The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.
This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice.NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation.In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as "positive and negative regulation."
Affiliation: From the The Department of Cell Biology, Shandong University School of Medicine, Jinan, China, 250012.
- Apoptosis/drug effects/genetics*
- Endoplasmic Reticulum Stress/drug effects/genetics*
- Inhibitor of Apoptosis Proteins/genetics*/metabolism
- Insulin-Secreting Cells/cytology/drug effects/metabolism*
- Nuclear Receptor Subfamily 4, Group A, Member 1/antagonists & inhibitors/genetics*/metabolism
- RNA, Messenger/antagonists & inhibitors/genetics*/metabolism
- Repressor Proteins/genetics*/metabolism
- Transcription Factor CHOP/genetics*/metabolism
- Base Sequence
- Binding Sites
- Caspase 3/genetics/metabolism
- Endoplasmic Reticulum/drug effects/metabolism
- Gene Expression Regulation
- Mice, Knockout
- Molecular Sequence Data
- Palmitic Acid/pharmacology
- Primary Cell Culture
- Promoter Regions, Genetic
- Protein Binding
- RNA, Small Interfering/genetics/metabolism
- Signal Transduction
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Figure 6: The effect of NR4A1 overexpression on the expression of NF-κB, Bcl-2, and Survivin in MIN6 cells and mouse islets.A, qPCR analysis of NF-κB, Bcl-2, and Survivin expression in NC and OV cells. B, NR4A1, NF-κB, Bcl-2, and Survivin protein levels in NC and OV cells were assessed by Western blotting. C, NR4A1 and Survivin protein expression in MIN6 cells after transient adenoviral expression. D, morphology of mouse islets after Ad-GFP and Ad-NR4A1 infection. Images were acquired using fluorescence microscopy. Both viruses expressed GFP. E, relative mRNA levels of NF-κB, Bcl-2, and Survivin in mouse islets infected with Ad-GFP or Ad-NR4A1 were determined with qPCR. Densitometric analyses of the Western blots are shown as histograms. F, diagram of Survivin promoters of different lengths. G, relative luciferase activity of Survivin promoters of different lengths in NC and OV cells. H, result of ChIP analysis. ChIP was used to pull down DNA fragments associated with exogenous NR4A1 with an HA tag, and a pair of primers was used to amplify the fragment of the Survivin promoter containing an NBRE binding site from the ChIP product. The data show the means of three independent experiments. IP, immunoprecipitate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NC or Ad-GFP.
The qPCR results show that NR4A1 overexpressing MIN6 cells (OV cells) had higher mRNA expression levels of NF-κB, Bcl-2, and Survivin than normal control cells (NC) (Fig. 6A). Western blotting revealed that NF-κB, BCL-2, and Survivin protein levels also increased in OV cells compared with NC cells (Fig. 6B). We found that transient adenoviral overexpression of NR4A1 in MIN6 cells also increased Survivin protein expression (Fig. 6C).