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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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Receptor-interacting protein 140 (RIP140) overexpression promotes neuro-2a (N2a) cell differentiation by activation of ERK1/2. (a) Levels of p-ERK1/2 were detected by western blot in N2a, N2a-M and N2a-RIP140 cells with (+retinoic acid [+RA]) or without (−RA) RA treatment. (b) Cell morphology of N2a-RIP140 cells cultured in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. (c) Bar graphs representing the percentage (%) of neurite-bearing cells of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in the presence or absence of U0126 (10 μmol/L) for 24 hours. (d) Bar graphs represent the numbers of neurites per cell of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. All data are representative of three independent experiments and are shown as mean ± SD.
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Figure 5: Receptor-interacting protein 140 (RIP140) overexpression promotes neuro-2a (N2a) cell differentiation by activation of ERK1/2. (a) Levels of p-ERK1/2 were detected by western blot in N2a, N2a-M and N2a-RIP140 cells with (+retinoic acid [+RA]) or without (−RA) RA treatment. (b) Cell morphology of N2a-RIP140 cells cultured in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. (c) Bar graphs representing the percentage (%) of neurite-bearing cells of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in the presence or absence of U0126 (10 μmol/L) for 24 hours. (d) Bar graphs represent the numbers of neurites per cell of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. All data are representative of three independent experiments and are shown as mean ± SD.

Mentions: Increasing evidence suggests that the activation of ERK1/2 plays an important role in neuronal differentiation.[1112] We thus investigated whether RIP140 overexpression promoted N2a cell differentiation via ERK1/2 activation. Western blot analysis showed that in the absence of RA, compared to N2a and N2a-M cells, N2a-RIP140 cells possessed high levels of p-ERK1/2. Following 24 hours of RA addition, p-ERK1/2 levels of N2a-RIP140 dramatically increased. However, p-ERK1/2 levels in N2a and N2a-M cells following RA treatment were still undetected [Figure 5a].


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 neuro-2a (N2a) cell differentiation by activation of ERK1/2. (a) Levels of p-ERK1/2 were detected by western blot in N2a, N2a-M and N2a-RIP140 cells with (+retinoic acid [+RA]) or without (−RA) RA treatment. (b) Cell morphology of N2a-RIP140 cells cultured in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. (c) Bar graphs representing the percentage (%) of neurite-bearing cells of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in the presence or absence of U0126 (10 μmol/L) for 24 hours. (d) Bar graphs represent the numbers of neurites per cell of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. All data are representative of three independent experiments and are shown as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Receptor-interacting protein 140 (RIP140) overexpression promotes neuro-2a (N2a) cell differentiation by activation of ERK1/2. (a) Levels of p-ERK1/2 were detected by western blot in N2a, N2a-M and N2a-RIP140 cells with (+retinoic acid [+RA]) or without (−RA) RA treatment. (b) Cell morphology of N2a-RIP140 cells cultured in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. (c) Bar graphs representing the percentage (%) of neurite-bearing cells of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in the presence or absence of U0126 (10 μmol/L) for 24 hours. (d) Bar graphs represent the numbers of neurites per cell of N2a-RIP140 cells grown in growth or differentiating media (containing 20 μmol/L RA) in presence or absence of U0126 (10 μmol/L) for 24 hours. All data are representative of three independent experiments and are shown as mean ± SD.
Mentions: Increasing evidence suggests that the activation of ERK1/2 plays an important role in neuronal differentiation.[1112] We thus investigated whether RIP140 overexpression promoted N2a cell differentiation via ERK1/2 activation. Western blot analysis showed that in the absence of RA, compared to N2a and N2a-M cells, N2a-RIP140 cells possessed high levels of p-ERK1/2. Following 24 hours of RA addition, p-ERK1/2 levels of N2a-RIP140 dramatically increased. However, p-ERK1/2 levels in N2a and N2a-M cells following RA treatment were still undetected [Figure 5a].

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