DIXDC1 Promotes Retinoic Acid-Induced Neuronal Differentiation and Inhibits Gliogenesis in P19 Cells

Department of Brain Protection & Plasticity Research, Beijing Institute of Basic Medical Sciences, Beijing, People's Republic of China.
Cellular and Molecular Neurobiology (Impact Factor: 2.51). 02/2009; 29(1):55-67. DOI: 10.1007/s10571-008-9295-9
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Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid.

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    • "P19 cells can be induced to differentiate into cell types of three germ layers (ectoderm, endoderm, or mesoderm) when exposed to the appropriate inducer and culture conditions [9]–[12]. P19 cells have been used extensively as an in vitro model system for the study of molecular mechanisms involved in cellular differentiation and early embryonic development [13]; moreover, several genes that play important roles in mammalian differentiation have been identified using P19 cells [14]–[18]. When P19 cells are grown as aggregates and exposed to 1 µM of ATRA, they differentiate into neurons and glial cells that exhibit characteristic neural morphology and express proteins commonly found in central nervous system (CNS) neurons, such as neuron-specific class III β-tubulin (Tuj1), neuronal nuclei (NeuN) and neurofilament proteins [9], [11], [13], [19], [20]. "
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    ABSTRACT: κ-Casein (CSN3) is known to play an essential role in controlling the stability of the milk micelles. We found that the expression of Csn3 was induced by all-trans retinoic acid (ATRA) during neural differentiation in P19 embryonal carcinoma cells from our study using DNA microarray. In this paper, we describe the detailed time course of Csn3 expression and the induction mechanism of Csn3 transcription activation in this process. The Csn3 expression was induced rapidly and transiently within 24 h of ATRA treatment. Retinoic acid receptor (RAR)-specific agonists were used in expression analysis to identify the RAR subtype involved upregulation of Csn3; a RARα-specific agonist mimicked the effects of ATRA on induction of Csn3 expression. Therefore, RARα may be the RAR subtype mediating the effects of ATRA on the induction of Csn3 gene transcription in this differentiation-promoting process of P19 cells. We found that the promoter region of Csn3 contained a typical consensus retinoic acid response element (RARE), and this RARE was necessary for ATRA-dependent transcriptional regulation. We confirmed that RARα bound to this RARE sequence in P19 cells. These findings indicated that the Csn3 expression is upregulated via ATRA-bound RARα and binding of this receptor to the RARE in the Csn3 promoter region. This will certainly serve as a first step forward unraveling the mysteries of induction of Csn3 in the process of neural differentiation.
    PLoS ONE 04/2013; 8(4):e61938. DOI:10.1371/journal.pone.0061938 · 3.23 Impact Factor
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    • "However, how Ccd1 positively regulates Wnt signaling and why overexpression of Ccd1 deregulates zebrafish neural patterning is unknown. Our previous work on DIXDC1 showed that overexpression could promote neuronal and inhibit gliogenous differentiation of P19 cells induced by retinoic acid (RA) (Jing et al., 2009). DIXDC1 protein has also been implicated as an inhibitor of "
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    ABSTRACT: DIXDC1 is a Dishevelled-Axin (DIX) domain-containing protein involved in neural development and Wnt signaling pathway. Besides the DIX domain, DIXDC1 also contains a coiled-coil domain (MTH domain), which is a common feature of centrosomal proteins. We have demonstrated that exogenously expressed GFP-tag fused DIXDC1 co-localize with gamma-tubulin both at interphase and mitotic phase in HEK293 cells. By immunostaining with anti-DIXDC1 and anti-gamma-tubulin antibody, endogenous DIXDC1 was also co-localized with gamma-tubulin at the centrosomes in HEK293 cells. We confirmed this interaction of DIXDC1 with gamma-tubulin by co-immunoprecipitation. The findings suggest that DIXDC1 might play an important role in chromosome segregation and cell cycle regulation.
    Cell Biology International 05/2009; 33(6):697-701. DOI:10.1016/j.cellbi.2009.04.001 · 1.93 Impact Factor
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    ABSTRACT: Previous observations suggest that Bis, a Bcl-2-binding protein, may play a role the neuronal and glial differentiation in vivo. To examine this further, we investigated Bis expression during the in vitro differentiation of P19 embryonic carcinoma cells induced by retinoic acid (RA). Western blotting and RT-PCR assays showed that Bis expression was temporarily decreased during the free floating stage and then began to increase on day 6 after the induction of differentiation. Double immunostaining indicated that Bis-expressing cells do not express several markers of differentiation, including NeuN, MAP-2 and Tuj-1. However, some of the Bis-expressing cells also were stained with GFAP-antibodies, indicating that Bis is involved glial differentiation. Using an shRNA strategy, we developed bis-knock down P19 cells and compared them with control P19 cells for the expression of NeuroD, Mash-1 and GFAP during RA-induced differentiation. Among these, only GFAP induction was significantly attenuated in P19-dnbis cells and the population showing GFAP immunoreactivity was also decreased. It is noteworthy that distribution of mature neurons and migrating neurons was disorganized, and the close association of migrating neuroblasts with astrocytes was not observed in P19-dnbis cells. These results suggest that Bis is involved in the migration-inducing activity of glial cells.
    Korean Journal of Physiology and Pharmacology 06/2009; 13(3):251-6. DOI:10.4196/kjpp.2009.13.3.251 · 1.38 Impact Factor
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