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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

Western blotting analysis shows receptor-interacting protein 140 (RIP140) protein levels in each cell line. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-RIP140 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments.
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Figure 2: Western blotting analysis shows receptor-interacting protein 140 (RIP140) protein levels in each cell line. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-RIP140 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments.

Mentions: To determine whether RIP140 played a role in neuronal differentiation, we created a stable RIP140-overexpressing N2a cell line. Cells were maintained under antibiotic selection, and by 15−20 days, transfected cells formed individual clones, while untransfected cells all died. RT-qPCR and western blot were used to quantify the mRNA and protein levels of RIP140 expressions. RT-qPCR showed that relative to the parental N2a cells, RIP140 mRNA expression of cells transfected with empty vector was 1.28 ± 0.07 (P = 0.081)-fold higher, while cells transfected with RIP140 overexpression plasmid were 2.38 ± 0.07 (P < 0.01)-fold higher. Western blots further indicated that RIP140 protein expression also increased in cells transfected with RIP140 overexpression plasmid, as compared to the other two control groups [Figure 2]. A single clone from a plate of RIPl40-overexpressing cells was selected for further investigation. We named this cell line N2a-RIP140, and the cell line transfected with empty vector was named N2a-M.


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)

Western blotting analysis shows receptor-interacting protein 140 (RIP140) protein levels in each cell line. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-RIP140 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Western blotting analysis shows receptor-interacting protein 140 (RIP140) protein levels in each cell line. Equal amounts of cell lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-RIP140 or anti-β-actin antibodies. Immunoblot shown is representative of three independent experiments.
Mentions: To determine whether RIP140 played a role in neuronal differentiation, we created a stable RIP140-overexpressing N2a cell line. Cells were maintained under antibiotic selection, and by 15−20 days, transfected cells formed individual clones, while untransfected cells all died. RT-qPCR and western blot were used to quantify the mRNA and protein levels of RIP140 expressions. RT-qPCR showed that relative to the parental N2a cells, RIP140 mRNA expression of cells transfected with empty vector was 1.28 ± 0.07 (P = 0.081)-fold higher, while cells transfected with RIP140 overexpression plasmid were 2.38 ± 0.07 (P < 0.01)-fold higher. Western blots further indicated that RIP140 protein expression also increased in cells transfected with RIP140 overexpression plasmid, as compared to the other two control groups [Figure 2]. A single clone from a plate of RIPl40-overexpressing cells was selected for further investigation. We named this cell line N2a-RIP140, and the cell line transfected with empty vector was named N2a-M.

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