Inactivation of Menin, the Product of the Multiple Endocrine Neoplasia Type 1 Gene, Inhibits the Commitment of Multipotential Mesenchymal Stem Cells into the Osteoblast Lineage*
Hideaki Sowa,
Hiroshi Kaji,
Lucie Canaff
,
Geoffrey N. Hendy
,
Tatsuo Tsukamoto,
Toru Yamaguchi,
Kohei Miyazono ¶ ||,
Toshitsugu Sugimoto ** and
Kazuo Chihara
From the
Division of Endocrinology/Metabolism, Neurology, and Hematology/Oncology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan the
Departments of Medicine, Physiology, and Human Genetics, McGill University and Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada, the ¶Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, 1-37-1, Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan, and the ||Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, February 26, 2003
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ABSTRACT
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The physiological roles of menin, the product of the multiple endocrine neoplasia type 1 gene, are not known. Homozygous menin knockout mice exhibit cranial and facial hypoplasia. We, therefore, investigated the role of menin in the regulation of osteoblastic differentiation. Menin antisense oligonucleotides (AS-oligo) reduced endogenous menin expression in the C3H10T1/2 (10T1/2) mouse mesenchymal stem cells and antagonized alkaline phosphatase (ALP) activity and the expression of type I collagen, Runx2/cbfa1 (Runx2), and osteocalcin (OCN) induced by bone morphogenetic protein 2 (BMP-2). AS-oligo did not affect adipogenic markers (Oil red staining and PPAR
expression) and chondrogenic markers (Alcian blue staining and type IX collagen) induced by BMP-2 in 10T1/2 cells. Menin co-immunoprecipitated with Smad1 and Smad5, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5. In osteoblastic MC3T3-E1 cells, AS-oligo affected neither BMP-2-stimulated ALP activity nor the expression of Runx2 and OCN. Stable inactivation of menin in MC3T3-E1 cells increased ALP activity, mineralization, and the expression of type I collagen and OCN. In 21-day cultures of MC3T3-E1 cells and BMP-2-treated 10T1/2 cells, endogenous menin expression increased up to day 14 and declined thereafter. These data indicate that menin inactivation specifically inhibits the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage, mediated by menin and Smad1/5 interactions. Menin is important for both early differentiation of osteoblasts and inhibition of their later differentiation, and it might be crucial for intramembranous ossification.
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INTRODUCTION
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Bone is a dynamic connective tissue, undergoing modeling and remodeling. During modeling, bone is formed in two different ways, intramembranous ossification and endochondral ossification. In intramembranous ossification, condensing cells differentiate into osteoblasts, whereas in endochondral ossification, condensing cells differentiate into chondrocytes to form cartilage, later replaced by bone. It has been suggested that osteoblasts, chondrocytes, adipocytes, and myoblasts are derived from common precursor cells (1), which are multipotential mesenchymal stem cells derived from mesoderm. The differentiation of multipotential mesenchymal stem cells into a distinct cell lineage is termed "commitment,"and their commitment to the osteoblast lineage as well as their differentiation and maturation that follow are necessary for both bone modeling and remodeling. These sequential events are regulated both systemically and locally by several hormones, growth factors, and cytokines. Moreover, transcription factors, such as Runx2/cbfa1 (Runx2), Fra-1, and Osx, are important for osteoblastic differentiation (2, 3, 4, 5) and modulate the expression of osteoblast-specific genes, such as type I collagen (COLI),1 alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN).
Menin is the product of the MEN1 gene (6), responsible for the multiple endocrine neoplasia type 1, an autosomal dominant cancer syndrome characterized by multiple tumors of the parathyroid, endocrine pancreas, and the anterior pituitary. Over 250 independent germ line and somatic mutations scattered throughout the protein-coding region have been identified (7, 8). Most of the mutations are clearly inactivating, leading to a truncated product. This would be consistent with menin acting as a tumor suppressor gene and a complete lack of menin, caused by the loss of both alleles, resulting in tumor development (6). The human MEN1 gene encodes a 610-amino acid protein with homology to no known protein. Two novel nuclear localization signal sequences have been identified in the carboxyl-terminal portion of menin protein, which has been demonstrated to be predominantly located in the nucleus (9, 10). Menin function may be related to transcriptional regulation (11) or cell cycle control (10). Moreover, recent studies revealed that menin interacts with a variety of proteins, including JunD, NF-
B, nm23, and Pem (11, 12, 13, 14), and that it represses JunD- and NF-
B-activated transcription, although the significance of these findings is unclear. We recently demonstrated that menin interacts with Smad3 and that inactivation of menin blocks transforming growth factor type
(TGF-
) signaling by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites (15). These data implicate a mechanism of tumorigenesis by menin inactivation. However, the physiological functions of menin remain unknown.
Several studies revealed that menin is widely expressed in mouse tissues and is found in both nonendocrine and endocrine tissues (16, 17). Recently, Crabtree et al. (18) reported that homozygous menin inactivation in mice was embryonic lethal and some fetuses exhibited clear defects in cranial and facial development, whereas the heterozygous phenotype was strikingly similar to that of the human disorder MEN1, and endocrine tumors developed later in life. Since cranial bones are formed by intramembranous ossification, these findings suggested the possibility that menin might play an important role in commitment from multipotential mesenchymal stem cells to osteoblast lineage and osteoblastic differentiation. We therefore investigated the effects of menin inactivation with anti-sense menin oligonucleotides (AS-oligo) on bone morphogenetic protein 2 (BMP-2)-induced differentiation in mouse C3H10T1/2 (10T1/2) cells as a model for the commitment of undetermined multipotential mesenchymal stem cells into osteoblast lineage. We also examined the osteoblast phenotype by using mouse osteoblastic MC3T3-E1 cells in which antisense-menin (AS) was stably expressed as a model for osteoblastic differentiation.
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EXPERIMENTAL PROCEDURES
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MaterialsAnti-type I collagen antibody was from Calbiochem, and anti-type IX collagen antibody was from LSL Co., Ltd. (Tokyo, Japan). Human recombinant BMP-2 was kindly provided by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). Retinoic acid and human recombinant TGF-
1 were from Sigma. The menin rabbit polyclonal antibody was raised against a decapentapeptide (synthesized by solid-phase chemistry at the Peptide Synthesis Facility of the Sheldon Biotechnology Centre of McGill University) corresponding to amino acids 476489 of menin (a sequence that is completely conserved between human and mouse) with an additional cysteine residue at the carboxyl-terminus. The peptide was coupled through the cysteine residue to keyhole limpet hemocyanin. The antiserum was immunoaffinity-purified before use, and, by Western blot of a variety of cell lines of different species, it recognized human and rodent menin with similar affinity (10). 24-base pair phosphorothioate-derivatized antisense and sense menin oligonucleotides (AS- and S-oligos) were synthesized on an automated solid phase synthesizer (Oligonucleotide Synthesis Facility of the Sheldon Biotechnology Centre of McGill University) by using standard phosphoramide chemistry. The sequence of the AS-oligo was 5'-GGCCTTC-AGCCCCATGGCGGCGGG-3', and that of the S-oligo was 5'-CCCGC-CGCCATGGGGCTGAAGGCC-3'. These sequences are identical in human and rodent menin. All chemicals used were of analytical grade.
Cell CultureC3H10T1/2 (10T1/2) murine mesenchymal progenitor cells were grown in Eagle's basal medium (Invitrogen) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). BMP-2 induced 10T1/2 cell adipogenic and chondrogenic differentiation without additional reagents specific to adipogenic and chondrogenic differentiation, as previously described by others (19, 20). MC3T3-E1 cells, a clonal osteoblastic cell line isolated from calvariae of late stage mouse embryo (21), and the two bone marrow stromal cell lines, ST2 and MC3T3-G2/PA6 (PA6) cells, were cultured in
-minimum essential medium (MEM) (containing 50 µg/ml ascorbic acid). These media were supplemented with 10% FBS and 1% penicillin-streptomycin (Invitrogen) and changed twice a week. For the mineralization assay, MC3T3-E1 cells were cultured in
-MEM containing 10% FBS, 1% penicillin-streptomycin, and 10 mM
-glycerophosphate (Sigma) for 2 weeks after reaching confluence. For immunoprecipitation experiments, African green monkey COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% FBS and 1% penicillin-streptomycin.
Construction of Expression Plasmids and Stable TransfectionThe human menin cDNA was constructed as previously described (10). Rat FLAG-tagged Smad1 and Smad5 were subcloned from pcDNA3 to pCMV-Tag 2A and pCMV-Tag 2B, respectively. For the antisense menin construct, human menin DNA was cloned in an antisense orientation into the EcoRI site of pcDNA3.1(+). Antisense menin cDNA (AS-DNA) and empty vector (V) (each 3 µg) were transfected into MC3T3-E1 cells with LipofectAMINE (Invitrogen). 6 h after transfection, the cells were fed with fresh
-MEM containing 10% FBS. After 48 h, cells were passaged, and clones were selected in
-MEM supplemented with G418 (0.3 mg/ml) (Invitrogen) and 10% FBS. Reduced expression of menin by AS-DNA was detected with Western blot analysis, using the polyclonal anti-menin antibody. To rule out the possibility of clonal variation, we characterized at least three independent clones for each transfection. V-transfected cells were used as the control. We constructed a heterologous luciferase reporter termed 3GC2-Lux, containing three copies of the GC-rich sequences flanked by two adenine residues inserted into 90COLXLUC (gift of S. Harada) containing the core promoter of the mouse collagen X gene in pGL2-Basic, as described (22). The transfection of 3GC2-Lux into ST2 and MC3T3-E1 cells was performed as described above.
Luciferase AssayCells were seeded at a density of 2 x 105/six-well plate. 24 h later, cells were transfected with 3 µg of the reporter plasmid (p3TP-Lux or 3GC2-Lux) and the pCH110 plasmid expressing
-galactosidase (1 µg) using LipofectAMINE (Invitrogen). 15 h later, the medium was changed to a 4% FBS-containing medium, and the cells were incubated for an additional 9 h. Thereafter, the cells were cultured for 24 h in the presence or absence of TGF-
in the medium containing 0.2% FBS. The cells were lysed, and the luciferase activity was measured and normalized to the relative
-galactosidase activity, as previously described (23).
Protein Extraction, Co-immunoprecipitation, and Western Blot AnalysisCells were lysed with radioimmunoprecipitation buffer containing 0.5 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture, 1% Triton X-100, and 1 mM sodium orthovanadate. Cell lysates were centrifuged at 12,000 x g for 20 min at 4 °C, and the supernatants were stored at -80 °C. Protein quantitation was performed with BCA protein assay reagent (Pierce). Equal amounts of proteins were denatured in SDS sample buffer and separated on 10% polyacrylamide-SDS gels. Proteins were transferred in 25 mM Tris, 192 mM glycine, and 20% methanol to polyvinylidene difluoride. Blots were blocked with TBS (20 mM Tris-HCl (pH 7.5) and 137 mM NaCl) plus 0.1% Tween 20 containing 3% dried milk powder. The antigen-antibody complexes were visualized using the appropriate secondary antibodies (Sigma) and the enhanced chemiluminescence detection system, as recommended by the manufacturer (Amersham Biosciences). For all experiments, 20 µg of protein was applied to each lane.
For co-immunoprecipitation experiments, cells were lysed with a buffer containing 1% Triton X-100, 1% deoxycholate, 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1.5 mM MgCl2, 2 mM EGTA, plus a protease inhibitor mixture for 30 min at 4 °C, and insoluble materials were separated by centrifugation at 4 °C for 30 min at 14,000 x g. The supernatant containing 1 mg of protein was clarified and incubated with anti-FLAG antibody (Sigma) on a rocking platform at 4 °C overnight. The immune complexes were collected with Protein G Plus/Protein A-agarose beads (Calbiochem) for 30 min at 4 °C. The beads were washed three times with the lysis buffer, resuspended in 2x sample buffer, and boiled for 5 min. Immunoprecipitated proteins were then analyzed by SDS-PAGE and subjected to Western blot analysis as described above.
RNA Extraction and Northern Blot AnalysisRNA was prepared with TrizolR reagent (Invitrogen). Northern blot analysis was performed, as previously described (24, 25). In brief, 20-µg aliquots of total RNA were denatured, electrophoresed on 1% agarose gels containing 2% formaldehyde, and then transferred to nitrocellulose membranes and fixed with UV light. Membranes were hybridized to a 32P (Amersham Biosciences)-labeled DNA probe overnight at 42 °C. The hybridization probes were type I collagen (COLI) (a gift from Dr. T. Kimura, Osaka University, Japan) and mouse OCN. After hybridization, the filters were washed twice with saline citrate containing SDS and exposed to x-ray film. All values were normalized for RNA loading by probing blots with human
-actin cDNA (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)Reverse transcription of 5 µg of cultured cell total RNA was carried out for 50 min at 42 °C and then 15 min at 70 °C, using the SuperScriptTM first strand synthesis system for RT-PCR (Invitrogen), which contained RT buffer, oligo(dT)1218, 5x first strand solution, 10 mM dNTP, 0.1 M dithiothreitol, SuperScript II (RT-enzyme), and RNase H (RNase inhibitor). PCR using primers to unique sequences in each cDNA was carried out in a volume of 10 µl of reaction mixture for PCR (as supplied by TaKaRa, Otsu, Japan), supplemented with 2.5 units of TaKaRa TaqTM, 1.5 mM each dNTP (TaKaRa), and PCR buffer (10x), which contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, and 15 mM MgCl2. 25 ng of each primer and 1 µl of template (from a 50-µl RT reaction) were used. Thermal cycling conditions and primer sequences are described below: 1) initial denaturation at 96 °C for 2 min; 2) cycling for cDNA-specific number of cycles (96 °C for 1 min, cDNA-specific annealing temperature for 2 min, and 72 °C for 2 min); and 3) final extension at 72 °C for 5 min. Primer sequences, annealing temperature, and cycle numbers were as follows: COLI, 5'- GCAATCGGGATCAGTACGAA-3' and 5'- CTTTCACGCCTTTGAAGCCA-3' (61 °C; 37 cycles); OPN, 5'- TCACCATTCGGATGAGTCTG-3' and 5'- ACTTGTGGCTCTGATGTTCC-3' (47 °C; 30 cycles); Runx2, 5'-CAGGAAGACTGCAAGAAGGCTCTGG-3' and 5'-ACACGGTGTCACTGCGCTGAAGA-3' (62 °C; 25 cycles); OCN, 5'-CTCTGTCTCTCTGACCTCACAG-3' and 5'-GGAGCTGCTGTGACATCCATAC-3' (53 °C; 28 cycles); PPAR
, 5'-GCTGTTATGGGTGAAACTCTG-3' and 5'-ATAAGGTGGAGATGCAGGTTC-3' (58 °C; 29 cycles); glyceraldehyde-3-phosphate dehydrogenase (GA-PDH), 5'-ATCCCATCACCATCTTCCAGGAG-3' and 5'-CCTGCTTCACCACCTTCTTGATG-3' (47 °C; 24 cycles) (19). For semiquantitative RT-PCR, the number of cycles was chosen so that amplification remained well within the linear range, as assessed by densitometry (NIH Image J, version 1.08i, public domain program). An equal volume from each PCR was analyzed by 6% nondenaturing polyacrylamide gel electrophoresis, and ethidium bromide-stained PCR products were evaluated. Marker gene expression was normalized to GAPDH expression in each sample.
Assay of ALP Activity and ALP StainALP activity was assayed by a method modified from that of Lowry et al. (26). In brief, the assay mixtures contained 0.1 M 2-amino-2-methyl-1-propanol (Sigma), 1 mM MgCl2, 8 mM p-nitrophenyl phosphate disodium, and cell homogenates. After a 3-min incubation at 37 °C, the reaction was stopped with 0.1 N NaOH, and the absorbance was read at 405 nm. A standard curve was prepared with p-nitrophenol (Sigma). Each value was normalized to the protein concentration. ALP staining was performed by a standard protocol. In brief, cultured cells were rinsed in PBS, fixed in 100% methanol, rinsed with PBS, and then overlaid with 1.5 ml of 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate (Invitrogen) plus 0.3 mg/ml nitro blue tetrazolium chloride (Invitrogen) in 0.1 M Tris-HCl, pH 9.5, 0.01 N NaOH, 0.05 M MgCl2, followed by incubation at room temperature for 2 h in the dark.
Oil Red Staining and Alcian Blue StainingIn order to evaluate adipogenic and chondrogenic differentiation, cell layers were rinsed with ice-cold PBS, fixed with 20% formaldehyde, and stained with Oil red and Alcian blue (Nacalai Tesque, Inc., Kyoto, Japan), respectively.
Mineralization AssayMineralization of MC3T3-E1 cells was determined in six-well and 12-well plates using von Kossa staining and Alizarin red staining, respectively. The cells were fixed with 95% ethanol and stained with AgNO3 by the von Kossa method. At the same time, the other plates were fixed with ice-cold 70% ethanol and stained with Alizarin red (Sigma). For quantitation, cells stained with Alizarin red were destained with ethylpyridinium chloride (Wako Pure Chemical Industries, Ltd.), and then the extracted stain was transferred to a 96-well plate, and the absorbance at 562 nm was measured using a microplate reader, as previously described (24, 25).
StatisticsData were expressed as means ± S.E. Statistical analysis was performed using an unpaired t test or analysis of variance.
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RESULTS
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Menin Expression and Effects of Menin AS-oligo in 10T1/2 CellsMenin is widely expressed in mammalian tissues (27). We examined the expression of menin in 10T1/2 mouse mesenchymal stem cells by Western blot analysis. Menin was expressed at the confluent stage, culture day 7, and the expression was increased by treatment with BMP-2 until culture day 14 and declined thereafter (Fig. 1A). Some osteoblastic phenotypes in 10T1/2 cells are shown in Fig. 1, B and C. The level of menin expression was higher when the levels of COLI and OPN expression were elevated, and it was lower when OCN expression was elevated. These results suggested that menin played an important role in the early differentiation stage of osteoblasts. The production of ALP was increased by the treatment with BMP-2 in a time-dependent manner (Fig. 1D). Treatment with AS-oligo for 12 h suppressed the expression of endogenous menin in 10T1/2 cells (Fig. 1E).

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FIG. 1. Expression of menin in 10T1/2 cells. A shows the expression of menin in 10T1/2 cells. After seeding at 2.5 x 104 cells/cm2, cells were grown in Eagle's basal medium containing 10% FBS for 7 days until reaching confluence. Cells were then grown in Eagle's basal medium with BMP-2 (100 ng/ml) until the day indicated, and protein extraction was performed as described under "Experimental Procedures." B shows the expression of COLI, OPN, OCN, and Runx2 mRNA in 10T1/2 cells cultured with BMP-2. Cells were grown in medium with 100 ng/ml BMP-2 for the indicated times. Total RNA was extracted, and semiquantitative RT-PCR analysis was performed, as described under "Experimental Procedures." C shows the quantitation of semiquantitative RT-PCR analysis in B. The RT-PCR signals were scanned and quantitated with NIH Image analyzer. Each value is expressed as relative level of target gene to GAPDH. D shows ALP activity in 10T1/2 cells cultured with BMP-2. Cells were grown in medium and 100 ng/ml BMP-2 for the indicated days of culture. ALP activity was measured, as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01 compared with the value at day 7. E shows the effects of AS- and S-oligos on endogenous expression of menin in 10T1/2 cells. When cells reached confluence at day 7 of culture, they were fed fresh medium with AS-oligo (100 ng/ml) or S-oligo (100 ng/ml). After 12 h of incubation, protein extraction was performed, as described under "Experimental Procedures."
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Effects of Menin Inactivation on BMP-2-induced Osteoblastic Differentiation in 10T1/2 CellsWhen treated with BMP-2, 10T1/2 cells undergo commitment into the osteoblastic lineage and osteoblastic differentiation (28). To investigate the effects of menin inactivation by AS-oligo on BMP-2-induced osteoblastic commitment in 10T1/2 cells, we first examined the effects of AS-oligo on the expression of COLI and ALP activity in 10T1/2 cells. As shown in Fig. 2, AS-oligo specifically antagonized the expression of COLI mRNA (Fig. 2A) and COLI protein (Fig. 2B) induced by BMP-2, although S-oligo did not affect these parameters. Moreover, AS-oligo concentration-dependently antagonized ALP activity stimulated by BMP-2 in 10T1/2 cells (Fig 3, AC). However, AS-oligo did not affect retinoic acid-induced ALP activity in 10T1/2 cells (Fig. 3D), suggesting that although menin was inactivated, retinoic acid signaling was intact and that menin specifically related to BMP-2-signaling.

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FIG. 2. Effects of AS-oligo on BMP-2-induced expression of COLI in 10T1/2 cells. A shows the expression of COLI mRNA in 10T1/2 cells. After reaching confluence at culture day 7, the cells were fed fresh medium and were pretreated with 100 ng/ml of AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml of AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and Northern blot analysis was performed, as described under "Experimental Procedures." B shows the protein level of COLI in 10T1/2 cells. After reaching confluence, the cells were fed fresh medium. The cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 48 h later, total protein was extracted, and Western blot analysis was performed as described under "Experimental Procedures."
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FIG. 3. Effects of menin inactivation on ALP activity and staining in 10T1/2 cells. A shows ALP staining in 10T1/2 cells. After reaching confluence, the cells were fed fresh medium. The cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 48 h later, ALP staining was performed, as described under "Experimental Procedures." B shows ALP activity in 10T1/2 cells. 10T1/2 cells were cultured as in A, and ALP activity was measured, as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with the BMP-2-untreated group. C shows ALP activity in 10T1/2 cells as in A. Treatment with AS-oligo was performed at the concentrations indicated, and ALP activity was measured as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with the BMP-2-untreated group. D shows ALP activity in 10T1/2 cells. The confluent cells were pretreated with 100 ng/ml of AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of retinoic acid (R.A.) (10-6 M). 48 h later, ALP activity was measured, as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with the retinoic acid-untreated group.
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Runx2 is an osteochondrogenic transcriptional factor (3, 5), and OCN is an osteoblast-specific marker (29). As shown in Fig. 4, BMP-2 promoted the levels of Runx2 and OCN mRNAs in 10T1/2 cells. The AS-oligo specifically antagonized BMP-2-induced expression of these genes, and the S-oligo did not affect them. These results indicated that menin positively regulated the commitment of mesenchymal stem cells into the osteoblast lineage.

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FIG. 4. Effects of AS-oligo on BMP-2-induced expression of Runx2 and OCN mRNA in 10T1/2 cells. A shows the expression of Runx2 mRNA in 10T1/2 cells. After reaching confluence, the cells were fed fresh medium. The cells were pretreated with 100 ng/ml of AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, as described under "Experimental Procedures." The expression of Runx2 mRNA was examined with semi-quantitative RT-PCR. B shows the quantitation of the RT-PCR analysis in A. The signals of the RT-PCR analysis were scanned and quantitated with NIH Image analyzer. Each value is the expression level relative to that for GAPDH. C shows the expression of OCN mRNA in 10T1/2 cells. Total RNA was extracted as in A, and then Northern blot analysis was performed as described under "Experimental Procedures."
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Effects of Menin AS-oligo on BMP-2-induced Chondrogenic and Adipogenic Differentiation in 10T1/2 CellsIt is also possible that menin might play some role in BMP-2-induced commitment of mesenchymal stem cells into the chondrocyte and adipocyte lineages, because BMP-2 possesses the ability to induce 10T1/2 cells into the chondrocyte and adipocyte lineages as well as the osteoblast lineage (30), and Runx2 has been considered to be required for chondrogenesis (3). We therefore examined the effects of menin inactivation on BMP-2-induced commitment of 10T1/2 cells into the chondrocyte and adipocyte lineages. BMP-2 increased the number of Alcian blue stainingpositive 10T1/2 cells (Fig. 5, A and B) and promoted the expression of COL IX, chondrocyte-specific markers in these cells (Fig. 5C). Inactivation of menin by AS-oligo did not affect the enhancement of these chondrocyte markers by BMP-2 (Figs. 5, AC). These results suggested that inactivation of menin did not affect the differentiation of mesenchymal stem cells to chondrocytes. AS-oligo did not affect the increased number of Oil red staining-positive 10T1/2 cells by BMP-2 (Fig. 6, A and B) as well as the BMP-2-induced PPAR
mRNA expression (Fig. 6, C and D). These findings suggested that inactivation of menin did not affect BMP-2-induced adipogenic differentiation. Taken together, these results indicated that menin promoted BMP-2-induced commitment of mesenchymal stem cells into the osteoblast lineage but not into the chondrocyte and adipocyte lineages.

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FIG. 5. Effects of AS-oligo on BMP-2-induced chondrocyte differentiation in 10T1/2 cells. A shows Alcian blue staining. After cells were grown with and without 100 ng/ml of AS- or S-oligo in the presence or absence of BMP-2 (100 ng/ml) for 14 days after seeding at 2.5 x 104/cm2, cell layers were stained with Alcian blue solution, as described under "Experimental Procedures." B shows the number of Alcian blue-positive cells. Each value is normalized to the control group (cell number ratio). *, p < 0.01, BMP-2-treated compared with the corresponding BMP-2-untreated group. C shows the protein levels of COL IX in 10T1/2 cells. The confluent cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 48 h later, total protein was extracted, and Western blot analysis was performed with anti-COL IX antibody, as described under "Experimental Procedures."
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FIG. 6. Effects of AS-oligo on BMP-2-induced adipocyte differentiation in 10T1/2 cells. A shows Oil red staining. Cells were grown with and without 100 ng/ml AS- or S-oligo in the presence or absence of BMP-2 (100 ng/ml) for 14 days after seeding at 2.5 x 104/cm2, and then cell layers were stained with Oil red, as described under "Experimental Procedures." B shows the number of Oil red-positive cells. Each value is normalized to the control group (cell number ratio). *, p < 0.01, BMP-2-treated compared with corresponding BMP-2-untreated group. C shows the expression of PPAR mRNA in 10T1/2 cells. The confluent cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence or absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and semi-quantitative RT-PCR analysis was performed as described under "Experimental Procedures." Each lane represents the product from 20 µg of RNA. D shows the quantitation of the RT-PCR analysis in C. The RT-PCR signals were scanned and quantitated with NIH Image analyzer. Each value is normalized to the relative level of GAPDH mRNA.
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Effects of Menin AS-oligo on BMP-2-induced Differentiation in ST-2 CellsPrimary cultures of mouse or human mesenchymal progenitor cells have not been established yet. We therefore employed two bone marrow stromal cell lines, ST2 and PA6. BMP-2 induces ALP activity in ST2 and PA6 cells (31). As shown in Fig. 7, A and B, AS-oligo antagonized BMP-2-induced ALP activity and the expression of OCN in ST2 cells. However, it did not affect the BMP-2-increased number of Alcian blue staining- and Oil red staining-positive cells (Fig. 7, C and D). Similar results were obtained in PA6 cells (data not shown). These data indicate that menin inactivation specifically inhibits the commitment of the mesenchymal stem cells to the osteoblast lineage.

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FIG. 7. Effects of menin inactivation on BMP-2-induced differentiation in ST2 cells. A shows ALP activity in ST2 cells. After reaching confluence, the cells were fed with fresh medium and were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed with fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 48 h later, ALP activity was measured, as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with the corresponding oligonucleotide-untreated group. B shows the expression of OCN in ST2 cells. After reaching confluence, the cells were fed fresh medium. The cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and the expression of OCN mRNA was examined by semiquantitative RT-PCR, as described under "Experimental Procedures." C and D show the number of Al-cian blue- and Oil red-positive cells. ST2 cells were grown with and without 100 ng/ml AS- or S-oligo in the presence or absence of BMP-2 (100 ng/ml) for 14 days after seeding at 2.5 x 104/cm2, and then cell layers were stained with Alcian blue or Oil red, as described under "Experimental Procedures." Each value is normalized to the control group (cell number ratio). *, p < 0.01, BMP-2-treated compared with corresponding BMP-2-untreated group.
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Effects of Menin Inactivation on the Response to BMP-2 in 10T1/2 Cells That Have Undergone Differentiation into Osteoblastic CellsWe examined the effects of inactivation of menin by AS-oligo on BMP-2-induced parameters after BMP-2 treatment for 14 days, at which time 10T1/2 cells are considered to have differentiated into osteoblastic cells (Fig. 1, BD). BMP-2 enhanced the expression of COLI and OCN mRNA as well as ALP activity in 10T1/2 cells pretreated with BMP-2 for 14 days (Fig. 8, AD). However, menin AS-oligo did not affect these BMP-2-stimulated osteoblast differentiation markers at this time (Fig. 8, AD). These findings suggested that menin inactivation did not affect the response to BMP-2 after commitment into the osteoblast lineage.

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FIG. 8. Effects of menin inactivation on the response to BMP-2 in 10T1/2 cells that have differentiated into osteoblastic cells. A shows the expression of COLI mRNA in 10T1/2 cells. After cells were grown with BMP-2 (100 ng/ml) for 14 days, they were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with 100 ng/ml AS- or S-oligo in the presence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and Northern blot analysis was performed as described under "Experimental Procedures." Each lane represents 20 µg of RNA. B shows ALP activity in 10T1/2 cells. After cells were grown with BMP-2 (100 ng/ml) for 14 days, cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence or absence of BMP-2 (100 ng/ml). 48 h later, ALP activity was measured as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, BMP-2-treated compared with the BMP-2-untreated group. C shows the expression of OCN mRNA in 10T1/2 cells. After cells were grown with and without BMP-2 (100 ng/ml) for 14 days, they were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and semiquantitative RT-PCR analysis was performed as described under "Experimental Procedures." D shows the quantitation of the RT-PCR analysis in C. The RT-PCR signals were scanned and quantitated with NIH Image analyzer. Each value is normalized to the relative level of GAPDH mRNA.
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Menin Expression and Effects of Menin Inactivation on the Differentiation of MC3T3-E1 CellsTo examine the role of menin in the later differentiation of osteoblastic cells, we employed MC3T3-E1 cells. First, we examined the expression of endogenous menin in MC3T3-E1 cells during their culture by Western blotting. Menin expression was increased until culture day 14 and declined thereafter until culture day 21, a period of mineralization (Fig. 9A). ALP activity increased with time of culture (Fig. 9B). The levels of COLI and OPN expression were high at culture days 7 and 14, respectively, whereas the expression of OCN mRNA, a terminal differentiation marker, was highest at culture day 21 (Fig. 9C). Although AS-oligo suppressed endogenous menin expression (Fig. 10A), AS-oligo did not affect BMP-2-induced ALP activity and the expression of OCN and Runx2 mRNA (Fig. 10, BE). These findings suggested the possibility that the role of menin as a differentiation factor decreases as osteoblastic differentiation progresses. In order to clarify this point, we made AS-stably expressing MC3T3-E1 (AS-MC) cells and compared the osteoblastic phenotype in AS-MC and V-transfected MC3T3-E1 (V-MC) cells as control. Western blot analysis showed that endogenous menin was suppressed in AS-MC cells, compared with V-MC cells (Fig. 11A), confirming the effectiveness of the menin AS construct. In AS-MC cells, the expression of COLI mRNA and production of COLI protein, ALP activity, and the expression of OCN mRNA were enhanced, compared with V-MC cells (Fig. 11, BF). The most important phenotype of osteoblasts, mineralization, was also enhanced in AS-MC cells (Fig. 11, G and H). These results indicated that menin served as an inhibitor of osteoblastic maturation after the commitment into osteoblasts.

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FIG. 9. Endogenous menin expression and effects of menin inactivation in MC3T3-E1 cells. A shows the endogenous expression of menin in MC3T3-E1 cells. MC3T3-E1 cells were grown until the indicated times, total protein was extracted, and Western blot analysis was performed with anti-menin antibody as described under "Experimental Procedures." B shows ALP activity in MC3T3-E1 cells cultured in -MEM containing 10% FBS for the indicated times. ALP activity was measured, as described under "Experimental Procedures." Each bar is expressed as the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with values at day 7. C shows the expression of COLI, OPN, and OCN mRNA in MC3T3-E1 cells grown until the indicated time. Total RNA was extracted, and Northern blot analysis was performed for COLI, OPN, and OCN mRNAs as described under "Experimental Procedures."
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FIG. 10. Effects of AS-oligo on BMP-2-induced differentiation in MC3T3-E1 cells. A shows effects of AS-oligo on the expression of menin in MC3T3-E1 cells. Cells were grown for 7 days after seeding at 2.5 x 104 cells/cm2. Fresh medium was added, and after treatment with 100 ng/ml AS-oligo or S-oligo for 6 h, total protein was extracted and Western blot analysis was performed as described under "Experimental Procedures." B shows ALP staining in MC3T3-E1 cells. Confluent cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h. Then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). After treatment for 48 h, ALP staining was performed as described under "Experimental Procedures." C shows ALP activity in MC3T3-E1 cells. Cells were cultured as in B. ALP activity was measured, as described under "Experimental Procedures." Each bar is expressed as the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with the BMP-2-untreated group. D shows the expression of OCN and Runx2 mRNA in MC3T3-E1 cells. Confluent cells were pretreated with 100 ng/ml AS- or S-oligo for 6 h, and then cells were fed fresh medium with or without 100 ng/ml AS- or S-oligo in the presence and absence of BMP-2 (100 ng/ml). 24 h later, total RNA was extracted, and the expression of Runx2 and OCN mRNA were examined with semiquantitative RT-PCR as described under "Experimental Procedures." E shows the quantitation of the RT-PCR analysis in D. The RT-PCR signals were scanned and quantitated with NIH Image analyzer. Each value is normalized for the relative level of GAPDH mRNA.
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FIG. 11. Effects of AS-DNA stable transfection on MC3T3-E1 cells. A shows effects of stable AS-DNA expression on endogenous menin in MC3T3-E1 cells. After V- and AS-transfected MC3T3-E1 cells were cultured for 7 days, total protein was extracted, and Western blot analysis was performed as described under "Experimental Procedures." B shows the expression of COLI mRNA in V- and AS-DNA-transfected MC3T3-E1 cells (V-MC and AS-MC). After cells were grown for 7 days, total RNA was extracted, and Northern blot analysis was performed, as described under "Experimental Procedures." C shows the expression of COLI protein in V-MC and AS-MC. After cells were grown for 7 days, total protein was extracted, and Western blot analysis was performed with anti-COLI antibody as described under "Experimental Procedures." D shows ALP staining of V-MC and AS-MC. After cells were grown for 7 days, ALP staining was performed as described under "Experimental Procedures." E shows ALP activity of V-MC and AS-MC. After cells were grown for 7 days, ALP activity was measured as described under "Experimental Procedures." Each value is the mean ± S.E. (nmol/min/mg protein) of four determinations. *, p < 0.01, compared with V-MC. F shows the expression of OCN mRNA in V-MC and AS-MC. After cells were grown for 7 days, total RNA was extracted, and Northern blot analysis was performed as described under "Experimental Procedures." G shows mineralization of V-MC and AS-MC. Confluent cells were cultured under the medium with 10 mM -glycerophosphate for 14 days. Then cells were stained with the von Kossa method or Alizarin red as described under "Experimental Procedures." H shows the quantitation of mineralization of V-MC and AS-MC. After cells were cultured as in G, cell layers stained with Alizarin red were destained, and the mineralization was quantitated as described under "Experimental Procedures." *, p < 0.01, compared with V-MC.
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Menin Interacts with Smad1 and Smad5 Both Physically and FunctionallyThe Smad family proteins are critical components of the BMP signaling pathway. Receptor-mediated phosphorylation of Smad1, Smad5, and Smad8 induces their association with the common partner Smad4, followed by translocation into the nucleus, where these complexes activate transcription of specific genes (32). In order to identify the target through which AS blocks the BMP signaling pathway, we examined whether menin would interact with FLAG-tagged Smad1 and Smad5. When menin was transfected with Smad1 or Smad5 into COS-7 cells, the complexes of menin-Smad1 and menin-Smad5 were detected (Fig. 12A). These data indicated that menin physically interacted with Smad1 or Smad5. In order to explore the functional relationship of Smad1/5 and menin, we examined whether inactivation of menin would affect the transcriptional activity of Smad1 and Smad5. In this experiment, we used a heterologous luciferase reporter termed 3GC2-Lux, containing three tandemly repeated GC-rich sequences fused to the collagen X promoter inserted into pGL2-Basic. This construct is a Smad1- and Smad5-responsive vector, as previously described (22). As shown in Fig. 12B, the transfection of Smad1 or Smad5 in the presence of BMP-2 increased the luciferase activity about 10 times by the control group in ST2 cells. AS DNA transfection significantly antagonized the transcriptional activity of Smad1 or Smad5. These findings indicated that menin functionally interacts with Smad1 or Smad5.

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FIG. 12. The physical and functional interaction of menin with Smad1 and Smad5. A shows the association of menin and Smad1/Smad5 in COS-7 cells. Menin was transfected into COS-7 cells with FLAG-tagged Smad1 or Smad5. Cell extracts were immunoprecipitated (IP) with anti-FLAG, followed by immunoblotting (IB) with menin, as described under "Experimental Procedures." B shows luciferase activity of 3GC2-Lux-transfected ST2 cells. 3GC2-Lux was transfected into ST2 cells together with V, AS, and FLAG-tagged Smad1 or Smad5, and then the cells were stimulated with BMP-2 (100 ng/ml). Relative luciferase activity was measured, as described under "Experimental Procedures." Each value is the mean ± S.E. of four determinations. *, p < 0.01, compared with the corresponding AS-untransfected group.
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DISCUSSION
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Menin is expressed in various tissues (16, 17) in addition to those affected in the MEN1 syndrome. The targeted disruption of menin caused a defect of the cranial and facial bones in fetal mice (18). We therefore hypothesized that menin might play an important role in membranous ossification. In this study, we investigated the effects of inactivation of menin by AS-oligo or stable AS expression in multipotential mesenchymal stem cells and osteoblasts. 10T1/2 cells, derived from mouse embryonic fibroblasts and a mesodermal progenitor cell line, can differentiate to several lineages (30). Treatment of 10T1/2 cells with azacytidine, which causes hypomethylation of the DNA, results in the differentiation of a portion of the cells to myoblasts, adipocytes, or chondrocytes (33). BMP-2 and BMP-7 induce 10T1/2 cells to differentiate into osteoblasts, chondrocytes, and adipocytes (28, 30, 34, 35). Thus, 10T1/2 cells are considered to be an appropriate model for multipotential mesenchymal stem cells. On the other hand, the MC3T3-E1 nontransformed cell line is derived from mouse embryonic calvaria (21) and is widely used to investigate the biology of osteoblast. We therefore used MC3T3-E1 cells as a model for the early stage osteoblast or preosteoblast after the commitment of multipotential mesenchymal stem cells into the osteoblastic lineage. We employed two approaches to inactivate menin in the cell lines, namely an antisense oligonucleotide strategy and an antisense RNA strategy. In the present study, AS-oligo suppressed the endogenous expression of menin in both 10T1/2 and MC3T3-E1 cells, and stable AS expression inhibited the endogenous expression of menin in MC3T3-E1 cells. Moreover, AS-oligo antagonized TGF-
-induced transcriptional activity in 10T1/2 cells and MC3T3-E1 cells (data not shown). These data indicated that AS-oligo and AS expression effectively inactivated endogenous menin in these cells.
BMP-2 was originally identified as a peptide that induces ectopic bone and cartilage formation in extraskeletal tissues in vivo (36, 37, 38). Extensive studies have demonstrated that BMP-2 plays important roles in bone formation and bone cell differentiation (39, 40). Several studies have shown that BMP-2 induces 10T1/2 cells to differentiate into osteoblasts (28, 41). In the present study, menin AS-oligo antagonized BMP-2-induced osteoblastic differentiation of 10T1/2 cells, whereas the AS-oligo did not affect the ALP activity and Runx2 expression stimulated by BMP-2 in 10T1/2 cells that had already been committed to the osteoblast lineage with sustained BMP-2 treatment. These results indicated that menin inactivation inhibited the differentiation of 10T1/2 cells into osteoblasts and that menin is a crucial factor for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage. We show in the present study that when menin was inactivated by AS-oligo, BMP-2 did not stimulate the expression of Runx2 as it normally does in 10T1/2 cells. Mice with a heterozygous mutation in the Runx2 locus showed a phenotype similar to that of cleidocranial dysplasia in humans, in whom mutations of Runx2 have been identified (3, 5, 42). A homozygous mutation of this gene in mice induced a complete lack of bone formation with arrest of osteoblastic differentiation (3, 42). Since the complete lack of osteoblasts and neonatal lethality made it impossible to examine the postnatal function of Runx2 in the knockout model, use of an alternative mouse model, in which a dominant negative form of Runx2 was expressed in osteoblasts only postnatally, demonstrated the importance of the gene for postnatal bone formation (43). A large number of in vitro studies also demonstrated that Runx2 is a positive regulator of bone matrix genes, including COLI, OPN, bone sialoprotein, OCN, and fibronectin (44, 45, 46, 47, 48, 49, 50, 51). These findings indicate that Runx2 functions in the commitment of multipotential mesenchymal stem cells into the osteoblast lineage. Liu et al. (52) reported that in Runx2 transgenic mice osteoblast number was increased at an early differentiation stage but that osteoblastic function, such as matrix production and mineralization, was impaired in these cells. That study suggested that Runx2 inhibited maturation at the late differentiation stage, restricting its positive effect on differentiation to an early stage in osteoblast development (52). Our present findings suggested that menin might play a similar important role in the commitment of multipotential mesenchymal stem cells into the osteoblast lineage like Runx2. Moreover, menin might be involved in BMP-2 signaling upstream of Runx2. Some of the functions of Runx2 might be related to the interaction of menin and the BMP pathway, since Smad3 as well as Smad1/5 directly bind to Runx2 (48). Further studies are ongoing into the relationship of menin and BMP/Runx2 pathways in our laboratories. In the present study, the endogenous expression of menin in 10T1/2 cells was increased until culture day 14 and declined thereafter. These findings support the notion that menin is important for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage and the early differentiation of osteoblasts. In addition, it is also possible that menin regulates transcriptional factors other than Runx2 that are important for the early differentiation of osteoblasts.
BMP-2 can induce multipotential mesenchymal stem cells to form chondrocytes (33). In the present study, in 10T1/2 cells inactivation of menin antagonized the BMP-2-induced expression of Runx2, which also has been shown to be important for the commitment of multipotential mesenchymal stem cells into the chondrocyte lineage (33). In Runx2 null mice, osteoblast differentiation is arrested in both the endochondral and intramembranous skeleton (3, 5). However, in the present study, menin AS-oligo did not affect chondrogenic markers such as Alcian blue staining and the expression of COL IX induced by BMP-2 in 10T1/2 cells, indicating that lack of menin did not alter BMP-2-induced chondrogenic differentiation in 10T1/2 cells. Recently, transcriptional factors that critically regulate chondrocyte differentiation were identified. These include three members of the Sox family, Sox9, L-Sox5, and Sox6 (53, 54). These alternative signaling pathways might have rescued chondrocyte differentiation when the Runx2 signal pathway was blocked by inactivation of menin.
PPAR
is a member of the steroid hormone receptor family expressed in adipose tissue and is activated by fatty acids. By itself, PPAR
is sufficient to activate the adipocyte-specific gene enhancer in nonadipocyte cell lines (55, 56). BMP-2 stimulates the expression of PPAR
in 10T1/2 cells (20) and induces 10T1/2 cells into the adipocyte lineage. The present study revealed that menin inactivation by AS-oligo did not affect adipogenic markers such as Oil red staining and PPAR
expression induced by BMP-2 in 10T1/2 cells. These findings indicated that menin was not involved in the BMP-2-induced commitment of multipotential mesenchymal stem cells into the adipocyte lineage.
Inactivation of menin with AS-oligo did not affect ALP activity and Runx2 expression stimulated by BMP-2 in 10T1/2 cells, which already had been committed to osteoblasts. Moreover, AS-oligo did not affect BMP-2-induced ALP activity and Runx2 expression in MC3T3-E1 cells. These results indicated that menin inactivation did not affect osteoblastic differentiation stimulated by BMP-2 in mature osteoblasts. On the other hand, inactivation of menin in MC3T3-E1 cells by stable expression of AS increased ALP activity, mineralization, and the expression of COLI and OCN, indicating that menin inhibits the differentiation of osteoblasts. These findings suggested that menin might be more important in the commitment into the osteoblast lineage and early differentiation of osteoblasts than in later stages during osteoblastic differentiation.
In the present study, menin inactivation antagonized BMP-2-induced osteoblast differentiation in 10T1/2, ST2, and PA6 cells. Since menin physically and functionally interacts with Smad3, but not with Smad2 and Smad4 (15), the possibility was raised that menin interacts with BMP-receptor-regulated Smads. We have now shown that menin physically and functionally interacts with Smad1 and Smad5. Ju et al. (41) reported that Smad1 transfection promoted the commitment of 10T1/2 cells to the osteoblast lineage. Moreover, activation of Smad5 and subsequent Smad5-DPC4 (Smad4) complex formation are key steps in the BMP signaling pathway, which mediates BMP-2-induced osteoblastic differentiation of C2C12 mesenchymal cells (57). Taking this into account, the present study suggests that the interaction of menin and Smad1/5 is important for the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage.
Menin interacted with Smad3, and menin inactivation antagonized TGF-
-mediated cell growth inhibition and transcriptional activity in our previous study (15). However, TGF-
did not induce ALP activity in 10T1/2 cells (28), and we have confirmed this (data not shown). Therefore, it is unlikely that the interaction of menin and Smad3 plays a key role in the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage. Our data showed that menin inactivation by AS-oligo affected neither BMP-2-stimulated ALP activity nor the expression of Runx2 and OCN in MC3T3-E1 cells. In our preliminary study, menin inactivation did not affect Smad1/5-mediated transcriptional activity in MC3T3-E1 cells (data not shown). On the other hand, TGF-
inhibits the expression of Runx2 and OCN presumably through Smad3 in MC3T3-E1 cells (58). Taking into account our previous findings that menin augments Smad3-mediated transcriptional activity (15), the interaction of menin and Smad3 might antagonize BMP-induced osteoblast differentiation in already committed osteoblasts, such as MC3T3-E1 cells.
We hypothesized a model by which menin may act to regulate differentiation at the molecular level, as shown in Fig. 13. In the mesenchymal stem cells, which are not yet committed into the osteoblast lineage, menin interacts with Smad1 and Smad5, which are crucial mediators of BMP-2 signaling, and the disruption of this interaction by menin inactivation antagonizes the transcriptional activity of Smad1 and Smad5, resulting in an inhibition of the differentiation into the osteoblast lineage. On the other hand, in the cells committed to osteoblast lineage, the interactions of menin and BMP/Smad pathway do not affect the ostoblast differentiation, and menin suppresses osteoblast maturation in well differentiated osteoblasts.

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FIG. 13. The role of menin in osteoblast differentiation. In mesenchymal stem cells that have not yet committed to the osteoblast lineage, menin interacts with Smad1 and Smad5, which are crucial mediators of BMP-2 signaling, and the disruption of this interaction by menin inactivation antagonizes the transcriptional activity of Smad1 and Smad5, resulting in an inhibition of the differentiation into the osteoblast lineage. On the other hand, in the cells committed to osteoblast lineage, the interactions of menin and BMP/Smad pathway do not affect the ostoblast differentiation, and menin suppresses further osteoblast maturation in well differentiated osteoblasts.
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In summary, the data indicated that menin inactivation specifically inhibited the commitment of multipotential mesenchymal stem cells to the osteoblast lineage. Menin seems to be a crucial factor for osteoblastic commitment and inhibits the further differentiation of osteoblasts after they are committed to the osteoblast lineage.
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FOOTNOTES
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* This work was supported in part by grants from the Uehara Memorial Foundation (to H. K.) and Canadian Institutes of Health Research Grant MOP-9315 (to G. N. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Recipient of a National Cancer Institute of Canada research studentship. 
** To whom correspondence should be addressed: Division of Endocrinology/Metabolism, Neurology, and Hematology/Oncology, Dept. of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5885; Fax: 81-78-382-5899; E-mail: sugimot{at}med.kobe-u.ac.jp.
1 The abbreviations used are: COLI, type I collagen; ALP, alkaline phosphatase; OPN, osteopontin; OCN, osteocalcin; TGF-
, transforming growth factor type
; BMP, bone morphogenic protein; AS, antisense; AS-oligo, antisense oligonucleotide; S-oligo, sense oligonucleotide; FBS, fetal bovine serum; MEM, minimum essential medium; RT, reverse transcriptase; PPAR
, peroxisome proliferator-activated receptor
; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; COLIX, type IX collagen; RDU, relative densitometry unit. 
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ACKNOWLEDGMENTS
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We greatly thank J. Massague for 3TP-Lux and Y. Higashimaki and C. Ogata for excellent technical support.
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