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The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

Yu C, Cui S, Zong C, Gao W, Xu T, Gao P, Chen J, Qin D, Guan Q, Liu Y, Fu Y, Li X, Wang X - J. Biol. Chem. (2015)

Bottom Line: 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."

View Article: PubMed Central - PubMed

Affiliation: From the The Department of Cell Biology, Shandong University School of Medicine, Jinan, China, 250012.

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The effects of NR4A1 knockdown on the expression of Survivin and Bcl-2 and on ER stress-induced apoptosis in MIN6 cells. Analyses of MIN6 cell stably knockdown NR4A1 clone (designated as KD) and normal control clone (designated as CON). A, protein expression of NR4A1, Bcl-2, and Survivin in CON and KD cells. B and C, the cell viability of CON and KD cells treated with TG (B) or PA (C) for 24 h was analyzed with an MTT assay. D, changes in protein expression of NR4A1, phosphorylated eif2α (P-eif2α), CHOP, and Caspase3 (17 kDa) in CON and KD cells treated with 0.5 μm TG. Densitometric analyses of the Western blots are shown as histograms or curves. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus CON.
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Figure 7: The effects of NR4A1 knockdown on the expression of Survivin and Bcl-2 and on ER stress-induced apoptosis in MIN6 cells. Analyses of MIN6 cell stably knockdown NR4A1 clone (designated as KD) and normal control clone (designated as CON). A, protein expression of NR4A1, Bcl-2, and Survivin in CON and KD cells. B and C, the cell viability of CON and KD cells treated with TG (B) or PA (C) for 24 h was analyzed with an MTT assay. D, changes in protein expression of NR4A1, phosphorylated eif2α (P-eif2α), CHOP, and Caspase3 (17 kDa) in CON and KD cells treated with 0.5 μm TG. Densitometric analyses of the Western blots are shown as histograms or curves. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus CON.

Mentions: To further confirm the above observations, we infected MIN6 cells with a lentivirus encoding siRNA against mouse NR4A1 or with a control lentivirus and selected cell clones using puromycin. We obtained several NR4A1 knockdown clones (KD) in which the NR4A1 expression level was <70% that in control cells (CON), and chose one pair of them as representation (Fig. 7A). Other clones were also tested to exclude false conclusion due to cloning variation. KD cells also exhibited reduced Survivin and Bcl-2 protein expression compared with CON cells (Fig. 7A). After TG or PA treatment of CON and KD cells, MTT results show that the viability of KD cells was reduced compared with CON cells (Fig. 7, B and C). Western blotting results showed that eif2α phosphorylation or activation increased in KD cells upon TG treatment, TG-induced CHOP protein expression was much higher in KD cells, and TG-induced active Caspase3 (17 kDa) was also increased in KD cells compared with CON cells (Fig. 7D).


The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

Yu C, Cui S, Zong C, Gao W, Xu T, Gao P, Chen J, Qin D, Guan Q, Liu Y, Fu Y, Li X, Wang X - J. Biol. Chem. (2015)

The effects of NR4A1 knockdown on the expression of Survivin and Bcl-2 and on ER stress-induced apoptosis in MIN6 cells. Analyses of MIN6 cell stably knockdown NR4A1 clone (designated as KD) and normal control clone (designated as CON). A, protein expression of NR4A1, Bcl-2, and Survivin in CON and KD cells. B and C, the cell viability of CON and KD cells treated with TG (B) or PA (C) for 24 h was analyzed with an MTT assay. D, changes in protein expression of NR4A1, phosphorylated eif2α (P-eif2α), CHOP, and Caspase3 (17 kDa) in CON and KD cells treated with 0.5 μm TG. Densitometric analyses of the Western blots are shown as histograms or curves. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus CON.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543630&req=5

Figure 7: The effects of NR4A1 knockdown on the expression of Survivin and Bcl-2 and on ER stress-induced apoptosis in MIN6 cells. Analyses of MIN6 cell stably knockdown NR4A1 clone (designated as KD) and normal control clone (designated as CON). A, protein expression of NR4A1, Bcl-2, and Survivin in CON and KD cells. B and C, the cell viability of CON and KD cells treated with TG (B) or PA (C) for 24 h was analyzed with an MTT assay. D, changes in protein expression of NR4A1, phosphorylated eif2α (P-eif2α), CHOP, and Caspase3 (17 kDa) in CON and KD cells treated with 0.5 μm TG. Densitometric analyses of the Western blots are shown as histograms or curves. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus CON.
Mentions: To further confirm the above observations, we infected MIN6 cells with a lentivirus encoding siRNA against mouse NR4A1 or with a control lentivirus and selected cell clones using puromycin. We obtained several NR4A1 knockdown clones (KD) in which the NR4A1 expression level was <70% that in control cells (CON), and chose one pair of them as representation (Fig. 7A). Other clones were also tested to exclude false conclusion due to cloning variation. KD cells also exhibited reduced Survivin and Bcl-2 protein expression compared with CON cells (Fig. 7A). After TG or PA treatment of CON and KD cells, MTT results show that the viability of KD cells was reduced compared with CON cells (Fig. 7, B and C). Western blotting results showed that eif2α phosphorylation or activation increased in KD cells upon TG treatment, TG-induced CHOP protein expression was much higher in KD cells, and TG-induced active Caspase3 (17 kDa) was also increased in KD cells compared with CON cells (Fig. 7D).

Bottom Line: 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."

View Article: PubMed Central - PubMed

Affiliation: From the The Department of Cell Biology, Shandong University School of Medicine, Jinan, China, 250012.

Show MeSH
Related in: MedlinePlus