Nogo-A inhibits necdin-accelerated neurite outgrowth by retaining necdin in the cytoplasm.

Nogo-A has been identified in the central nervous system as an inhibitor for axonal regeneration. Previous works have mainly focused on Nogo-A in oligodendrocytes and the roles of neuronal intracellular Nogo-A remain elusive. To gain deep insight into the physiological functions of Nogo-A, a yeast two-hybrid screening was performed with Nogo-66 as bait. We identified a new interaction between Nogo-66 and necdin. Mutagenesis analysis revealed that the central region of necdin was indispensable for the interaction of necdin with Nogo-66. The interaction was further confirmed by co-immunoprecipitation in neural tissues and cultured cortical neurons. Morphological evidence showed that Nogo-A and necdin highly colocalized in rat cortical and dorsal root ganglia neurons. Ectopic expression of Nogo-A in HEK293 cells led to retention of necdin from the nucleus to the cytoplasm. Furthermore, overexpression of Nogo-A in PC12 cells and cultured cortical neurons inhibited necdin-accelerated neurite outgrowth. Meanwhile, necdin was found to be significantly sequestered in the cytoplasm of PC12 cells stably overexpressing Nogo-A. Together, these data suggest that Nogo-A is a novel necdin binding protein and inhibits necdin-accelerated neuronal neurite outgrowth by sequestering necdin in the cytoplasm.

Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II Mage proteins.

In mammals, the type II melanoma antigen (Mage) protein family is constituted by at least 10 closely related members that are expressed in different tissues, including the nervous system. These proteins are believed to regulate cell cycle withdrawal, neuronal differentiation, and apoptosis. However, the analysis of their specific function has been complicated by functional redundancy. In accordance with previous studies in teleosts and Drosophila, we present evidence that only one mage gene exists in genomes from protists, fungi, plants, nematodes, insects, and nonmammalian vertebrates. We have identified the chicken mage gene and cloned the cDNA encoding the chick Mage protein (CMage). CMage shares close homology with the type II Mage protein family, and, as previously shown for the type II Mage proteins Necdin and Mage-G1, it can interact with the transcription factor E2F-1. CMage is expressed in specific regions of the developing nervous system including the retinal ganglion cell layer, the ventral horn of the spinal cord, and the dorsal root ganglia, coinciding with the expression of the neurotrophin receptor p75 (p75(NTR)) in these regions. We show that the intracellular domain of p75(NTR) can interact with both CMage and Necdin, thus preventing the binding of the latter proteins to the transcription factor E2F-1, and facilitating the proapoptotic activity of E2F-1 in N1E-115 differentiating neurons. The presence of a single mage gene in the chicken genome, together with the close functional resemblance between CMage and Necdin, makes this species ideal to further analyze signal transduction through type II Mage proteins.

Overexpression of SOCS3 inhibits astrogliogenesis and promotes maintenance of neural stem cells.

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.

Necdin and E2F4 are modulated by rosiglitazone therapy in diabetic human adipose and muscle tissue.

To identify novel pathways mediating molecular mechanisms of thiazolidinediones (TZDs) in humans, we assessed gene expression in adipose and muscle tissue from six subjects with type 2 diabetes before and after 8 weeks of treatment with rosiglitazone. mRNA was analyzed using Total Gene Expression Analysis (TOGA), an automated restriction-based cDNA display method with quantitative analysis of PCR products. The expression of cell cycle regulatory transcription factors E2F4 and the MAGE protein necdin were similarly altered in all subjects after rosiglitazone treatment. E2F4 expression was decreased by 10-fold in muscle and 2.5-fold in adipose tissue; necdin was identified in adipose tissue only and increased 1.8-fold after TZD treatment. To determine whether changes were related to an effect of the drug or adipogenesis, we evaluated the impact of rosiglitazone and differentiation independently in 3T3-L1 adipocytes. While treatment of differentiated adipocytes with rosiglitazone did not alter E2F4 or necdin, expression of both genes was significantly altered during differentiation. Differentiation was associated with increased cytosolic localization of E2F4. Moreover, necdin overexpression potently inhibited adipocyte differentiation and cell cycle progression. These data suggest that changes in necdin and E2F4 expression after rosiglitazone exposure in humans are associated with altered adipocyte differentiation and may contribute to improved insulin sensitivity in humans treated with TZDs.

Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin.

The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differentiation via complex signalling networks. Here, using microarray analysis of brown preadipocytes that are derived from wild-type and insulin receptor substrate (Irs) knockout animals that exhibit progressively impaired differentiation, we define 374 genes/expressed-sequence tags whose expression in preadipocytes correlates with the ultimate ability of the cells to differentiate. Many of these genes, including preadipocyte factor-1 (Pref-1) and multiple members of the Wnt signalling pathway, are related to early adipogenic events. Necdin is also markedly increased in Irs knockout cells that cannot differentiate, and knockdown of necdin restores brown adipogenesis with downregulation of Pref-1 and Wnt10a expression. Insulin receptor substrate proteins regulate a necdin-E2F4 interaction that represses peroxisome-proliferator-activated receptor gamma (PPARgamma) transcription via a cyclic AMP response element binding protein (CREB)-dependent pathway. Together these define a key signalling network that is involved in brown preadipocyte determination.

Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas.

Affymetrix and spotted oligonucleotide microarrays were used to assess global differential gene expression comparing normal human melanocytes with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the overexpression in melanoma cells relative to normal melanocytes of several genes in the growth factor/receptor family that confer growth advantage and metastasis. In addition, novel pathways and patterns of associated expression in melanoma cells not reported before emerged, including the following: (a) activation of the NOTCH pathway; (b) increased Twist expression and altered expression of additional transcriptional regulators implicated in embryonic development and epidermal/mesenchymal transition; (c) coordinated activation of cancer/testis antigens; (d) coordinated down-regulation of several immune modulation genes, in particular in the IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, such as the Prader-Willi gene NECDIN, whose function was confirmed by overexpression of ectopic Flag-necdin. Validation of differential expression using melanoma tissue microarrays showed that reduced ubiquitin COOH-terminal esterase L1 in primary melanoma is associated with worse outcome and that increased expression of the basic helix-loop-helix protein Twist is associated with worse outcome. Some differentially expressed genes reside on chromosomal regions displaying common loss or gain in melanomas or are known to be regulated by CpG promoter methylation. These results provide a comprehensive view of changes in advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of melanoma patients.

Msx2 and necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells.

Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for "stem" cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor beta. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor beta, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.

Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal differentiation and p75NTR expression.

The expression pattern of Necdin, a gene involved in the etiology of Prader-Willi syndrome and a member of the MAGE family of genes, is described during mouse nervous system development. Using RNA in situ hybridization, immunohistochemical staining, and colocalization with neuronal differentiation markers, we found that Necdin RNA and protein are expressed within post-mitotic neurons at all stages studied. From E10 to E12, Necdin is detected in all developing neurons, in both central and peripheral nervous system, with the highest expression levels in the diencephalon and the hindbrain. After E13, Necdin is expressed in specific structures of the nervous system, in particular the hypothalamus, the thalamus, and the pons, suggesting a specific developmental role therein. In addition, Necdin expression is also detected in non-neural tissues, such as the somites, the developing limb buds, the first branchial arches, the tong, and the axial muscles. Recently, Necdin and other MAGE proteins were found to interact in vitro with the intracellular domain of the p75NTR neurotrophin receptor, but this interaction has not been validated in vivo. We report here that the spatial and temporal expression of p75NTR is included in Necdin expression domain. These results are in agreement with Necdin proposed role on cell cycle arrest, inhibition of apoptosis and facilitation of neuronal differentiation in vitro, and with hypothalamic cellular deficiencies reported in mice with abrogation of the Necdin gene. Furthermore, they are also consistent with the putative role of Necdin in signaling events promoted by p75NTR during mouse nervous system development.

The p75 neurotrophin receptor interacts with multiple MAGE proteins.

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.