Necdin interacts with the Msx2 homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells.

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation.

Necdin is a 325-amino acid residue protein encoded in a cDNA sequence isolated from the library of neurally differentiated P19 embryonal carcinoma cells (1). The necdin gene is expressed predominantly in postmitotic neurons (2,3). Necdin is also expressed in non-neuronal cells such as skeletal myocytes, chondrocytes, adipocytes, and skin fibroblasts (4 -7). Ectopic expression of necdin strongly suppresses the growth of proliferative cells (8 -10) and promotes differentiation of neuroblastoma cells (11). In primary cultures of dorsal root ganglia from mouse embryos, down-regulation of endogenous necdin expression results in a severe impairment of neuronal maturation and an increase in the number of apoptotic cells (12). Necdin knockout mice show a phenotype resembling Prader-Willi syndrome, a genomic imprinting-associated neurobehav-ioral disorder, suggesting that the absence of necdin impairs neuronal differentiation or maturation (5,13,14). These findings suggest that necdin facilitates terminal differentiation and prevents apoptosis in neurons and that necdin has a similar function in terminally differentiated non-neuronal cells.
Necdin shows a significant homology to MAGE family proteins, the remarkable feature of which is a large central region termed MAGE homology domain (MHD). 1 MHD is consistent with the functional region through which necdin interacts with various proteins such as SV40 large T antigen, adenovirus E1A, E2F1, E2F4, p53, NEFA, heterogeneous nuclear ribonucleoprotein U, and p75 neurotrophin receptor (4, 9 -11, 15-17). Furthermore, the MHDs of necdin/MAGE family proteins are thought to be responsible for their functions. For example, necdin, MAGE-D1 (18), MAGE-G1 (17), and magphinin (MAGE-D4) (19), whose MHDs have close similarities to necdin, suppress cell proliferation. The fact that most of the necdin interactors are related to cell cycle regulation, differentiation, and apoptosis suggests that necdin exerts its diverse biological activities through its MHD.
Recently, the necdin homologous protein MAGE-D1(also designated NRAGE or Dlxin-1) has been characterized as a regulator of apoptosis and transcriptional modulators. NRAGE, a rat homolog of human MAGE-D1, binds to p75 neurotrophin receptor via the MHD and confers nerve growth factor-dependent apoptosis through a c-Jun N-terminal kinase-dependent mitochondrial pathway (18,20). NRAGE also interacts with inhibitors of apoptosis proteins (21) and the axon guidance receptor UNC5H which mediates apoptosis (22). Dlxin-1, a mouse homolog of MAGE-D1, interacts with Dlx/Msx homeodomain proteins (23) and Ror receptor kinases (24). Because necdin forms a homodimer (16), it seems likely that necdin and MAGE-D1 form a complex via their MHDs, and that these two proteins cooperate to modulate the activities of MAGE-D1 interactors.
In this study, we attempted to examine the association between necdin and MAGE-D1 in vivo and in vitro. We demonstrate that necdin interacts with Msx homeodomain proteins via MAGE-D1, releases Msx-dependent transcriptional repression, and promotes muscle differentiation by canceling Msxinduced repression. These indicate that two types of MAGE proteins cooperate to regulate the function of Msx homeodomain proteins in cellular differentiation.

EXPERIMENTAL PROCEDURES
Immunoblotting-Tissues of ICR mouse embryos were homogenized with a lysis buffer containing 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 1 mM EGTA, 0.5% Triton X-100, and 1ϫ protease inhibitor mixture (Complete, Roche Applied Science) and ul-* This work was supported in part by grants-in-aid from the Japan Society for the Promotion of Science (Scientific Research B2) (to K. Y.) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (the National Project on Protein Structure and Functional Analysis). 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  tracentrifuged at 100,000 ϫ g for 1 h at 4°C to obtain the supernatant. C2C12, COS-7, and N1E-115 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, harvested, and suspended in the lysis buffer. P19 embryonal carcinoma cells were cultured and induced to differentiate as described (25). The equal amounts of proteins (20 g) were separated by 10% SDS-PAGE, blotted onto Immobilon membrane (Millipore), and incubated with anti-MAGE-D1 antibody (1:1,000) or anti-necdin antibody (NC243) (26). Anti-mouse MAGE-D1 polyclonal antibody was raised in New Zealand rabbit against purified maltose-binding protein (MBP)-MAGE-D1 (amino acids 1-775) fusion protein. After incubation with peroxidaseconjugated anti-rabbit IgG (Cappel), necdin and MAGE-D1 were detected by the chemiluminescence method (Renaissance, PerkinElmer Life Sciences). The protein concentration was determined by the Bradford method (Bio-Rad).
Fluorescence Immunohistochemistry-Tissues in ICR mice at E12.5 and E14.5 were fixed by transcardial perfusion of 4% paraformaldehyde solution in phosphate buffer (pH 7.4). The whole embryos were immersed in the same fixative followed by cryoprotection with 20% sucrose and cut at a thickness of 20 m by a cryostat. The sections mounted on glass slides coated with gelatin were incubated with antinecdin antibody (NC243) (1:500) and anti-MAGE-D1 antibody (1:300) in PBS containing 0.05% Tween 20 and 5% normal goat serum at room temperature for 3 h. The tissues were then incubated at room temperature for 2 h with anti-rabbit IgG conjugated with fluorescein isothiocyanate (1:500) (Cappel). Fluorescent images were observed with a fluorescence microscope (BX60 -34-FLAD1, Olympus), taken by CCD camera system (M-3204C, Olympus), and processed using Adobe Photoshop 5.0 software.
Immunocytochemistry-C2C12 myoblast cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as a growth medium. p3xFLAG-Msx2 and p3xFLAG (4 g each) were transfected into C2C12 cells by LipofectAMINE Plus reagent (Invitrogen). G418-resistant colonies were obtained after a 14-day incubation, and C2C12 cells expressing FLAG-Msx2 and FLAG were subcultured in the growth medium for 7-14 days. Myogenic differentiation was initiated by incubating confluent cloned cells for 3 days in Dulbecco's modified Eagle's medium containing 2% horse serum as a differentiation medium (28). Cells were fixed with 4% formaldehyde in PBS (pH 7.4) at 4°C for 20 min and permeabilized with methanol/ acetone (1:1) at Ϫ20°C for 20 min. Fixed cells were incubated with anti-FLAG M2 antibody (1:300) and an antibody against MHC (MF-20) (1:2) (a gift from Dr. T. Komiya, Osaka City University) in PBS containing 0.05% Tween 20 and 5% normal goat serum at 4°C overnight. The cells were then incubated at room temperature for 120 min with anti-mouse IgG conjugated with fluorescein isothiocyanate (1:500), treated with 3.3 M Hoechst 33342 for chromosomal DNA detections, and observed with the fluorescence microscope.

Interactions between Necdin and MAGE-D1 in Vitro and in
Vivo-We first analyzed the expression patterns of necdin and MAGE-D1 in mouse embryo by Western blotting and immunohistochemistry (Fig. 1). Endogenous ϳ43-kDa necdin protein was detected almost exclusively in the brain and skeletal muscle at embryonic day (E) 18.5 (Fig. 1A). In contrast, endogenous ϳ85-kDa MAGE-D1 protein was expressed in a ubiquitous manner, although the levels in the brain, skeletal muscle, heart, lung, and kidney were higher than those in the small intestine and liver. In various cell lines, necdin was detected only in neurally differentiated P19 embryonal carcinoma cells, whereas MAGE-D1 was expressed in C2C12 myoblast cells, COS-7 kidney cells, N1E-115 neuroblastoma cells, and P19 cells (Fig. 1B). Immunohistochemistry revealed that necdin and MAGE-D1 were concentrated in the preplate (i.e. a structure composed of early differentiated postmitotic cells) of the forebrain at E12.5 and skeletal muscle tissues in the hind limb at E14.5 (Fig. 1C). In developing neural tube, MAGE-D1 immunoreactivity was distributed in the ventricular zone containing a mixed population of neuroepithelial stem cells and neuronal progenitors as well as in postmitotic cells at the marginal zone. Because expression of necdin and MAGE-D1 overlapped in these regions, we speculated that necdin and MAGE-D1 potentially form a heterodimer in vivo.
Interactions between Necdin and MAGE-D1-To investigate whether necdin and MAGE-D1 form a heterodimer in vitro, we constructed various MAGE-D1 deletion mutants fused to MBP and examined their interactions with His-tagged necdin (Fig. 2, A-C). Full-length MAGE-D1 (amino acids 1-775) and deletion mutants encompassing the MHD (amino acids 466 -775 and 553-688) interacted with His-tagged necdin, indicating that necdin and MAGE-D1 form a heterodimer via the MHD. We then examined the interaction between necdin and MAGE-D1 in vivo by co-immunoprecipitation analysis (Fig. 2, D and E). Full-length MAGE-D1 and MAGE-D1⌬N was co-immunoprecipitated with necdin, but MAGE-D1⌬C failed to bind necdin (Fig. 2E, upper panel). Necdin was also co-immunoprecipitated with MAGE-D1 (Fig. 2E, lower panel). These results together suggest that necdin complexes with MAGE-D1 in vitro and in vivo.
Formation of a Complex Containing Necdin, MAGE-D1, and Msx-We next examined the formation of a complex containing necdin, MAGE-D1, and Msx in vivo by co-immunoprecipitation assay (Fig. 4). Msx1 was co-immunoprecipitated with necdin in the presence and absence of MAGE-D1 (Fig. 4A). The complex formation between necdin and Msx1 in the absence of MAGE-D1 is likely because of the presence of endogenous MAGE-D1 in COS-7 cells. Similarly, Msx2 was co-immunopre-cipitated with necdin (Fig. 4B). Neither Myc-Msx1 nor Myc-Msx2 was co-immunoprecipitated with a necdin deletion mutant (necdin⌬243-306), which was defective in MAGE-D1 binding activity, when co-expressed with MAGE-D1 in this assay system (data not shown). These results suggest that necdin interacts with Msx1 and Msx2 via MAGE-D1.
We then attempted to detect an endogenous complex containing necdin, MAGE-D1, and Msx2 in tissue lysates by immunoaffinity purification. However, we were unable to find normal tissues or primary cells that contain high levels of Msx and necdin for biochemical analyses. Furthermore, we failed to detect the Msx protein in skeletal muscle tissues of the hind limb at E14.5 and E18.5 by Western blotting (data not shown). We alternatively used P19 embryonal carcinoma cells, whose endogenous Msx2 expression is up-regulated upon cell aggregation (29). Expression of necdin and Msx2 was up-regulated in retinoic acid-treated P19 cells, but MAGE-D1 expression levels remained almost unchanged during the course of neural differentiation (Fig. 4C). For detection of the endogenous complex, extracts of retinoic acid-treated P19 cells were subjected to the immunoaffinity purification using antibodies against necdin and MAGE-D1 (Fig. 4, D and E). Both MAGE-D1 and Msx2 bound to immunopurified necdin, and both necdin and Msx2 bound to immunopurified MAGE-D1. About 2% of the amounts of necdin and MAGE-D1 in the lysate were recovered after immunoaffinity purification. Under these conditions, 0.9 and 0.3% of the amounts of Msx2 and MAGE-D1, respectively, in the lysate bound to necdin, whereas 0.7 and 0.5% of the amounts of Msx2 and necdin, respectively, in the lysate bound to MAGE-D1. These results suggest that the endogenous complex containing necdin, MAGE-D1, and Msx2 is present in neurally differentiating P19 cells in vivo.
Release from Msx-dependent Transcriptional Repression by Necdin and MAGE-D1-Because Msx1 and Msx2 are potent repressors of the Wnt1 promoter (30), we examined whether a complex of necdin and MAGE-D1 modulates the Msx-dependent transcriptional activity (Fig. 5). We carried out the Msxdependent transcriptional assay in N1E-115 neuroblastoma cells, which express endogenous MAGE-D1 but not necdin as shown in Fig. 1B. Transfection of expression vectors for Gal4-Msx1 and Gal4-Msx2 resulted in the repression (44 and 32% of control values, respectively) of GAL4 site-containing reporter, whereas co-transfection with necdin and MAGE-D1 released the repression to 95 and 97% of the control levels, respectively (Fig. 5A). Necdin alone released the repression by Gal4-Msx1 and Gal4-Msx2 to 70 and 80%, respectively, presumably due to endogenous MAGE-D1 present in N1E-115 cells. We next examined the effects of these proteins on the transcription of the Wnt1 promoter (WIP) containing the Msx-binding site (30) (Fig.  5B). Msx1 and Msx2 also repressed the transcription of the WIP promoter (41 and 30% of the control level, respectively), whereas the activity was recovered to 80 and 67% of the control level, respectively, by co-expression of necdin and MAGE-D1. Necdin alone released the repression by Msx1 and Msx2 to 65 and 50%, respectively. These observations indicate that necdin releases Msx-dependent transcriptional repression via MAGE-D1.
Release from Msx2-induced Myogenic Repression by Necdin and MAGE-D1-Because Msx2 inhibits myogenic differentiation of C2C12 myoblasts (28,31), we used this cell line as a model system to examine the effects of necdin and MAGE-D1 on Msx-dependent myogenic repression. We established C2C12 myoblast cells that stably express FLAG-Msx2. In wild-type C2C12 cells, MHC (i.e. a marker for skeletal muscle differentiation) and MAGE-D1 were distributed predominantly in the cytosol of multinucleated differentiated C2C12 cells (Fig. 6A). Western blot analysis revealed that differentiated C2C12 cells expressed MHC and MAGE-D1, whereas necdin was undetected in C2C12 cells even under differentiated conditions (Fig.  6B). We then analyzed myogenic differentiation of FLAG-Msx2-expressing C2C12 clones. Under differentiation conditions, only a few MHC-positive mononucleated cells appeared in Msx2-expressing C2C12 cultures, indicating an impairment in myotube formation (Fig. 6C). C2C12 cells expressing FLAG-Msx2 differentiated into myocytes at much lower efficiency than control C2C12 cells as judged by MHC expression levels (Fig. 6D).
We then examined the effects of necdin and MAGE-D1 on myogenic differentiation of Msx2-expressing C2C12 cells (Fig.  7). Expression of necdin by adenovirus vector-mediated gene transfer up-regulated MHC expression and promoted myotube formation, whereas infection of adenoviruses expressing LacZ and MAGE-D1 had little effect on MHC expression (Fig. 7A). Necdin and MAGE-D1 increased MHC expression ϳ9 times the control level (Fig. 7, B and C). Necdin alone up-regulated the MHC expression 3 times the control level, presumably because of the presence of endogenous MAGE-D1. Similarly, necdin and MAGE-D1 up-regulated the expression of the myogenic transcription factor MyoD ϳ5 times the control level, and necdin alone increased the MyoD level ϳ3 times the control level (Fig.  7B). The complex of necdin, MAGE-D1, and Msx2 in the nuclear fraction was detected by co-immunoprecipitation assay (Fig. 7D). Co-expression of necdin and Myc-tagged MAGE-D1 formed the complex efficiently. This may be because exogenous MAGE-D1, when co-expressed with necdin, tends to accumulate preferentially in the nucleus. Expression of MAGE-D1, , and FLAG-tagged necdin (1.5 g) (Necdin) were transfected along with pG5luc (0.5 g) into N1E-115 neuroblastoma cells. The luciferase activity driven by the promoter carrying 5xGal4-binding sites (5xGal4) was measured with a luminometer (mean Ϯ S.E., n ϭ 3). The transfection efficiency was normalized with the activity of co-transfected pRc-LacZ (0.5 g). The total amount of plasmids was adjusted to 5 g/assay by adding the empty vector. B, Wnt1 promoter-reporter system. Combinations of pGL2-WIP (0.5 g), Gal4-Msx (1 g), Myc-tagged MAGE-D1 (1.5 g), and FLAG-tagged necdin (1.5 g) were transfected into N1E-115 cells. The luciferase activity driven by the Wnt1 promoter (WIP) was measured and presented as in A.
necdin, or both in control C2C12 cells, which express no endogenous Msx2, failed to promote the myogenesis (data not shown). These data indicate that necdin and MAGE-D1 released Msx2-induced myogenic repression through formation of the complex with Msx2. DISCUSSION The present study has shown that MAGE family members cooperate to modulate the function of Msx homeodomain proteins, which are key transcription factors for cellular differentiation (32). The MAGE family proteins are grouped into two types based on their sequence similarities in the MHDs (33). Type I MAGE members (MAGE-A-C subfamilies) are expressed in undifferentiated cells such as transformed cells and testicular germ cells, whereas type II MAGE members are expressed in differentiated cells such as neurons. Necdin and MAGE-D1, whose functions have been best documented among MAGE proteins, are classified as type II MAGE proteins. Type II MAGE proteins may be divided further into two subgroups based on the similarities in the MHD sequences and molecular sizes; A subgroup ("necdin subgroup") of type II MAGE proteins consists of relatively short proteins (Ͻ350 amino acid residues) whose MHD sequences are more homologous to that of necdin. This subgroup includes necdin, MAGE-F1, G1, and H1 in mammals, Drosophila MAGE (34), and zebrafish MAGE (35). The other subgroup ("MAGE-D subgroup") consists of larger proteins (Ͼ650 amino acid residues) bearing N-terminal extensions. This subgroup includes MAGE-D1-D4, MAGE-E1 (DAM-AGE) (36), and MAGEL2 (NDNL1). Earlier studies have shown that MAGE-D1 interacts, via its MHD, with the death domain receptors p75NTR (18) and UNC5H1 (22), the receptor tyrosine kinase Ror2 (24), and the RING finger protein Praja1 (37) two homologs of Drosophila muscle segment homeobox (msh) genes that are expressed in mouse embryos at critical stages of neural tube, neural crest, and craniofacial development (32). Therefore, Msx1 and Msx2 are thought to play important roles in organogenesis and cellular differentiation. For example, forced expression of Msx1 in myoblasts blocks terminal differentiation, and Msx1-expressing cells acquire a transformed phenotype (38). Msx1 inhibits transcription of the myogenic transcription factor MyoD, which is a target for homeobox gene regulation (39). Msx proteins serve as transcriptional repres-sors and negative regulators of differentiation by preventing cell cycle exit and blocking terminal differentiation of mesenchymal progenitor cells (28). Furthermore, Msx2 prevents differentiation and stimulates cell proliferation at the extreme ends of osteogenesis (40). These findings suggest that Msx homeodomain proteins generally repress terminal differentiation of mesenchymal cells. The present findings showed that necdin releases Msx2-induced repression of myogenic differentiation in C2C12 myoblasts (Fig. 7). This supports the notion that necdin promotes terminal differentiation of postmitotic cells by repressing cell proliferation (11,12).
The physiological relevance of endogenous regulation of Msx function by necdin/MAGE proteins in cellular differentiation remains to be elucidated. It has most recently been reported (41) that Msx2 and necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells, a class of vessel-derived stem cells that can differentiate into different mesodermal cell types. In a mesoangioblast clone that spontaneously expresses a smooth muscle phenotype, Msx2 and necdin are expressed at least 100 times the stem cell level. Co-expression of Msx2 and necdin, but not either alone, induces a number of smooth muscle markers, and their down-regulation through RNA interference results in a decreased expression of smooth muscle markers. These findings suggest that Msx2 and necdin, presumably in the presence of endogenous MAGE-D1, cooperate to promote the smooth muscle differentiation of mesoangioblast stem cells. Furthermore, these raise the possibility that Msx-dependent differentiation of specific cell types is modulated physiologically by necdin and necdinrelated MAGE proteins via MAGE-D1.
In the nervous system, necdin is expressed predominantly in postmitotic neurons, whereas MAGE-D1 is in proliferative neural progenitors and young postmitotic neurons (18,42). In the present study, we confirmed that MAGE-D1 is expressed in postmitotic cells such as neurons and skeletal myocytes as well as in neuronal progenitors (Fig. 1C). Furthermore, we detected overlapping expression of necdin and MAGE-D1 in the preplate, which consists of early differentiated postmitotic cells, in developing mouse brain. These observations suggest that these MAGE proteins form a heteromeric complex in vivo during terminal differentiation and maturation of neurons. In differentiating P19 embryonal carcinoma cells, Msx2 mediates death signals from bone morphogenetic proteins (BMPs) (29). Msx2 also mediates the proapoptotic signals from BMP-4 in ventricular zone neural progenitor cells (43). We have identified the complex containing necdin, MAGE-D1, and Msx in neurally differentiating P19 cells in vivo (Fig. 4, C-E). Therefore, necdin likely suppresses the Msx-mediated proapoptotic signals by forming a complex with Msx and MAGE-D1 during neuronal differentiation. This is consistent with the idea that necdin has an anti-apoptotic function in neuronal differentiation (11,12).
Necdin and MAGE-D1 may also form a complex with Dlx family homeodomain proteins, which have been reported to interact with the murine MAGE-D1 ortholog Dlxin-1 (23). Both necdin and MAGE-D1 were highly expressed in the preplate (Fig. 1C) and the ganglionic eminence in developing mouse brain (3). Cells in these regions express Dlx family homeodomain proteins such as Dlx1, Dlx2, Dlx5, and Dlx6 (44). Forced expression of Dlx2 and Dlx5 induces the phenotype of GABAergic neurons (44). These observations suggest that the complex of necdin and MAGE-D1 modulates the function of Dlx homeodomain proteins in terminal differentiation and specification of GABAergic neurons. Further information about the functional interactions of MAGE proteins with Msx/Dlx homeodomain proteins will lead to a better understanding of molecular mechanisms underlying organogenesis and cell type specification in mammals.