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Unspliced X-box-binding protein 1 (XBP1) protects endothelial cells from oxidative stress through interaction with histone deacetylase 3.

Martin D, Li Y, Yang J, Wang G, Margariti A, Jiang Z, Yu H, Zampetaki A, Hu Y, Xu Q, Zeng L - J. Biol. Chem. (2014)

Bottom Line: In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner.Knockdown of HDAC3 ablated XBP1u-mediated effects.Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

View Article: PubMed Central - PubMed

Affiliation: From the Cardiovascular Division, King's College London, London SE5 9NU, United Kingdom.

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XBP1u protected cell survival under oxidative stress.A, overexpression of XBP1u increased EC survival ex vivo under 50 μmol/liter H2O2. The left panel shows the X-gal staining images, whereas the right panel indicates the relative cell numbers that were defined as cells/mm2 with that of uninfected (CTL)/PBS group set as 1.0. B, overexpression of XBP1u attenuated H2O2-induced cell loss in HUVECs. C, knockdown of XBP1 enhanced H2O2 (20 μmol/liter)-induced cell loss in HUVECs. D, H2O2 induced significant cell apoptosis in XBP1−/− mouse embryonic fibroblasts. The left panel shows the morphology of mouse embryonic fibroblasts isolated from wild type (XBP1+/+) and XBP1- (XBP1−/−) embryos and the PCR strategy to verify the disruption of the XBP1 gene. The right panel indicates the effect of 20 μmol/liter H2O2 on cell apoptosis. Data presented are representatives or average of three independent experiments. *, p < 0.05.
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Figure 2: XBP1u protected cell survival under oxidative stress.A, overexpression of XBP1u increased EC survival ex vivo under 50 μmol/liter H2O2. The left panel shows the X-gal staining images, whereas the right panel indicates the relative cell numbers that were defined as cells/mm2 with that of uninfected (CTL)/PBS group set as 1.0. B, overexpression of XBP1u attenuated H2O2-induced cell loss in HUVECs. C, knockdown of XBP1 enhanced H2O2 (20 μmol/liter)-induced cell loss in HUVECs. D, H2O2 induced significant cell apoptosis in XBP1−/− mouse embryonic fibroblasts. The left panel shows the morphology of mouse embryonic fibroblasts isolated from wild type (XBP1+/+) and XBP1- (XBP1−/−) embryos and the PCR strategy to verify the disruption of the XBP1 gene. The right panel indicates the effect of 20 μmol/liter H2O2 on cell apoptosis. Data presented are representatives or average of three independent experiments. *, p < 0.05.

Mentions: HDAC3 has been demonstrated to protect cells from oxidative stress (19, 25). To assess whether up-regulation of XBP1u has a similar protective effect, arterial segments were isolated from Tie2-LacZ/ApoE−/− mice and infected with Ad- or Ad-XBP1u viruses followed by 50 μmol/liter H2O2 challenge. In these mice, the β-galactosidase is selectively expressed in endothelial cells and some progenitor cells driven by the Tie2 promoter. X-gal staining reveals the endothelium. Overexpression of XBP1u significantly reduced H2O2-induced EC loss from the vessel wall (Fig. 2A), which was further confirmed by in vitro experiments challenging HUVECs with 50 μmol/liter H2O2 (Fig. 2B). In contrast, knockdown of XBP1 via shRNA lentiviral infection slightly increased the basal level of cell apoptosis but significantly augmented H2O2-induced HUVECs apoptosis even at a low concentration (20 μmol/liter) (Fig. 2C). Wild type and XBP1 (XBP1−/−) embryonic fibroblasts were isolated from XBP1+/− cross-bred mouse embryonic day 8.5 embryos and verified by PCR (Fig. 2D). Spontaneously apoptotic cells were higher in XBP1 cells than that in wild type cells (4% versus 1%), which dramatically increased after 20 μmol/liter H2O2 challenge (35% versus 2.5%, Fig. 2E). These results suggest that XBP1u is essential for EC survival especially under oxidative stress.


Unspliced X-box-binding protein 1 (XBP1) protects endothelial cells from oxidative stress through interaction with histone deacetylase 3.

Martin D, Li Y, Yang J, Wang G, Margariti A, Jiang Z, Yu H, Zampetaki A, Hu Y, Xu Q, Zeng L - J. Biol. Chem. (2014)

XBP1u protected cell survival under oxidative stress.A, overexpression of XBP1u increased EC survival ex vivo under 50 μmol/liter H2O2. The left panel shows the X-gal staining images, whereas the right panel indicates the relative cell numbers that were defined as cells/mm2 with that of uninfected (CTL)/PBS group set as 1.0. B, overexpression of XBP1u attenuated H2O2-induced cell loss in HUVECs. C, knockdown of XBP1 enhanced H2O2 (20 μmol/liter)-induced cell loss in HUVECs. D, H2O2 induced significant cell apoptosis in XBP1−/− mouse embryonic fibroblasts. The left panel shows the morphology of mouse embryonic fibroblasts isolated from wild type (XBP1+/+) and XBP1- (XBP1−/−) embryos and the PCR strategy to verify the disruption of the XBP1 gene. The right panel indicates the effect of 20 μmol/liter H2O2 on cell apoptosis. Data presented are representatives or average of three independent experiments. *, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4215241&req=5

Figure 2: XBP1u protected cell survival under oxidative stress.A, overexpression of XBP1u increased EC survival ex vivo under 50 μmol/liter H2O2. The left panel shows the X-gal staining images, whereas the right panel indicates the relative cell numbers that were defined as cells/mm2 with that of uninfected (CTL)/PBS group set as 1.0. B, overexpression of XBP1u attenuated H2O2-induced cell loss in HUVECs. C, knockdown of XBP1 enhanced H2O2 (20 μmol/liter)-induced cell loss in HUVECs. D, H2O2 induced significant cell apoptosis in XBP1−/− mouse embryonic fibroblasts. The left panel shows the morphology of mouse embryonic fibroblasts isolated from wild type (XBP1+/+) and XBP1- (XBP1−/−) embryos and the PCR strategy to verify the disruption of the XBP1 gene. The right panel indicates the effect of 20 μmol/liter H2O2 on cell apoptosis. Data presented are representatives or average of three independent experiments. *, p < 0.05.
Mentions: HDAC3 has been demonstrated to protect cells from oxidative stress (19, 25). To assess whether up-regulation of XBP1u has a similar protective effect, arterial segments were isolated from Tie2-LacZ/ApoE−/− mice and infected with Ad- or Ad-XBP1u viruses followed by 50 μmol/liter H2O2 challenge. In these mice, the β-galactosidase is selectively expressed in endothelial cells and some progenitor cells driven by the Tie2 promoter. X-gal staining reveals the endothelium. Overexpression of XBP1u significantly reduced H2O2-induced EC loss from the vessel wall (Fig. 2A), which was further confirmed by in vitro experiments challenging HUVECs with 50 μmol/liter H2O2 (Fig. 2B). In contrast, knockdown of XBP1 via shRNA lentiviral infection slightly increased the basal level of cell apoptosis but significantly augmented H2O2-induced HUVECs apoptosis even at a low concentration (20 μmol/liter) (Fig. 2C). Wild type and XBP1 (XBP1−/−) embryonic fibroblasts were isolated from XBP1+/− cross-bred mouse embryonic day 8.5 embryos and verified by PCR (Fig. 2D). Spontaneously apoptotic cells were higher in XBP1 cells than that in wild type cells (4% versus 1%), which dramatically increased after 20 μmol/liter H2O2 challenge (35% versus 2.5%, Fig. 2E). These results suggest that XBP1u is essential for EC survival especially under oxidative stress.

Bottom Line: In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner.Knockdown of HDAC3 ablated XBP1u-mediated effects.Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

View Article: PubMed Central - PubMed

Affiliation: From the Cardiovascular Division, King's College London, London SE5 9NU, United Kingdom.

Show MeSH
Related in: MedlinePlus