to equalize the amounts of total DNA. B, Transfected menin cDNA leads to expression of menin. On d 0, 0.8 million per well 293T cells were seeded in six-well plates. On d 1, cells were transfected with increasing amounts of Flag-tagged menin cDNA as indicated in Fig. 3AGo. The transfected cells were harvested 48 h after transfection, and the cell lysates (400 µg) were subjected to Western blotting using an anti-Flag antibody. C, The K119{Delta} mutant fails to suppress the IGFBP-2 promoter activity. The cells were seeded, and then transfected with pIGFBP2-Luc and various menin constructs for luciferase assays as described for Fig. 3AGo.

 
Menin alters the promoter structure of the IGFBP-2 gene
In the promoter of the mouse and rat IGFBP-2 genes, more than three transcription factor Sp1 binding sites are clustered approximately 200 bp upstream of the initiator ATG (24, 25). These sites were shown to bind Sp1 by both gelshift and DNA footprinting assays and were found to be crucial for Sp1-mediated activation of the IGFBP-2 promoter (24). To determine whether menin can alter the chromatin structure surrounding these Sp1 binding sites, we sought to determine the sensitivity of the DNA spanning the Sp1 binding sites to DNase I digestion. If menin represses the sensitivity of this stretch of DNA to DNase I, it means that menin inhibits the activation of the promoter, at least in part by repressing the opening of the chromatin encompassing the Sp1 binding sites. Figure 4AGo illustrates the gene structure of murine IGFBP-2, including the promoter sequence, the first exon containing the initiator ATG, and the first intron. The probe used for the Southern blot analysis is approximately 3.9 kb away from the cluster of the Sp1 sites.



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  		FIG. 4. Menin expression decreases the sensitivity of the IGFBP-2 promoter to DNase I digestion. A, A diagram for the portion of murine IGFBP-2 genomic DNA that was analyzed for DNase I hypersensitivity. B, Menin-null and menin-expressing cells (5 x 106) were harvested, and treated with increasing concentrations of DNase I as described in Materials and Methods (0, 0.1, 0.5 or 1 µg/ml). The purified DNA from the DNase I-digested cells was further digested with BstXI, and subjected to Southern blotting analysis using the probe as indicated in Fig. 4AGo. This is a representative of three independent and consistent experiments. C, The signals of DNase Isensitive or -resistant DNA fragments in Fig. 4BGo were quantitated using a phosphorimager software, and the values are the ratio of the DNase I-sensitive fragment signal to the DNase I-resistant fragment signal. This is a representative of three independent experiments.

 
Thus, we treated the nuclei from the menin-null cells and menin-expressing cells with increasing concentrations of DNase I, and then used Southern blotting analysis to examine the sensitivity of the IGFBP-2 promoter to DNase I. If a region in the promoter is activated and the surrounding chromatin structure is more open, this region should be more sensitive to DNase I digestion. Figure 4BGo indicates that in both control menin-null cells and menin-expressing cells, a low concentration of DNase I generates a DNase I-hypersensitive band, 3.7 kb in length (lanes 2 and 6). This is consistent with the existence of a hypersensitive site, which overlaps with a cluster of the Sp1 sites (25). However, with increasing concentrations of DNase I, a slightly more DNase I-sensitive fragment (~3.7-kb fragment) and a less DNase I-resistant fragment (~9.6-kb fragment) are generated (Fig. 4BGo, lanes 1–4 and 5–8). The ratio of the intensity of the two fragments reflects the normalized sensitivity of the promoter DNA to DNase I, which should correlate with the level of the promoter activation. Although with increasing concentrations of DNase I, the intensity of the DNase I-sensitive fragments slightly increases for both the menin-null control cells (Fig. 4BGo, lanes 1–4) and menin-expressing cells (Fig. 4BGo, lanes 5–8), the normalized sensitivity (ratio) of the promoter in the menin-null control cells (lanes 1–4) is consistently higher than that in the menin-expressing cells (lanes 5–8) (Fig. 4BGo, bottom). The normalized sensitivity of the IGFBP-2 promoter for both the control cells and menin-expressing cells is plotted in Fig. 4CGo. Collectively, these results suggest that complementation of menin-null cells with menin represses the activation of the promoter of the IGFBP-2 containing the Sp1 binding sites, albeit to a modest extent. However, it is unclear whether menin physically associates with this region.

Mutation of nuclear localization signals in menin compromises the role of menin in repressing expression of IGFBP-2
We previously showed that menin is primarily localized in the nucleus, especially in chromatin and the nuclear matrix (20). Two independent NLSs have been shown to target menin to the nucleus (26). If these NLSs are crucial for the targeting of menin to the nucleus, mutation of the NLSs in menin would likely interfere with menin’s translocation into the nucleus and consequently with its role in regulating the transcription of IGFBP-2. Two NLSs, from amino acids 479–497 and 588–608, were deleted individually or in combination (Fig. 5AGo). The resulting proteins were expressed, as determined by the in vitro translation analysis (Fig. 5BGo). Figure 5CGo shows that wild-type menin is localized to the nucleus, as shown by immunofluorescent staining. Deletion of either NLS1 or NLS2 does not block the translocation of menin into the nucleus (Fig. 5CGo). However, deletion of both NLS1 and NLS2 compromises the nuclear translocation of menin (Fig. 5CGo, bottom panel), although there is still significant amount of menin in the nucleus. To further confirm the results obtained with fluorescent staining, the above cell lines were fractionated, and the nuclear and cytoplasmic fractions were subjected to Western blot analysis for the distribution of menin in the cytoplasmic and the nuclear extracts. Figure 5DGo shows that in the cells expressing the wild-type menin and the single NLS deletion mutants (NLS1{Delta} and NLS2 {Delta}), the majority of menin is localized in the nucleus (lanes 3–8). In contrast, almost an equal amount of double NLS deletion menin mutant is distributed between the nucleus and the cytosol (lanes 9 and 10). Together, these results indicate that a single NLS is sufficient for the nuclear targeting and deletion of both NLS1and NLS2 compromises, but does not abolish, the nuclear translocation, suggesting an additional NLS in menin.



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  		FIG. 5. Mutation of two NLSs in menin compromises the role of menin in suppressing IGFBP-2. A, A diagram of various constructs expressing various NLS deletion mutants. B, In vitro translation of menin and its NLS deletion mutants. wild-type menin and its various NLS mutants were translated in vitro in the presence of 35S-methionine. The translated products were separated on an SDS-PAGE gel and detected on a Molecular Dynamics PhosphorImager. C, Nuclear localization of menin and its NLS mutants. Menin-null cells infected with the retroviruses expressing menin or its various NLS mutants were seeded and processed for immunofluorescent staining with an anti-Flag antibody. D, Detection of menin in the nuclear and cytoplasmic fractions using Western blotting. The nuclear and cytoplasmic fractions (88 µg) were isolated from the cells used for the immunofluorescent staining, and subjected to Western blotting analysis using an anti-Flag antibody. E, Menin NLS{Delta} mutants fail to repress the expression of IGFBP-2. Total RNA was isolated from the various indicated cells and hybridized with the IGFBP-2 probe (top panel). As a control, the membrane was also hybridized with the control GAPDH probe (bottom panel).

 
Next, we determined whether mutation of either one NLS or both NLSs of menin interferes with the role for menin in suppressing IGFBP-2 expression. Menin-null cells or cells expressing either wild-type menin or one of the mutants, NLS1{Delta}, NLS2{Delta}, and NLS1&2{Delta}, were analyzed for expression of IGFBP-2. Figure 5EGo shows that wild-type menin reduces the level of the IGFBP-2 mRNA, as expected. In contrast, menin NLS1{Delta} and NLS2{Delta} mutants lose their ability to fully repress the expression of IGFBP-2 (Fig. 5EGo), whereas their ability to translocate into the nucleus is not affected (Fig. 5CGo). Mutation of both NLS1 and NLS2 further decreases the ability of menin to suppress IGFBP-2 expression (Fig. 5EGo). As a control, the level of expression of GAPDH is similar for all cell lines. These results indicate that each NLS in menin has a crucial role in repressing IGFBP-2, in addition to its role in targeting menin to the nucleus.

Ectopic expression of IGFBP-2 fails to enhance cell proliferation
To examine whether ectopic expression of IGFBP-2 enhances the proliferation of menin-null cells, a retroviral construct expressing murine IGFBP-2 cDNA was generated, and expression of IGFBP-2 from the construct was verified in 293T cells after transient transfection, followed by Western blot analysis (Fig. 6AGo, lane 2). The retroviruses expressing IGFBP-2 or menin were used to infect the menin-null cells individually or in combination. The resulting infected cells were subjected to stable selection with puromycin and the expression of menin (Fig. 6BGo, top panel) or IGFBP-2 (Fig. 6BGo, middle panel) was confirmed by Western blot analysis. The equal loading of all the samples was confirmed by Western blot analysis for the amount of actin protein (Fig. 6BGo, bottom panel). These results indicate that the expression of ectopic IGFBP-2 in IGFBP-2 retrovirus-infected menin-null cells is higher than that of the control menin-null cells (Fig. 6BGo, middle panel, lane 1 vs. 3). The IGFBP-2 expression in the control cells is higher than that in the menin-expressing cells (Fig. 6BGo, lanes 1 and 2), consistent with the findings in Fig. 1CGo.



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  		FIG. 6. Ectopic expression of IGFBP-2 fails to enhance cell proliferation. A, Ectopic expression of IGFBP-2 in 293T cells. 293T cells (3 x 106) were transfected with control vector DNA or a retroviral construct expressing murine IGFBP-2. The lysates from the transfected cells (160 µg) were subjected to Western blot analysis using an anti-IGFBP-2 antibody (M-18, Santa Cruz Biotechnology). B, Expression of IGFBP-2 from the ectopic IGFBP-2 cDNA. Men1–/– cells were infected with control retroviruses, or retroviruses expressing either menin or IGFBP-2 alone or in combination, as indicated. The whole cell lysates were isolated from the resulting cells, followed by Western blotting analysis using an antimenin antibody (top panel), an anti-IGFBP-2 antibody (middle panel), or an antiactin antibody for a loading control (bottom panel). This is representative of two independent experiments. The asterisk denotes a nonspecific cross-reactive protein. C, Ectopic expression of IGFBP-2 fails to significantly enhance cell proliferation. Menin-null cells infected with the indicated constructs analyzed in Fig. 6BGo were seeded at a density of 2 x 105 per 100-mm dish on d 0, and counted on d 4 of culture. This is a representative of three independent experiments.

 
We further examined the effects of ectopic expression of menin and/or IGFBP-2 in menin-null cells on cell proliferation. To this end, the above various cell lines expressing IGFBP-2 and/or menin were seeded in culture medium and cell number was counted on d 4 of culture. Figure 6CGo shows that menin indeed suppresses cell proliferation, but infection of the menin-null cells with the IGFBP-2 viruses only slightly increases cell proliferation. However, coexpression of menin and IGFBP-2 still leads to inhibition of cell proliferation. Together, these results suggest that ectopic expression of IGFBP-2 fails to induce proliferation of the menin-null cells. It is possible that IGFBP2 is not the major target for menin in menin-induced cell inhibition. Alternatively, menin-mediated repression of the IGFBP-2 expression may be crucial only in certain types of cells or only in the context of other certain activated oncogenes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although a crucial role for menin in suppression of tumorigenesis is well documented, little is known about its biochemical function (27). Menin interacts with a number of nuclear proteins, including JunD, NF-{kappa}b, Smad3, and Pem (6, 7, 8, 9) and represses the activities of some of these factors in reporter gene assays. Overexpression of menin suppresses insulin-induced transcription of c-Fos (10). Ectopic expression of menin has also been reported to repress the promoters of prolactin and gastrin (28, 29), based on luciferase reporter assays. However, so far little is known about whether menin is essential for repression of endogenous genes. Using menin-null cells generated by gene targeting, we show that menin is essential for repressing the expression of an endogenous gene, IGFBP-2 (Figs. 1Go and 2Go). Complementation of the menin-null cells with menin inhibits the expression of IGFBP-2. Moreover, a common menin mutation from MEN1 patients, K119{Delta}, fails to repress the IGFBP-2 expression, although the level of expression of K119{Delta} is even higher than that of wild-type menin. In addition, mutations of each NLS1 and NLS2 in menin fail to block the nuclear translocation of menin but compromise the ability to repress IGFBP-2 expression, indicating an additional role of the NLSs in repressing IGFBP-2.

These findings have several important implications for understanding the role of menin in regulation of gene transcription. First, these studies indicate that menin is essential for repression of the endogenous IGFBP-2 gene because targeted disruption of Men1 in MEFs leads to activation of the IGFBP-2, a gene involved in both positive and negative regulation of cell proliferation, depending on cell type (13). Second, the menin-mediated repression of IGFBP-2 is at least in part executed through the promoter region (Figs. 3Go and 4Go). Third, the fact that a point mutant, K119{Delta}, fails to repress expression of IGFBP-2 strongly suggest that menin-mediated regulation of IGFBP-2 may be in part related to the function of menin in suppressing the development of MEN1. Finally, the fact that the NLS1 and NLS2 double mutant menin is still able to enter the nucleus indicates the existence of additional NLSs in menin. Currently it is not known where the additional NLS is located. Furthermore, the NLS1 and NLS2 are not only important for the nuclear localization, but also crucial for repressing the expression of IGFBP-2 (Fig. 5Go). These findings provide an opportunity to further investigate how menin regulates endogenous genes.

IGFBP-2 is a member of the IGFBP family (11). It inhibits cell proliferation induced by IGFs (11). In support of this function, transgenic mice overexpressing IGFBP-2 display significantly reduced body weight (11). Consistent with this, IGFBP-2 mediates inhibition of cell proliferation that is induced by TGF-ß (14). This is particularly interesting because TGF-ß usually inhibits proliferation of epithelial cells but stimulates proliferation of cancer cells that harbor TGF-ß-resistant oncogenes (15). On the other hand, IGFBP-2 also possesses IGF-independent activity. For example, expression of IGFBP-2 increases the tumorigenesis of adrenal cortical cancer cells (12). In addition, IGFBP-2 also stimulates the growth of prostate cancer cells (13) and the metastasis of glioblastoma invasion (30). Furthermore, it is also a highly expressed marker for many cancers including prostate cancer and colon cancer (11, 13, 23). It is still unclear how exactly IGFBP-2 induces tumorigenesis.

In the current study, we fail to observe IGFBP-2-induced cell proliferation in the menin-null MEFs. Several explanations may exist for this observation. First, it is possible that the MEFs may not be a cell type in which IGFBP-2 induces cell proliferation. Second, IGFBP-2 may act similar to TGF-ß, and inhibit proliferation of normal cells but stimulate proliferation only in cancer cells that harbor certain types of oncogenes, which are not present in the MEFs used in these studies. Third, it is also formally possible that IGFBP-2 is not the major target gene that is crucial for menin-induced inhibition of cell proliferation. However, studying menininduced expression of IGFBP-2 will provide novel insights into how menin regulates gene expression.

It is unclear how menin represses the expression of IGFBP-2. We previously showed that a majority of menin closely associates with chromatin (20). Here we show that menin inhibits the promoter of IGFBP-2 in a dose-dependent manner (Fig. 3AGo), and a common mutation from MEN1 patients fails to inhibit the promoter of IGFBP-2 (Fig. 3CGo). In addition, menin also alters the status of the chromatin structure surrounding the IGFBP-2 promoter (Fig. 4Go). It has recently been shown that menin inhibits the expression of human telomere reverse transcriptase, by binding to the putative activator protein 1 and NF-{kappa}b binding sites in the promoter of human telomere reverse transcriptase (31). Menin could inhibit these transcription factors by recruiting mSin3A and histone deacetylase (32, 33). Menin is also recently shown to associate with a histone methyltransferase complex and activate transcription of the endogenous Hoxc-8 gene (34). Thus, it is possible that menin interacts with other coregulators such as the mSin3A-histone deacetylase complex to repress the expression of IGFBP-2, at least in part, through the promoter containing multiple Sp1 binding sites.

Further supporting the role of menin in regulating IGFBP-2, NLS1, and NLS2 in menin are each crucial for repressing the IGFBP-2 expression (Fig. 5EGo), although deletion of NLS1 or NLS2 alone does not block the nuclear translocation of menin. These results imply that these NLSs are essential for repressing IGFBP-2 in addition to targeting menin to the nucleus. Perhaps the positively charged NLSs may mediate menin’s association with other factors or nuclear structures that modulate transcription. Although the detailed mechanism for repression of IGFBP-2 by menin remains to be determined, the current studies have established that menin is essential for optimal repression of IGFBP-2, a protein that plays crucial roles in positive and negative regulation of cell proliferation.


   Acknowledgments
 
We thank Dr. Don Baldwin of the Penn Microarray Facility for technical advice and discussions, Angela Desmond for technical assistance, and Dr. Steve Reiner at Department of Medicine at University of Pennsylvania for helpful discussions on DNase I-hypersensitivity assays.


   Footnotes
 
This work was supported in part by a Howard Temin Award (K01CA78592 to X.H.), a Burroughs Wellcome Career Award (No. 1676; to X.H.), an award from the Rita Allen Foundation (to X.H.), and a Research Scholar award from American Cancer Society.

Abbreviations: DNase, deoxyribonuclease; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP, IGF binding protein; MEF, mouse embryonic fibroblasts; MEN1, multiple endocrine neoplasia type I; NF, nuclear factor; NLS, nuclear localization sequence.

Received February 2, 2004.

Accepted for publication March 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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