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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) differentiation into Tuj1-positive neurons. (a) Bar graphs showing mRNA levels of Tuj1 expressed in N2a-M and N2a-RIP140 cells, relative to N2a parental cells, in the absence (retinoic acid [RA]) and presence of RA (+RA). Data were normalized to β-actin mRNA, and differences were considered significant when P < 0.05. (b) Western blotting analysis showing Tuj1 protein levels under each condition for each cell line. Samples were collected after 24 hours RA treatment. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-Tuj1 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments. (c) Detection of Tuj1 (red) by immunocytochemistry in N2a, N2a-M and N2a-RIP140 cells treated with (+RA) or without (−RA) RA. Blue color corresponds to DAPI (*P < 0.05).
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Figure 4: Receptor-interacting protein 140 (RIP140) overexpression promotes neuro-2a (N2a) differentiation into Tuj1-positive neurons. (a) Bar graphs showing mRNA levels of Tuj1 expressed in N2a-M and N2a-RIP140 cells, relative to N2a parental cells, in the absence (retinoic acid [RA]) and presence of RA (+RA). Data were normalized to β-actin mRNA, and differences were considered significant when P < 0.05. (b) Western blotting analysis showing Tuj1 protein levels under each condition for each cell line. Samples were collected after 24 hours RA treatment. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-Tuj1 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments. (c) Detection of Tuj1 (red) by immunocytochemistry in N2a, N2a-M and N2a-RIP140 cells treated with (+RA) or without (−RA) RA. Blue color corresponds to DAPI (*P < 0.05).

Mentions: Because Tuj1 is one of the earliest cytoskeletal proteins specifically associated with neuronal development, it is widely used as a specific differentiation marker.[710] To further confirm the stimulatory effect of RIP140 overexpression on neuronal differentiation, we measured Tuj1 expression levels by RT-qPCR, western blot, and immunofluorescence staining. Figure 4a and b shows that both the mRNA and protein levels of Tuj1 were enhanced significantly (P < 0.05) in N2a-RIP140 cells, as compared to N2a cells and N2a-M cells, regardless of the absence or presence of RA.


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) differentiation into Tuj1-positive neurons. (a) Bar graphs showing mRNA levels of Tuj1 expressed in N2a-M and N2a-RIP140 cells, relative to N2a parental cells, in the absence (retinoic acid [RA]) and presence of RA (+RA). Data were normalized to β-actin mRNA, and differences were considered significant when P < 0.05. (b) Western blotting analysis showing Tuj1 protein levels under each condition for each cell line. Samples were collected after 24 hours RA treatment. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-Tuj1 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments. (c) Detection of Tuj1 (red) by immunocytochemistry in N2a, N2a-M and N2a-RIP140 cells treated with (+RA) or without (−RA) RA. Blue color corresponds to DAPI (*P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Receptor-interacting protein 140 (RIP140) overexpression promotes neuro-2a (N2a) differentiation into Tuj1-positive neurons. (a) Bar graphs showing mRNA levels of Tuj1 expressed in N2a-M and N2a-RIP140 cells, relative to N2a parental cells, in the absence (retinoic acid [RA]) and presence of RA (+RA). Data were normalized to β-actin mRNA, and differences were considered significant when P < 0.05. (b) Western blotting analysis showing Tuj1 protein levels under each condition for each cell line. Samples were collected after 24 hours RA treatment. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-Tuj1 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments. (c) Detection of Tuj1 (red) by immunocytochemistry in N2a, N2a-M and N2a-RIP140 cells treated with (+RA) or without (−RA) RA. Blue color corresponds to DAPI (*P < 0.05).
Mentions: Because Tuj1 is one of the earliest cytoskeletal proteins specifically associated with neuronal development, it is widely used as a specific differentiation marker.[710] To further confirm the stimulatory effect of RIP140 overexpression on neuronal differentiation, we measured Tuj1 expression levels by RT-qPCR, western blot, and immunofluorescence staining. Figure 4a and b shows that both the mRNA and protein levels of Tuj1 were enhanced significantly (P < 0.05) in N2a-RIP140 cells, as compared to N2a cells and N2a-M cells, regardless of the absence or presence of RA.

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