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Published on: April 22, 2017
[Mechanisms regulating p21 gene expression by retinoic acid-induced gene G protein]
Qingping Zou1, Guiping Xu1, Likun Zhuang1
1Faculty of Medical Laboratory Science, State Key Laboratory of Medical Genomics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
Objective:
To investigate the mechanisms by which retinoic acid-induced gene G (RIG-G) protein regulates p21 gene expression.
Methods:
Western blot was used to detect the effects of RIG-G protein overexpression on p21 protein expression level in leukemia cell line NB4 cells and the phosphorylation of both c-Jun and JNK in U937 cells. The c-Jun expression plasmid and p21 gene promoter-containing reporter plasmid were co-transfected into 293T cells, to explore the regulatory effect of c-Jun protein on p21 gene expression by luciferase reporter assay.
Results:
Western blot showed that the overexpression of RIG-G protein significantly upregulated p21 protein level in the NB4 cells, and the level of p21 protein largely increased along with the induction of endogenous RIG-G protein during the differentiation of NB4 cells treated by all-trans retinoic acid (ATRA). Moreover, the phosphorylation of both c-Jun and JNK decreased in RIG-G-overexpressing U937 cells while total c-Jun and JNK proteins remained unchanged. After using the JNK inhibitor SP600125 to block JNK phosphorylation, the level of c-Jun phosphorylation was still dramatically reduced in the RIG-G-overexpressing U937T-RIG-G cells, compared with the control U937T-pTRE cells. These results indicated that the inhibitory effect of Rig-G protein on c-Jun phosphorylation could not only be through the JNK pathway, but also via some JNK-independent pathways. Luciferase reporter assay showed that when 0.1, 0.5, 1.0 and 2.0 µg c-Jun-expressing plasmids were respectively transfected into 293T cells, compared with the empty vector-transfected group, the relative luciferase activities were (83.0 ± 1.7)%, (73.7 ± 0.7)%, (68.9 ± 0.9)% and (64.1 ± 0.9)%, indicating that the transcriptional activity of p21 gene could be inhibited by c-Jun protein.
Conclusions:
RIG-G protein may suppress the phosphorylation of c-Jun protein through different signal pathways, thereby increasing the expression of p21 gene, arresting the cell cycle and inhibiting the cell growth in U937 cells.
Insights
Retinoic acid-induced gene G (RIG-G) protein upregulates p21 expression by inhibiting c-Jun phosphorylation through both JNK-dependent and independent pathways. This mechanism arrests the cell cycle and inhibits cell growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Retinoic acid is crucial for cell differentiation and has implications in cancer therapy.
- The p21 gene is a key regulator of cell cycle arrest and is often dysregulated in cancer.
- Understanding the regulatory mechanisms of p21 expression is vital for developing targeted cancer treatments.
Purpose of the Study:
- To elucidate the molecular mechanisms by which retinoic acid-induced gene G (RIG-G) protein influences p21 gene expression.
- To investigate the role of RIG-G protein in regulating c-Jun and JNK phosphorylation.
- To determine the impact of RIG-G on the transcriptional activity of the p21 gene promoter.
Main Methods:
- Western blotting was employed to assess protein levels of RIG-G, p21, c-Jun, and JNK, as well as the phosphorylation status of c-Jun and JNK in NB4 and U937 cell lines.
- Luciferase reporter assays were conducted to evaluate the effect of c-Jun on p21 gene promoter activity in 293T cells.
- Pharmacological inhibition of JNK phosphorylation was utilized to differentiate between JNK-dependent and independent pathways.
Main Results:
- Overexpression of RIG-G protein significantly increased p21 protein levels in NB4 cells.
- RIG-G overexpression led to decreased phosphorylation of c-Jun and JNK in U937 cells, independent of total protein levels.
- Luciferase assays demonstrated that c-Jun protein inhibits the transcriptional activity of the p21 gene promoter.
Conclusions:
- RIG-G protein suppresses c-Jun phosphorylation via multiple signaling pathways, including JNK-dependent and independent routes.
- The observed downregulation of c-Jun phosphorylation by RIG-G results in increased p21 gene expression.
- This RIG-G-mediated regulation of p21 contributes to cell cycle arrest and inhibition of cell proliferation.
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