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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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Related in: MedlinePlus

Receptor-interacting protein 140 (RIP140) overexpression promotes neurite outgrowth. (a) Cell morphology of neuro-2a (N2a), N2a-M, and N2a-RIP140 cells in the absence (retinoic acid [RA]) and presence of RA (+RA). (b) Bar graphs represent the percentage of neurite-bearing cells for each cell line grown in the absence (−RA) and presence of RA (+RA). (c) Bar graphs represent the numbers of neurites per cell for each cell line grown in the absence (−RA) and presence of RA (+RA). All data are representative of three independent experiments and are shown as mean ± standard deviation (*P < 0.05).
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Figure 3: Receptor-interacting protein 140 (RIP140) overexpression promotes neurite outgrowth. (a) Cell morphology of neuro-2a (N2a), N2a-M, and N2a-RIP140 cells in the absence (retinoic acid [RA]) and presence of RA (+RA). (b) Bar graphs represent the percentage of neurite-bearing cells for each cell line grown in the absence (−RA) and presence of RA (+RA). (c) Bar graphs represent the numbers of neurites per cell for each cell line grown in the absence (−RA) and presence of RA (+RA). All data are representative of three independent experiments and are shown as mean ± standard deviation (*P < 0.05).

Mentions: We observed no significant difference in cell morphology between N2a cells and N2a-M cells in the absence of RA, with scarcely 4% of the cells developing neurites. However, nearly 26% of the N2A-RIP140 cells had already differentiated even prior to the addition of RA [Figure 3a and b]. We also quantified the number of neurites per cell body in each case. N2a-RIP140 showed an increased number of neurites per cell compared to N2A cells and N2A-M cells [Figure 3c].


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)

Receptor-interacting protein 140 (RIP140) overexpression promotes neurite outgrowth. (a) Cell morphology of neuro-2a (N2a), N2a-M, and N2a-RIP140 cells in the absence (retinoic acid [RA]) and presence of RA (+RA). (b) Bar graphs represent the percentage of neurite-bearing cells for each cell line grown in the absence (−RA) and presence of RA (+RA). (c) Bar graphs represent the numbers of neurites per cell for each cell line grown in the absence (−RA) and presence of RA (+RA). All data are representative of three independent experiments and are shown as mean ± standard deviation (*P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Receptor-interacting protein 140 (RIP140) overexpression promotes neurite outgrowth. (a) Cell morphology of neuro-2a (N2a), N2a-M, and N2a-RIP140 cells in the absence (retinoic acid [RA]) and presence of RA (+RA). (b) Bar graphs represent the percentage of neurite-bearing cells for each cell line grown in the absence (−RA) and presence of RA (+RA). (c) Bar graphs represent the numbers of neurites per cell for each cell line grown in the absence (−RA) and presence of RA (+RA). All data are representative of three independent experiments and are shown as mean ± standard deviation (*P < 0.05).
Mentions: We observed no significant difference in cell morphology between N2a cells and N2a-M cells in the absence of RA, with scarcely 4% of the cells developing neurites. However, nearly 26% of the N2A-RIP140 cells had already differentiated even prior to the addition of RA [Figure 3a and b]. We also quantified the number of neurites per cell body in each case. N2a-RIP140 showed an increased number of neurites per cell compared to N2A cells and N2A-M cells [Figure 3c].

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