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Receptor-interacting protein 140 overexpression promotes neuro-2a neuronal differentiation by ERK1/2 signaling.

Feng X, Yu W, Liang R, Shi C, Zhao Z, Guo J - Chin. Med. J. (2015)

Bottom Line: The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05).RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University People's Hospital, Beijing 100044, China.

ABSTRACT

Background: Abnormal neuronal differentiation plays an important role in central nervous system (CNS) development abnormalities such as Down syndrome (DS), a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140) is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.

Methods: Stably RIP140-overexpressing N2a (N2a-RIP140) cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA) was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.

Results: Compared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05). In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) levels in N2a-RIP140 cells were dramatically increased, while differentiation was inhibited by the ERK1/2-specific inhibitor U0126.

Conclusions: RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

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Exposure of neuro-2a (N2a) cells to retinoic acid (RA) leads to neurite outgrowth and Receptor-interacting protein 140 (RIP140) upregulation. (a) Phase-contrast micrographs of N2a cells treated with RA (+RA) or without RA (−RA). (b) Western blotting analysis shows RIP140 expression in the absence (−RA) and presence of RA (+RA).
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Figure 1: Exposure of neuro-2a (N2a) cells to retinoic acid (RA) leads to neurite outgrowth and Receptor-interacting protein 140 (RIP140) upregulation. (a) Phase-contrast micrographs of N2a cells treated with RA (+RA) or without RA (−RA). (b) Western blotting analysis shows RIP140 expression in the absence (−RA) and presence of RA (+RA).

Mentions: Retinoic acid has been shown to promote neurite outgrowth from a variety of neuronal cell types in culture.[9] Therefore, we chose RA to induce N2a cells differentiation. As shown in Figure 1a, after exposure to 20 μmol/L RA for 24 hours, N2a cells assumed a neuronal-like phenotype by activating neurite production, concurrent with upregulation of endogenous RIP140 [Figure 1b].


Receptor-interacting protein 140 overexpression promotes neuro-2a neuronal differentiation by ERK1/2 signaling.

Feng X, Yu W, Liang R, Shi C, Zhao Z, Guo J - Chin. Med. J. (2015)

Exposure of neuro-2a (N2a) cells to retinoic acid (RA) leads to neurite outgrowth and Receptor-interacting protein 140 (RIP140) upregulation. (a) Phase-contrast micrographs of N2a cells treated with RA (+RA) or without RA (−RA). (b) Western blotting analysis shows RIP140 expression in the absence (−RA) and presence of RA (+RA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837806&req=5

Figure 1: Exposure of neuro-2a (N2a) cells to retinoic acid (RA) leads to neurite outgrowth and Receptor-interacting protein 140 (RIP140) upregulation. (a) Phase-contrast micrographs of N2a cells treated with RA (+RA) or without RA (−RA). (b) Western blotting analysis shows RIP140 expression in the absence (−RA) and presence of RA (+RA).
Mentions: Retinoic acid has been shown to promote neurite outgrowth from a variety of neuronal cell types in culture.[9] Therefore, we chose RA to induce N2a cells differentiation. As shown in Figure 1a, after exposure to 20 μmol/L RA for 24 hours, N2a cells assumed a neuronal-like phenotype by activating neurite production, concurrent with upregulation of endogenous RIP140 [Figure 1b].

Bottom Line: The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05).RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University People's Hospital, Beijing 100044, China.

ABSTRACT

Background: Abnormal neuronal differentiation plays an important role in central nervous system (CNS) development abnormalities such as Down syndrome (DS), a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140) is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.

Methods: Stably RIP140-overexpressing N2a (N2a-RIP140) cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA) was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.

Results: Compared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05). In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) levels in N2a-RIP140 cells were dramatically increased, while differentiation was inhibited by the ERK1/2-specific inhibitor U0126.

Conclusions: RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

Show MeSH
Related in: MedlinePlus