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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 role of NR4A1 on β-cell apoptosis.A, Western blotting verification of MIN6 cell lines stably overexpressing NR4A1 (designated as OV) and normal control clones (designated as NC). B and C, NC and OV MIN6 cell viability upon TG treatment (B) or PA treatment (C) for various time periods was analyzed using MTT assays. D, NC and OV cells were treated with 0.5 μm TG for 24 h. Then the percentage of apoptotic cells was analyzed by TUNEL staining. In this representative image, the cells stained red are apoptotic. E, the localization analysis of NR4A1 in OV cells. OV cells were treated with 0.5 μm TG for various lengths of time. Nuclear (N) and cytoplasmic (C) proteins were separated with a protein extraction kit as described under “Experimental Procedures.” GAPDH is a marker for the cytoplasm, and lamin A is a marker for the nucleus. Densitometric analyses of the Western blots and cell death rate are shown as histograms. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NC.
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Figure 3: The role of NR4A1 on β-cell apoptosis.A, Western blotting verification of MIN6 cell lines stably overexpressing NR4A1 (designated as OV) and normal control clones (designated as NC). B and C, NC and OV MIN6 cell viability upon TG treatment (B) or PA treatment (C) for various time periods was analyzed using MTT assays. D, NC and OV cells were treated with 0.5 μm TG for 24 h. Then the percentage of apoptotic cells was analyzed by TUNEL staining. In this representative image, the cells stained red are apoptotic. E, the localization analysis of NR4A1 in OV cells. OV cells were treated with 0.5 μm TG for various lengths of time. Nuclear (N) and cytoplasmic (C) proteins were separated with a protein extraction kit as described under “Experimental Procedures.” GAPDH is a marker for the cytoplasm, and lamin A is a marker for the nucleus. Densitometric analyses of the Western blots and cell death rate are shown as histograms. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NC.

Mentions: It is well known that sustained ER stress can ultimately lead to apoptosis. To investigate whether NR4A1 plays a role in ER stress-induced apoptosis in MIN6 cells, we constructed a lentiviral vector encoding NR4A1 in which both NR4A1 and GFP are expressed. We infected MIN6 cells with the NR4A1-expressing lentivirus or control lentivirus (lentivirus only expressing GFP) and obtained several NR4A1 OV and NC clones after puromycin selection. We selected one pair of these clones for further experiments. Other clones were also tested to exclude the biased conclusion due to the cloning variation. Upon examining the NR4A1 protein levels in OV cells and NC cells (Fig. 3A), we did not observe any significant changes in morphology between OV cells and NC cells (data not shown). However, when the OV cells and NC cells were treated with 0.5 μm TG or 0.4 mm PA, the number of NC cells decreased significantly, whereas the number of OV cells decreased more slowly. The cell survival rates detected using MTT revealed that OV cells were more viable than NC cells after treatment with TG (Fig. 3B) or PA (Fig. 3C) over the series of time points examined, and this was consistent with TUNEL assays (Fig. 3D).


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 role of NR4A1 on β-cell apoptosis.A, Western blotting verification of MIN6 cell lines stably overexpressing NR4A1 (designated as OV) and normal control clones (designated as NC). B and C, NC and OV MIN6 cell viability upon TG treatment (B) or PA treatment (C) for various time periods was analyzed using MTT assays. D, NC and OV cells were treated with 0.5 μm TG for 24 h. Then the percentage of apoptotic cells was analyzed by TUNEL staining. In this representative image, the cells stained red are apoptotic. E, the localization analysis of NR4A1 in OV cells. OV cells were treated with 0.5 μm TG for various lengths of time. Nuclear (N) and cytoplasmic (C) proteins were separated with a protein extraction kit as described under “Experimental Procedures.” GAPDH is a marker for the cytoplasm, and lamin A is a marker for the nucleus. Densitometric analyses of the Western blots and cell death rate are shown as histograms. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: The role of NR4A1 on β-cell apoptosis.A, Western blotting verification of MIN6 cell lines stably overexpressing NR4A1 (designated as OV) and normal control clones (designated as NC). B and C, NC and OV MIN6 cell viability upon TG treatment (B) or PA treatment (C) for various time periods was analyzed using MTT assays. D, NC and OV cells were treated with 0.5 μm TG for 24 h. Then the percentage of apoptotic cells was analyzed by TUNEL staining. In this representative image, the cells stained red are apoptotic. E, the localization analysis of NR4A1 in OV cells. OV cells were treated with 0.5 μm TG for various lengths of time. Nuclear (N) and cytoplasmic (C) proteins were separated with a protein extraction kit as described under “Experimental Procedures.” GAPDH is a marker for the cytoplasm, and lamin A is a marker for the nucleus. Densitometric analyses of the Western blots and cell death rate are shown as histograms. The data show the means of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NC.
Mentions: It is well known that sustained ER stress can ultimately lead to apoptosis. To investigate whether NR4A1 plays a role in ER stress-induced apoptosis in MIN6 cells, we constructed a lentiviral vector encoding NR4A1 in which both NR4A1 and GFP are expressed. We infected MIN6 cells with the NR4A1-expressing lentivirus or control lentivirus (lentivirus only expressing GFP) and obtained several NR4A1 OV and NC clones after puromycin selection. We selected one pair of these clones for further experiments. Other clones were also tested to exclude the biased conclusion due to the cloning variation. Upon examining the NR4A1 protein levels in OV cells and NC cells (Fig. 3A), we did not observe any significant changes in morphology between OV cells and NC cells (data not shown). However, when the OV cells and NC cells were treated with 0.5 μm TG or 0.4 mm PA, the number of NC cells decreased significantly, whereas the number of OV cells decreased more slowly. The cell survival rates detected using MTT revealed that OV cells were more viable than NC cells after treatment with TG (Fig. 3B) or PA (Fig. 3C) over the series of time points examined, and this was consistent with TUNEL assays (Fig. 3D).

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