Functional domains of necdin for protein-protein interaction, nuclear matrix targeting, and cell growth suppression

Necdin is a growth suppressor expressed predominantly in postmitotic neurons. The necdin gene is involved in the etiology of the genomic imprinting-associated neurodevelopmental disorder Prader-Willi syndrome and belongs to the MAGE gene family. All the MAGE family proteins contain a large homology domain termed the MAGE homology domain (MHD). We here characterize the regions of necdin required for the protein-protein interaction, nuclear matrix targeting, and cell growth suppression. The region including entire MHD (amino acids 116-280) of necdin was required for its interaction with p53, while the regions amino acids 144-184 and 191-222 within the MHD were required for both the nuclear matrix targeting and the cell growth suppression of osteosarcoma SAOS-2 cells. The amino-terminal proline-rich acidic region (amino acids 60-100) was also necessary for cell growth suppression. Tetracycline-regulatable overexpression of necdin induced growth arrest of SAOS-2 cells in a reversible manner, and the necdin-overexpressing cells showed a large, flattened morphology with double nuclei. In contrast, a necdin mutant lacking amino acids 191-222 did not induce such changes. These findings suggest that different functions of necdin are mediated via its distinct domains.     

Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.     

Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis

The MAGE (melanoma antigen) family is characterized by a large conserved domain termed MAGE homology domain. Originally identified MAGE genes encoding tumor rejection antigens are expressed only in cancers and male germ cells. Necdin, which contains the MAGE homology domain, is highly expressed in postmitotic cells such as neurons and skeletal muscle cells. The human necdin gene NDN is transcribed only from the paternal allele through genomic imprinting, and its deficiency is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Although over 30 MAGE genes have been identified in humans, fruit fly (Drosophila melanogaster) has only a single MAGE gene that encodes a protein similar to necdin homologous MAGE proteins. In this study, we analyzed the spatiotemporal expression patterns of MAGE mRNA and the encoded protein during fly development. Whole-mount embryo in situ hybridization analysis revealed that MAGE mRNA was highly expressed at the syncytial blastoderm stage and in the ventral and procephalic neurogenic regions of the ectoderm during gastrulation. In contrast, MAGE expression was nearly undetectable in postmitotic neurons of the central nervous system at late embryonic stages. During postembryonic neurogenesis, MAGE was highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages. MAGE was also expressed in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood. These results indicate that MAGE expression lasts throughout the postembryonic neurogenesis in Drosophila.     

Inhibition of apoptosis by the retinoblastoma gene product.

Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.     

Necdin controls proliferation of white adipocyte progenitor cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development.     

Regulation of tumor suppressor p53 and HCT116 cell physiology by histone demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53(-/-) HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53(-/-) compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of osteosarcoma

This study investigated the role of miR-143 in the chemoresistance of osteosarcoma tumor cells and the associated mechanisms. Real-time PCR was used to measure miR-143 levels. Western blot was used to detect protein expression. Cell proliferation was analyzed by MTT assay and Matrigel colony formation assay. Forced miR-143 expression was established by adenoviral vector infection. Cell death was detected by Hoechst33342 staining. Loss of miR-143 expression was observed in osteosarcomas, which correlated with shorter survival of patients with osteosarcomas underlying chemotherapy. In chemoresistant SAOS-2 and U2OS osteosarcomas cells, miR-143 levels were significantly downregulated and accompanied by increases in ATG2B, Bcl-2, and/or LC3-II protein levels, high rate of ALDH1⁺CD133⁺ cells, and an increase in Matrigel colony formation ability. H₂O₂ upregulated p53 and miR-143, but downregulated ATG2B, Bcl-2, and LC3-I expression in U2OS cells (wild-type p53) but not in SAOS-2 (p53-null) cells. Forced miR-143 expression significantly reversed chemoresistance as well as downregulation of ATG2B, LC3-I, and Bcl-2 expression in SAOS-2- and U2OS-resistant cells. Forced miR-143 expression significantly inhibited tumor growth in xenograft SAOS-2-Dox and U2OS-Dox animal models. Loss of miR-143 expression is associated with poor prognosis of patients with osteosarcoma underlying chemotherapy. The chemoresistance of osteosarcoma tumor cells to doxorubicin is associated with the downregulation of miR-143 expression, activation of ALDH1⁺CD133⁺ cells, activation of autophagy, and inhibition of cell death. miR-143 may play a crucial role in the chemoresistance of osterosarcoma tumors.