JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404143200 on July 21, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40484-40493, September 24, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/39/40484    most recent
M404143200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuwajima, T.
Right arrow Articles by Yoshikawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuwajima, T.
Right arrow Articles by Yoshikawa, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Necdin Interacts with the Msx2 Homeodomain Protein via MAGE-D1 to Promote Myogenic Differentiation of C2C12 Cells*

Takaaki Kuwajima, Hideo Taniura, Isao Nishimura, and Kazuaki Yoshikawa{ddagger}

From the Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan

Received for publication, April 14, 2004 , and in revised form, June 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 neurobehavioral 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 Msx-induced repression. These indicate that two types of MAGE proteins cooperate to regulate the function of Msx homeodomain proteins in cellular differentiation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 MgCl2, 1 mM EGTA, 0.5% Triton X-100, and 1x protease inhibitor mixture (Complete, Roche Applied Science) and ultracentrifuged at 100,000 x 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 µg) 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 peroxidase-conjugated 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 µm by a cryostat. The sections mounted on glass slides coated with gelatin were incubated with anti-necdin 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.

In Vitro Binding—cDNA encoding MAGE-D1 (amino acids 1–775) and MAGE-D1 deletion mutants were generated by PCR using synthetic oligonucleotide primers and inserted directionally into pMALC2 (New England Biolabs). MBP-MAGE-D1 fusion proteins were affinity-purified with amylose resin (New England Biolabs). Purified fusion proteins (5 µg) bound to amylose resin (40 µl) were incubated with purified His-tagged necdin (amino acids 1–325) (50 ng) at 4 °C for 30 min in 0.5 ml of the binding buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 1 mM EDTA (10), and the bound proteins were eluted with 20 mM maltose. His-tagged necdin was separated by 10% SDS-PAGE, blotted, and detected with an anti-necdin antibody (C2) (1:1,000) (1) and peroxidase-conjugated goat anti-rabbit IgG (Cappel) by the chemiluminescence method. For detection of the complex of MAGE-D1 and Msx, cDNAs encoding mouse Msx1 (amino acids 1–297) (a gift from Dr. C. Abate-Shen, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School) and Msx2 (amino acids 1–267), which was isolated from the library of neurally differentiated P19 embryonal carcinoma cells, were cloned into pGEX-5X-1 (Amersham Biosciences) to produce glutathione S-transferase (GST) fusion proteins. MBP-MAGE-D1 fusion proteins (5 µg) bound to amylose resin (40 µl) were incubated with GST-Msx1 or GST-Msx2 fusion proteins (1 µg) at 4 °C for 30 min in 0.5 ml of the binding buffer. Bound proteins were eluted and detected by Western blotting using an anti-GST antibody (1:2,000) (Sigma) as above.

Co-immunoprecipitation—cDNAs encoding Myc-tagged MAGE-D1 and its deletion mutants were subcloned into pcDNA3.1 (Invitrogen) as described previously (10). Combinations of the expression vectors p3xFLAG-necdin (amino acids 1–325), Myc-tagged MAGE-D1, and Myc-tagged MAGE-D1 deletion mutants were transfected into COS-7 cells and harvested 48 h after transfection as described previously (11). Cell extracts (400 µg) were incubated at 4 °C for 2 h with an anti-Myc antibody (9E10) (1:5) or antibody NC243 (1:100) in 200 µl of a lysis buffer containing 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, and 1x protease inhibitor mixture. The complexes were pelleted with protein A-Sepharose (Amersham Biosciences), separated by 10% SDS-PAGE, transferred to Immobilon membrane, and blotted with antibody 9E10 (1:10) or antibody NC243 (1:3,000) and peroxidase-conjugated goat anti-rabbit IgG (Cappel) or peroxidase-conjugated goat anti-mouse IgG (Cappel). For detection of the complexes containing Msx1 (or Msx2), cDNAs encoding Msx1 and Msx2 were inserted into p3xFLAG expression vector (Sigma). Combinations of the expression vectors encoding FLAG-tagged Msx1 and Msx2 were transfected into COS-7 cells, and Msx-containing complexes were immunoprecipitated and detected with anti-FLAG antibody M2 and anti-MAGE-D1 antibody. For analysis of the ternary complex of necdin, MAGE-D1, and Msx1 (Msx2), combinations of expression vectors for FLAG-tagged necdin, Myc-tagged MAGE-D1, and Myc-tagged Msx1(Msx2) were transfected into COS-7. The complex was precipitated with antibody NC243 (1:100) and detected with antibody 9E10 (1:10) as above.

Immunoaffinity Purification—To detect the complex containing necdin, MAGE-D1, and Msx2, P19 embryonal carcinoma cells were cultured and induced to differentiate by retinoic acid treatment for 4 days as described previously (25). Cell extracts were applied to HiTrap N-hydroxysuccinimide-activated affinity column (Amersham Biosciences) coupled with IgG fractions of anti-necdin (NC243) antibody, anti-MAGE-D1 antibody, or preimmune antiserum. Bound proteins were eluted with 2 M glycine-HCl (pH 2.5). Fractions were precipitated with 10% trichloroacetic acid, rinsed with cold acetone, separated by 10% SDS-PAGE, and immunoblotted with antibody NC243 (1:3,000), anti-MAGE-D1 antibody (1:1,000), and anti-Msx2 polyclonal antibody (H-70, 1:300) (Sigma). Integrated signal intensities were quantified with a chemiluminescence image analyzer (LAS-1000, FujiFilm).

Msx-dependent Transcription—N1E-115 neuroblastoma cells were cultured and transfected as described (11). For Gal4-mediated luciferase assay, cDNAs encoding Msx1 (amino acids 1–297) and Msx2 (amino acids 1–267) were inserted into pBIND (Promega) to make Gal4-Msx1 (pBIND-Msx1) and Gal4-Msx2 (pBIND-Msx2), respectively. Combinations of pBIND-Msx1, pBIND-Msx2, Myc-MAGE-D1, and FLAG-necdin were transfected into N1E-115 cells along with pG5luc reporter vector (Promega). For Wnt1 enhancer luciferase reporter assay, combinations of expression vectors encoding FLAG-Msx1 (FLAG-Msx2), Myc-MAGE-D1, and FLAG-necdin were infected into N1E-115 cells along with the Wnt1 luciferase reporter construct (pGL2-WIP) (provided by Dr. C. Abate-Shen) (27). Luciferase activity was measured with a luminometer (Lumat LB9501, Berthold) using a reagent kit (Toyo Ink, Tokyo, Japan). Transfection efficiency was normalized with co-transfected LacZ reporter plasmid (pRc-LacZ).

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 µg 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 –20 °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 µM Hoechst 33342 for chromosomal DNA detections, and observed with the fluorescence microscope.

Recombinant Adenoviruses—Adenoviruses expressing LacZ, necdin (amino acids 1–325), and Myc-tagged MAGE-D1 (amino acids 1–775) were constructed using AdEasy adenoviral system (Stratagene). DNAs encoding LacZ, necdin, and Myc-MAGE-D1 were inserted into pShuttle-CMV, amplified, and purified according to the manufacturer's instructions. Cloned C2C12 cells were infected with each adenovirus at 1.5 x 106 plaque-forming units/ml and cultured in the growth medium for 24 h and then in differentiation medium for 72 h. Efficiency of muscle differentiation was judged by MHC expression with antibody MF-20 (1:2) and MyoD (M-318, Santa Cruz Biotechnology) (1:500). MHC expression levels relative to tubulin expression levels were quantified with the image analyzer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 ~43-kDa necdin protein was detected almost exclusively in the brain and skeletal muscle at embryonic day (E) 18.5 (Fig. 1A). In contrast, endogenous ~85-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.



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 1.
Expression of necdin and MAGE-D1 in various tissues and cells. A, Western blot analysis of necdin and MAGE-D1 in mouse embryonal tissues. Tissue lysates (20 µg) from E18.5 embryos were separated by SDS-PAGE and immunoblotted with antibodies against necdin (Necdin), MAGE-D1 (D1), and tubulin (Tubulin). B, Western blot analysis of necdin and MAGE-D1 in cell lines. Cell lysates (20 µg) from undifferentiated C2C12 cells, COS-7 cells, undifferentiated N1E-115 cells, and neurally differentiated P19 cells were analyzed as above. C, fluorescence immunohistochemistry for necdin and MAGE-D1 in mouse embryos. Adjacent sections of the forebrain at E12.5 (upper panels) and the hind limb at E14.5 (lower panels) were stained with antibodies against necdin (Necdin) and MAGE-D1 (D1). The arrows point to the preplate (upper panels) and the skeletal muscles (lower panels). The arrowheads indicate the ventricular proliferative zone; V, ventricle; C, bone cavity. Scale bars, 100 µm.

 
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{Delta}N was co-immunoprecipitated with necdin, but MAGE-D1{Delta}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.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2.
Interactions between necdin and MAGE-D1 in vitro and in vivo. A, MBP-MAGE-D1 deletion mutants. IRD, interspersed hexapeptide repeat domain; MHD, MAGE homology domain. B, purified MBP-MAGE-D1 fusion proteins. The deletion mutants separated by SDS-PAGE were visualized by Coomassie Brilliant Blue staining. C, in vitro binding assay. Purified MBP-MAGE-D1 fusion proteins immobilized on amylose resin were incubated with purified His-tagged necdin. Bound His-tagged necdin (His-Necdin) was detected by immunoblotting with anti-necdin antibody. The results are summarized in A. D, Myc-tagged MAGE-D1 and necdin expressed in COS-7 cells. Expression vectors for Myc-tagged MAGE-D1 (amino acids 1–775), MAGE-D1{Delta}N (amino acids 466–775), MAGE-D1{Delta}C (amino acids 1–479), and necdin (amino acids 1–325) were co-transfected into COS-7 cells. Equal amounts (20 µg) of cell lysates were immunoblotted (IB) with antibodies to Myc ({alpha}Myc) (upper panel) and necdin ({alpha}Necdin) (lower panel). E, co-immunoprecipitation assay. The cell lysates were immunoprecipitated (IP) with anti-necdin antibody ({alpha}Necdin) and immunoblotted (IB) with anti-Myc antibody ({alpha}Myc) (upper panel), or conversely immunoprecipitated with anti-Myc antibody and immunoblotted with anti-necdin antibody (lower panel).

 
Interactions of MAGE-D1 with Msx1 and Msx2—Because MAGE-D1 (Dlxin-1) possesses a specific domain that interacts with Dlx/Msx family homeodomain proteins (23), we examined whether MAGE-D1 interacts in vitro with Msx1 and Msx2 (Fig. 3, A–C). Msx1 and Msx2 bound to MBP-MAGE-D1 fusion proteins containing the WQXPXX repeats (amino acids 1–775, 1–479, and 291–441), but the deletion mutants lacking the repeat domain (amino acids 466–775 and 1–291) failed to interact with Msx1 and Msx2. We next examined whether MAGE-D1 binds to Msx1 and Msx2 in vivo by co-immunoprecipitation assay using COS-7 cells, which express endogenous MAGE-D1 (Fig. 3D). FLAG-Msx1 and FLAG-Msx2 were co-immunoprecipitated with endogenous MAGE-D1. Conversely, endogenous MAGE-D1 was co-immunoprecipitated with FLAG-Msx1 and FLAG-Msx2. These data indicate that MAGE-D1 directly binds to Msx1 and Msx2.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3.
Interactions of MAGE-D1 with Msx1 and Msx2. A, MBP-MAGE-D1 deletion mutants. B, purified MBP-MAGE-D1 fusion proteins. MBP-MAGE-D1 deletion mutants separated by SDS-PAGE were visualized by Coomassie Brilliant Blue staining. C, in vitro binding assay. MBP-MAGE-D1 fusion proteins immobilized on amylose resin were incubated with GST-Msx1 and GST-Msx2. Bound GST-Msx1 and GST-Msx2 were detected by immunoblotting using anti-GST antibody. The results are summarized in A. D, co-immunoprecipitation assay. FLAG-tagged Msx1 (amino acids 1–297) and Msx2 (amino acids 1–267) were transfected into COS-7 cells. Cell lysates were immunoprecipitated (IP) with anti-MAGE-D1 antibody ({alpha}D1) and immunoblotted (IB) with anti-FLAG antibody ({alpha}FLAG) for FLAG-Msx (FLAG-Msx) (top panel) or, conversely, with anti-FLAG antibody and immunoblotted with anti-MAGE-D1 antibody for endogenous MAGE-D1 (End. D1)(2nd panel). Expression levels in the lysates (20 µg) were analyzed with antibodies to FLAG ({alpha}FLAG) (3rd panel) and MAGE-D1 ({alpha}D1) (bottom panel).

 
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-immunoprecipitated with necdin (Fig. 4B). Neither Myc-Msx1 nor Myc-Msx2 was co-immunoprecipitated with a necdin deletion mutant (necdin{Delta}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.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4.
Formation in vivo of a stable complex of Necdin, MAGE-D1. A, co-immunoprecipitation assay for Msx1. Combinations of expression vectors for Myc-tagged MAGE-D1, Myc-tagged Msx1, and necdin were transfected into COS-7 cells. The cell lysates were immunoprecipitated (IP) with anti-necdin antibody ({alpha}Necdin) and immunoblotted (IB) with anti-Myc antibody ({alpha}Myc)(top panel). Equal amounts (20 µg) of transfected cell lysates were separated by 10% SDS-PAGE and immunoblotted (IB) with antibodies against Myc ({alpha}Myc) and necdin ({alpha}Necdin)(middle and bottom panels, respectively). B, co-immunoprecipitation assay for Msx2. The transfection and immunoprecipitation were carried out as in A except Myc-tagged Msx2 was used instead of Myc-tagged Msx1. C, Western blot analysis of necdin, MAGE-D1, and Msx2 in P19 cells. P19 cells were neurally differentiated by retinoic acid treatment, and whole cell extracts (40 µg for Msx2 and 20 µg for the others) were separated by SDS-PAGE and immunoblotted for necdin (Necdin), MAGE-D1 (D1), Msx2 (Msx2), and tubulin (Tubulin). UD, undifferentiated stem cells; RA, aggregated cells treated with retinoic acid; PN, enriched postmitotic neurons. D, immunoaffinity purification with anti-necdin IgG. The lysate (Lysate) of retinoic acid-treated P19 cells (1 mg) was subjected to the immunoaffinity purification with anti-necdin IgG ({alpha}Necdin IgG) and preimmune IgG (Preimmune IgG). Bound proteins were separated by SDS-PAGE and immunoblotted for Msx2 (Msx2), MAGE-D1 (D1), and necdin (Necdin). Signal intensities of different amounts of the lysate (shown in µg of protein per lane) and the purified sample (equivalent to 1 mg of lysate) were quantified with a chemiluminescence image analyzer. The signal intensities of the purified proteins ({alpha}Necdin IgG) are presented as the amounts of lysate with equal signal intensities. E, immunoaffinity purification with anti-MAGE-D1 IgG. The analysis was carried out as in D except anti-MAGE-D1 IgG was used instead of anti-necdin IgG.

 
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 Msx-dependent 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.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5.
Release from Msx-induced transcriptional repression by necdin and MAGE-D1. A, GAL4-dependent transcription assay. Combinations of expression vectors for Gal4 (Vector), Gal4-Msx (1 µg) (Msx1 or Msx2), Myc-tagged MAGE-D1 (1.5 µg)(D1), and FLAG-tagged necdin (1.5 µg) (Necdin) were transfected along with pG5luc (0.5 µg) into N1E-115 neuroblastoma cells. The luciferase activity driven by the promoter carrying 5xGal4-binding sites (5xGal4) was measured with a luminometer (mean ± S.E., n = 3). The transfection efficiency was normalized with the activity of co-transfected pRc-LacZ (0.5 µg). The total amount of plasmids was adjusted to 5 µg/assay by adding the empty vector. B, Wnt1 promoter-reporter system. Combinations of pGL2-WIP (0.5 µg), Gal4-Msx (1 µg), Myc-tagged MAGE-D1 (1.5 µg), and FLAG-tagged necdin (1.5 µg) were transfected into N1E-115 cells. The luciferase activity driven by the Wnt1 promoter (WIP) was measured and presented as in A.

 
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).



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 6.
Repression of myogenic differentiation in stably Msx2-expressing C2C12 cells. A, immunocytochemistry of C2C12 cells. C2C12 cells were cultured under undifferentiated conditions (UD) or under differentiation conditions for 4 days (4D). Cells were fixed and triply stained for myosin heavy chain (MHC), MAGE-D1 (D1), and nuclear DNA with Hoechst33342 (Hoechst). The arrowheads point to differentiated multinucleated myocytes. Scale bar, 20 µm. B, Western blot analysis. C2C12 cells were cultured under undifferentiated (UD) or differentiation conditions for 2 (2D) and 4 days (4D). MHC (MHC), necdin (Necdin), MAGE-D1 (D1), and tubulin (Tubulin) in cell extracts (20 µg) were analyzed. P19, the extract of P19-derived neurons. C, immunocytochemistry of Msx2-expressing C2C12 cells. C2C12 cells that constitutively express FLAG-tagged Msx2 were cultured for 3 days under undifferentiated (UD) or differentiated conditions (D). C2C12 cells were stably transfected with FLAG-Msx2 expression vector (Msx2) or empty vector (Vector). Transfectants were stained for FLAG-Msx2 (FLAG) and MHC (MHC). Note the nuclear localization of FLAG-Msx2. The arrowheads point to mononucleated MHC-positive cells. Scale bar, 20 µm. D, Western blot analysis. Cell extracts were prepared from control (Vector) and Msx2-expressing C2C12 cells (Msx2) cultured for 3 days under undifferentiated (UD) or differentiated (D) conditions. MHC (MHC), FLAG-tagged Msx2 (Msx2), and tubulin (Tubulin) in cell extracts (15 µg) were analyzed.

 
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 ~9 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 ~5 times the control level, and necdin alone increased the MyoD level ~3 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, 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.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 7.
Release from Msx2-induced myogenic repression by necdin and MAGE-D1. A, immunohistochemistry for MHC expression. Msx2-expressing C2C12 cells were infected with the recombinant adenovirus expressing LacZ (LacZ), Myc-tagged-MAGE-D1(D1), necdin (Necdin), or both (Necdin+D1). Infected cells were cultured in the growth medium for 24 h and the differentiation medium for 72 h. Note the appearance of multinucleated MHC-positive cells showing myotube formation in necdin expressing cultures (arrowheads). Scale bar, 20 µm. B, Western blot analysis. Cell lysates (40 µg) were prepared from FLAG-Msx2-expressing C2C12 cells infected with the adenovirus expressing LacZ (LacZ), Myc-tagged MAGE-D1 (D1), necdin (Necdin), or both (Necdin+D1). MHC (MHC), MyoD (MyoD), necdin (Necdin), MAGE-D1 (D1), and tubulin (Tubulin) in cell extracts were detected by Western blotting. C, quantification of MHC levels. Expression levels of MHC and tubulin were quantified by chemiluminescence image analysis and expressed as relative values (MHC/Tubulin). Each value represents the mean ± S.E. (n = 3). D, co-immunoprecipitation of the complex containing necdin, MAGE-D1, and Msx2. Nuclear extracts (60 µg) of FLAG-Msx2-expressing C2C12 cells infected with the adenoviruses were immunoprecipitated (IP) with anti-necdin antibody ({alpha}Necdin) and immunoblotted (IB) with anti-FLAG antibody ({alpha}FLAG) for FLAG-Msx2 (FLAG-Msx2) and anti-Myc antibody ({alpha}Myc) for Myc-tagged MAGE-D1 (Myc-D1). Cell extracts (10 µg) were immunoblotted with the above antibodies for expression levels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (<350 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 (>650 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). The present study has shown that necdin is another MAGE-D1 MHD interactor. Because MAGE-D1 also associates with Msx/Dlx homeodomain proteins via the interspersed repeat domain district from the MHD (Ref 23 and present study), MAGE-D1 serves as an adapter protein that links these MHD interactors with Msx/Dlx homeodomain proteins.

Msx1 and Msx2 (previous Hox-7 and Hox-8, respectively) are 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 repressors 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 necdin-related 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.


   FOOTNOTES
 
* 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 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. Tel.: 81-66879-8621; Fax: 81-66879-8623; E-mail: yoshikaw{at}protein.osaka-u.ac.jp.

1 The abbreviations used are: MHD, MAGE homology domain; MBP, maltose-binding protein; GST, glutathione S-transferase; MHC, myosin heavy chain; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; E, embryonic day. Back


   ACKNOWLEDGMENTS
 
We thank Dr. C. Abate-Shen for the generous provision of research materials and Drs. M. Niinobe and T. Uetsuki for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maruyama, K., Usami, M., Aizawa, T., and Yoshikawa, K. (1991) Biochem. Biophys. Res. Commun. 178, 291–296[CrossRef][Medline] [Order article via Infotrieve]
  2. Aizawa, T., Maruyama, K., Kondo, H., and Yoshikawa, K. (1992) Brain Res. Dev. Brain Res. 68, 265–274[Medline] [Order article via Infotrieve]
  3. Uetsuki, T., Takagi, K., Sugiura, H., and Yoshikawa, K. (1996) J. Biol. Chem. 271, 918–924[Abstract/Free Full Text]
  4. Taniguchi, N., Taniura, H., Niinobe, M., Takayama, C., Tominaga-Yoshino, K., Ogura, A., and Yoshikawa, K. (2000) J. Biol. Chem. 275, 31674–31681[Abstract/Free Full Text]
  5. Gerard, M., Hernandez, L., Wevrick, R., and Stewart, C. L. (1999) Nat. Genet. 23, 199–202[CrossRef][Medline] [Order article via Infotrieve]
  6. Yoshikawa, K. (2000) Neurosci. Res. 37, 1–14[CrossRef][Medline] [Order article via Infotrieve]
  7. Hu, B., Wang, S., Zhang, Y., Feghali, C. A., Dingman, J. R., and Wright, T. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10008–10013[Abstract/Free Full Text]
  8. Hayashi, Y., Matsuyama, K., Takagi, K., Sugiura, H., and Yoshikawa, K. (1995) Biochem. Biophys. Res. Commun. 213, 317–324[CrossRef][Medline] [Order article via Infotrieve]
  9. Taniura, H., Taniguchi, N., Hara, M., and Yoshikawa, K. (1998) J. Biol. Chem. 273, 720–728[Abstract/Free Full Text]
  10. Taniura, H., Matsumoto, K., and Yoshikawa, K. (1999) J. Biol. Chem. 274, 16242–16248[Abstract/Free Full Text]
  11. Kobayashi, M., Taniura, H., and Yoshikawa, K. (2002) J. Biol. Chem. 277, 42128–42135[Abstract/Free Full Text]
  12. Takazaki, R., Nishimura, I., and Yoshikawa, K. (2002) Exp. Cell Res. 277, 220–232[CrossRef][Medline] [Order article via Infotrieve]
  13. Muscatelli, F., Abrous, D. N., Massacrier, A., Boccaccio, I., Moal, M. L., Cau, P., and Cremer, H. (2000) Hum. Mol. Genet. 9, 3101–3110[Abstract/Free Full Text]
  14. Ren, J., Lee, S., Pagliardini, S., Gerard, M., Stewart, C. L., Greer, J. J., and Wevrick, R. (2003) J. Neurosci. 23, 1569–1573[Abstract/Free Full Text]
  15. Taniura, H., and Yoshikawa, K. (2002) J. Cell. Biochem. 84, 545–555[CrossRef][Medline] [Order article via Infotrieve]
  16. Tcherpakov, M., Bronfman, F. C., Conticello, S. G., Vaskovsky, A., Levy, Z., Niinobe, M., Yoshikawa, K., Arenas, E., and Fainzilber, M. (2002) J. Biol. Chem. 277, 49101–49104[Abstract/Free Full Text]
  17. Kuwako, K., Taniura, H., and Yoshikawa, K. (2004) J. Biol. Chem. 279, 1703–1712[Abstract/Free Full Text]
  18. Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279–288[CrossRef][Medline] [Order article via Infotrieve]
  19. Saburi, S., Nadano, D., Akama, T. O., Hirama, K., Yamanouchi, K., Naito, K., Tojo, H., Tachi, C., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 49378–49389[Abstract/Free Full Text]
  20. Salehi, A. H., Xanthoudakis, S., and Barker, P. A. (2002) J. Biol. Chem. 277, 48043–48050[Abstract/Free Full Text]
  21. Jordan, B. W., Dinev, D., LeMellay, V., Troppmair, J., Gotz, R., Wixler, L., Sendtner, M., Ludwig, S., and Rapp, U. R. (2001) J. Biol. Chem. 276, 39985–39989[Abstract/Free Full Text]
  22. Williams, M. E., Strickland, P., Watanabe, K., and Hinck, L. (2003) J. Biol. Chem. 278, 17483–17490[Abstract/Free Full Text]
  23. Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., and Watanabe, K. (2001) J. Biol. Chem. 276, 5331–5338[Abstract/Free Full Text]
  24. Matsuda, T., Suzuki, H., Oishi, I., Kani, S., Kuroda, Y., Komori, T., Sasaki, A., Watanabe, K., and Minami, Y. (2003) J. Biol. Chem. 278, 29057–29064[Abstract/Free Full Text]
  25. McBurney, M. W., Reuhl, K. R., Ally, A. I., Nasipuri, S., Bell, J. C., and Craig, J. (1988) J. Neurosci. 8, 1063–1073[Abstract]
  26. Niinobe, M., Koyama, K., and Yoshikawa, K. (2000) Dev. Neurosci. 22, 310–319[CrossRef][Medline] [Order article via Infotrieve]
  27. Moroni, M. C., Hickman, E. S., Denchi, E. L., Caprara, G., Colli, E., Cecconi, F., Muller, H., and Helin, K. (2001) Nat. Cell Biol. 3, 552–558[CrossRef][Medline] [Order article via Infotrieve]
  28. Hu, G., Lee, H., Price, S. M., Shen, M. M., and Abate-Shen, C. (2001) Development (Camb.) 128, 2373–2384
  29. Marazzi, G., Wang, Y., and Sassoon, D. (1997) Dev. Biol. 186, 127–138[CrossRef][Medline] [Order article via Infotrieve]
  30. Iler, N., Rowitch, D. H., Echelard, Y., McMahon, A. P., and Abate-Shen, C. (1995) Mech. Dev. 53, 87–96[CrossRef][Medline] [Order article via Infotrieve]
  31. Takahashi, K., Nuckolls, G. H., Takahashi, I., Nonaka, K., Nagata, M., Ikura, T., Slavkin, H. C., and Shum, L. (2001) Dev. Dyn. 222, 252–262[CrossRef][Medline] [Order article via Infotrieve]
  32. Davidson, D. (1995) Trends Genet. 11, 405–411[CrossRef][Medline] [Order article via Infotrieve]
  33. Barker, P. A., and Salehi, A. (2002) J. Neurosci. Res. 67, 705–712[CrossRef][Medline] [Order article via Infotrieve]
  34. Pold, M., Pold, A., Ma, H. J., Sjak-Shieb, N. N., Vescio, R. A., and Berensonb, J. R. (2000) Dev. Comp. Immunol. 24, 719–731[CrossRef][Medline] [Order article via Infotrieve]
  35. Bischof, J. M., Ekker, M., and Wevrick, R. (2003) Dev. Dyn. 228, 475–479[CrossRef][Medline] [Order article via Infotrieve]
  36. Albrecht, D. E., and Froehner, S. C. (2004) J. Biol. Chem. 279, 7014–7023[Abstract/Free Full Text]
  37. Sasaki, A., Masuda, Y., Iwai, K., Ikeda, K., and Watanabe, K. (2002) J. Biol. Chem. 277, 22541–22546[Abstract/Free Full Text]
  38. Song, K., Wang, Y., and Sassoon, D. (1992) Nature 360, 477–481[CrossRef][Medline] [Order article via Infotrieve]
  39. Woloshin, P., Song, K., Degnin, C., Killary, A. M., Goldhamer, D. J., Sassoon, D., and Thayer, M. J. (1995) Cell 82, 611–620[CrossRef][Medline] [Order article via Infotrieve]
  40. Dodig, M., Tadic, T., Kronenberg, M. S., Dacic, S., Liu, Y. H., Maxson, R., Rowe, D. W., and Lichtler, A. C. (1999) Dev. Biol. 209, 298–307[CrossRef][Medline] [Order article via Infotrieve]
  41. Brunelli, S., Tagliafico, E., De Angelis, F. G., Tonlorenzi, R., Baesso, S., Ferrari, S., Niinobe, M., Yoshikawa, K., Schwartz, R. J., Bozzoni, I., and Cossu, G. (2004) Circ. Res. 94, 1571–1578[Abstract/Free Full Text]
  42. Kendall, S., Goldhawk, D., Kubu, C., Barker, P., and Verdi, J. (2002) Mech. Dev. 117, 187–200[CrossRef][Medline] [Order article via Infotrieve]
  43. Israsena, N., and Kessler, J. A. (2002) J. Neurosci. Res. 69, 803–809[CrossRef][Medline] [Order article via Infotrieve]
  44. Stuhmer, T., Anderson, S. A., Ekker, M., and Rubenstein, J. L. (2002) Development (Camb.) 129, 245–252[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
K. Hasegawa and K. Yoshikawa
Necdin Regulates p53 Acetylation via Sirtuin1 to Modulate DNA Damage Response in Cortical Neurons
J. Neurosci., August 27, 2008; 28(35): 8772 - 8784.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
D. Deponti, S. Francois, S. Baesso, C. Sciorati, A. Innocenzi, V. Broccoli, F. Muscatelli, R. Meneveri, E. Clementi, G. Cossu, et al.
Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation
J. Cell Biol., October 22, 2007; 179(2): 305 - 319.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. G. Di Certo, N. Corbi, T. Bruno, S. Iezzi, F. De Nicola, A. Desantis, M. T. Ciotti, E. Mattei, A. Floridi, M. Fanciulli, et al.
NRAGE associates with the anti-apoptotic factor Che-1 and regulates its degradation to induce cell death
J. Cell Sci., June 1, 2007; 120(11): 1852 - 1858.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. A. Timmons, K. Wennmalm, O. Larsson, T. B. Walden, T. Lassmann, N. Petrovic, D. L. Hamilton, R. E. Gimeno, C. Wahlestedt, K. Baar, et al.
Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages
PNAS, March 13, 2007; 104(11): 4401 - 4406.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Hayashi, S. Nakamura, W. Nishida, and K. Sobue
Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription
Mol. Cell. Biol., December 15, 2006; 26(24): 9456 - 9470.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. Kurita, T. Kuwajima, I. Nishimura, and K. Yoshikawa
Necdin Downregulates Cdc2 Expression to Attenuate Neuronal Apoptosis.
J. Neurosci., November 15, 2006; 26(46): 12003 - 12013.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T. Kuwajima, I. Nishimura, and K. Yoshikawa
Necdin promotes GABAergic neuron differentiation in cooperation with Dlx homeodomain proteins.
J. Neurosci., May 17, 2006; 26(20): 5383 - 5392.
[Abstract] [Full Text] [PDF]