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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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XBP1 was essential for disturbed flow-induced up-regulation of HO-1.A, overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad-XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad- was included to compensate the MOI. B, overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad-XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad- was included as control. FLAG indicates the exogenous XBP1u. C, knockdown of XBP1 via shRNA lentivirus (XBP1sh) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. D, knockdown of XBP1 via shRNA lentivirus (XBP1sh) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. E, Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA (CTLsi) or Nrf2 siRNA (Nrf2si) for 72 h, followed by disturbed flow for 4 h. F, flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D (AD) or 30 mg/liter cycloheximide (CH) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO (DM) was included as vehicle control. G, AZD2014 abolished Ad-XBP1u (X1u) or Ad-HDAC3 (HD3)-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H, AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR (red) and anti-pAkt Ser-473 (green) antibodies. I, AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR (red) and anti-Nrf2 (green) or pAkt Ser-473 (green) antibodies. Data presented are representatives of three independent experiments.
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Figure 4: XBP1 was essential for disturbed flow-induced up-regulation of HO-1.A, overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad-XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad- was included to compensate the MOI. B, overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad-XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad- was included as control. FLAG indicates the exogenous XBP1u. C, knockdown of XBP1 via shRNA lentivirus (XBP1sh) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. D, knockdown of XBP1 via shRNA lentivirus (XBP1sh) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. E, Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA (CTLsi) or Nrf2 siRNA (Nrf2si) for 72 h, followed by disturbed flow for 4 h. F, flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D (AD) or 30 mg/liter cycloheximide (CH) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO (DM) was included as vehicle control. G, AZD2014 abolished Ad-XBP1u (X1u) or Ad-HDAC3 (HD3)-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H, AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR (red) and anti-pAkt Ser-473 (green) antibodies. I, AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR (red) and anti-Nrf2 (green) or pAkt Ser-473 (green) antibodies. Data presented are representatives of three independent experiments.

Mentions: The PI3K/Akt pathway plays a key role in HO-1 expression (31). Our previous study has demonstrated that overexpression of HDAC3 increases Akt phosphorylation (19). In this study, Western blot analysis revealed that overexpression of XBP1u induced simultaneous increase in Akt phosphorylation and HO-1 protein in a dose- and time-dependent manner (Fig. 4, A and B). Knockdown of XBP1 decreased the basal level of Akt phosphorylation and HO-1 protein (Fig. 4C). Disturbed flow is reported to activate HO-1 expression (32). We also detected the up-regulation of HO-1, Nrf2, and Akt1 phosphorylation by disturbed flow (Fig. 4D). As expected, these effects were totally abolished by XBP1 knockdown via shRNA lentiviral infection (Fig. 4D). Further experiments confirmed that Nrf2 was necessary for flow-induced HO-1 up-regulation, as siRNA-mediated knockdown of Nrf2 abolished flow-induced HO-1 expression (Fig. 4E). The addition of the transcription inhibitor (actinomycin D) or translation inhibitor (cycloheximide) also abolished flow-induced HO-1 up-regulation (Fig. 4F). The addition of actinomycin D and especially cycloheximide reduced the basal level of Nrf2. However, disturbed flow still up-regulated Nrf2 at the protein level (Fig. 4F). These results suggest that the increase in observed Nrf2 protein is due to post-translational modification, whereas the increase in observed HO-1 protein is due to de novo biosynthesis. The phosphorylation of the Ser-473 site in Akt1 protein is reported to be activated by the Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR complex (mTORC2) (33, 34). To test whether XBP1u or HDAC3 induced Akt1 phosphorylation in a similar manner, the Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR complex inhibitor, AZD2014 (35) was added to Ad-XBP1u or Ad-HDAC3-infected cells. Cellular fractionation was performed to analyze Akt1 phosphorylation and Nrf2 nuclear translocation. Overexpression of XBP1u or HDAC3 increased Akt1 Ser-473 phosphorylation, the nuclear translocation of phosphorylated Akt1 and Nrf2 and up-regulated HO-1 (Fig. 4G). However, in the presence of 5 μmol/liter of AZD2014, all of these effects were diminished (Fig. 4G). The presence of XBP1u or HDAC3-induced pAkt1 Ser-473 in the nucleus was confirmed by immunofluorescence staining. This was significantly attenuated by AZD2014 (Fig. 4H). The presence of AZD2014 also abolished flow-induced Nrf2 nuclear translocation (Fig. 4I). These results suggest that XBP1 is essential for basal and disturbed flow-induced HO-1 expression via regulation of the Akt1/Nrf2 pathway in a Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR dependent manner.


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)

XBP1 was essential for disturbed flow-induced up-regulation of HO-1.A, overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad-XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad- was included to compensate the MOI. B, overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad-XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad- was included as control. FLAG indicates the exogenous XBP1u. C, knockdown of XBP1 via shRNA lentivirus (XBP1sh) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. D, knockdown of XBP1 via shRNA lentivirus (XBP1sh) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. E, Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA (CTLsi) or Nrf2 siRNA (Nrf2si) for 72 h, followed by disturbed flow for 4 h. F, flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D (AD) or 30 mg/liter cycloheximide (CH) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO (DM) was included as vehicle control. G, AZD2014 abolished Ad-XBP1u (X1u) or Ad-HDAC3 (HD3)-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H, AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR (red) and anti-pAkt Ser-473 (green) antibodies. I, AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR (red) and anti-Nrf2 (green) or pAkt Ser-473 (green) antibodies. Data presented are representatives of three independent experiments.
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Figure 4: XBP1 was essential for disturbed flow-induced up-regulation of HO-1.A, overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad-XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad- was included to compensate the MOI. B, overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad-XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad- was included as control. FLAG indicates the exogenous XBP1u. C, knockdown of XBP1 via shRNA lentivirus (XBP1sh) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. D, knockdown of XBP1 via shRNA lentivirus (XBP1sh) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. E, Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA (CTLsi) or Nrf2 siRNA (Nrf2si) for 72 h, followed by disturbed flow for 4 h. F, flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D (AD) or 30 mg/liter cycloheximide (CH) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO (DM) was included as vehicle control. G, AZD2014 abolished Ad-XBP1u (X1u) or Ad-HDAC3 (HD3)-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H, AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad- or Ad-XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR (red) and anti-pAkt Ser-473 (green) antibodies. I, AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR (red) and anti-Nrf2 (green) or pAkt Ser-473 (green) antibodies. Data presented are representatives of three independent experiments.
Mentions: The PI3K/Akt pathway plays a key role in HO-1 expression (31). Our previous study has demonstrated that overexpression of HDAC3 increases Akt phosphorylation (19). In this study, Western blot analysis revealed that overexpression of XBP1u induced simultaneous increase in Akt phosphorylation and HO-1 protein in a dose- and time-dependent manner (Fig. 4, A and B). Knockdown of XBP1 decreased the basal level of Akt phosphorylation and HO-1 protein (Fig. 4C). Disturbed flow is reported to activate HO-1 expression (32). We also detected the up-regulation of HO-1, Nrf2, and Akt1 phosphorylation by disturbed flow (Fig. 4D). As expected, these effects were totally abolished by XBP1 knockdown via shRNA lentiviral infection (Fig. 4D). Further experiments confirmed that Nrf2 was necessary for flow-induced HO-1 up-regulation, as siRNA-mediated knockdown of Nrf2 abolished flow-induced HO-1 expression (Fig. 4E). The addition of the transcription inhibitor (actinomycin D) or translation inhibitor (cycloheximide) also abolished flow-induced HO-1 up-regulation (Fig. 4F). The addition of actinomycin D and especially cycloheximide reduced the basal level of Nrf2. However, disturbed flow still up-regulated Nrf2 at the protein level (Fig. 4F). These results suggest that the increase in observed Nrf2 protein is due to post-translational modification, whereas the increase in observed HO-1 protein is due to de novo biosynthesis. The phosphorylation of the Ser-473 site in Akt1 protein is reported to be activated by the Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR complex (mTORC2) (33, 34). To test whether XBP1u or HDAC3 induced Akt1 phosphorylation in a similar manner, the Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR complex inhibitor, AZD2014 (35) was added to Ad-XBP1u or Ad-HDAC3-infected cells. Cellular fractionation was performed to analyze Akt1 phosphorylation and Nrf2 nuclear translocation. Overexpression of XBP1u or HDAC3 increased Akt1 Ser-473 phosphorylation, the nuclear translocation of phosphorylated Akt1 and Nrf2 and up-regulated HO-1 (Fig. 4G). However, in the presence of 5 μmol/liter of AZD2014, all of these effects were diminished (Fig. 4G). The presence of XBP1u or HDAC3-induced pAkt1 Ser-473 in the nucleus was confirmed by immunofluorescence staining. This was significantly attenuated by AZD2014 (Fig. 4H). The presence of AZD2014 also abolished flow-induced Nrf2 nuclear translocation (Fig. 4I). These results suggest that XBP1 is essential for basal and disturbed flow-induced HO-1 expression via regulation of the Akt1/Nrf2 pathway in a Rapamycin-insensitive companion of mammalian target of rapamycin-mTOR dependent manner.

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