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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 physically interacted with HDAC3.A, XBP1u and HDAC3 synergistically activated HO-1 expression. HUVECs were co-infected with Ad-XBP1u and Ad-HDAC3 at 10 MOI each for 24 h, followed by Western blot analysis. Ad- virus was included as control and to compensate the MOI. FLAG antibody was used to detect exogenous XBP1u and HDAC3. B, knockdown of HDAC3 via shRNA lentivirus (HDAC3sh) attenuated Ad-XBP1u-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. C, XBP1u physically interacted with HDAC3. HEK293 cells were co-transfected with HA-XBP1u and FLAG-HDAC3 plasmids, followed by immunoprecipitation with anti-HA antibody and Western blot analysis with anti-FLAG and anti-HA antibodies. D, XBP1u bound to amino acid 201–323 region in HDAC3 molecule. The left panel indicates the schematic illustration of HDAC3 truncated mutants. The right panel shows the interaction of XBP1u and truncated HDAC3 as revealed by immunoprecipitation assays. E, disturbed flow increased XBP1u association with HDAC3/Akt1. Co-immunoprecipitation with anti-XBP1u antibody was performed on static and disturbed flow (4 h)-treated cells, followed by Western blot with anti-HDAC3 or Akt1 and anti-XBP1u antibodies. F, disturbed flow induced mTOR/Akt1/HDAC3/XBP1u complex formation in the cytoplasm. Double immunofluorescence staining was performed on static and disturbed flow (4 h)-treated cells. Antibodies are indicated with red or green letters reflecting the color in the images. Data presented are representatives of three independent experiments.
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Figure 5: XBP1 physically interacted with HDAC3.A, XBP1u and HDAC3 synergistically activated HO-1 expression. HUVECs were co-infected with Ad-XBP1u and Ad-HDAC3 at 10 MOI each for 24 h, followed by Western blot analysis. Ad- virus was included as control and to compensate the MOI. FLAG antibody was used to detect exogenous XBP1u and HDAC3. B, knockdown of HDAC3 via shRNA lentivirus (HDAC3sh) attenuated Ad-XBP1u-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. C, XBP1u physically interacted with HDAC3. HEK293 cells were co-transfected with HA-XBP1u and FLAG-HDAC3 plasmids, followed by immunoprecipitation with anti-HA antibody and Western blot analysis with anti-FLAG and anti-HA antibodies. D, XBP1u bound to amino acid 201–323 region in HDAC3 molecule. The left panel indicates the schematic illustration of HDAC3 truncated mutants. The right panel shows the interaction of XBP1u and truncated HDAC3 as revealed by immunoprecipitation assays. E, disturbed flow increased XBP1u association with HDAC3/Akt1. Co-immunoprecipitation with anti-XBP1u antibody was performed on static and disturbed flow (4 h)-treated cells, followed by Western blot with anti-HDAC3 or Akt1 and anti-XBP1u antibodies. F, disturbed flow induced mTOR/Akt1/HDAC3/XBP1u complex formation in the cytoplasm. Double immunofluorescence staining was performed on static and disturbed flow (4 h)-treated cells. Antibodies are indicated with red or green letters reflecting the color in the images. Data presented are representatives of three independent experiments.

Mentions: As described above, both XBP1u and HDAC3 up-regulate HO-1 expression, whereas flow-induced HDAC3 is XBP1-dependent. Therefore, we hypothesized that there was cross-talk between XBP1u and HDAC3 during the regulation of HO-1. To test this, co-expression of HDAC3 and XBP1u was first introduced into HUVECs by co-infection with two viruses. As shown in Fig. 5A, overexpression of either XBP1u or HDAC3 alone up-regulated Akt1 phosphorylation, Nrf2 and HO-1, whereas co-expression of XBP1u and HDAC3 had a synergistic effect. Further experiments revealed that knockdown of HDAC3 attenuated XBP1u-induced Akt1 phosphorylation and HO-1 expression (Fig. 5B). Co-immunoprecipitation assays revealed that XBP1u physically bound to HDAC3 in transfected cells (Fig. 5C). Using truncated HDAC3 mutants, the binding domain in HDAC3 molecule could be defined to the amino acid 201∼323 region (Fig. 5D). Immunoprecipitation with antibody against endogenous XBP1u revealed that XBP1u bound to HDAC3 and Akt1 under disturbed flow (Fig. 5E). Double immunofluorescence staining showed that mTOR/Akt1, Akt1/HDAC3, Akt1/XBP1u, and HDAC3/XBP1u co-localized in the cytoplasm (Fig. 5F). These results suggest XBP1u/HDAC3/Akt1/mTOR may form a complex to regulate Akt1 phosphorylation, leading to Nrf2 stabilization and HO-1 expression.


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 physically interacted with HDAC3.A, XBP1u and HDAC3 synergistically activated HO-1 expression. HUVECs were co-infected with Ad-XBP1u and Ad-HDAC3 at 10 MOI each for 24 h, followed by Western blot analysis. Ad- virus was included as control and to compensate the MOI. FLAG antibody was used to detect exogenous XBP1u and HDAC3. B, knockdown of HDAC3 via shRNA lentivirus (HDAC3sh) attenuated Ad-XBP1u-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. C, XBP1u physically interacted with HDAC3. HEK293 cells were co-transfected with HA-XBP1u and FLAG-HDAC3 plasmids, followed by immunoprecipitation with anti-HA antibody and Western blot analysis with anti-FLAG and anti-HA antibodies. D, XBP1u bound to amino acid 201–323 region in HDAC3 molecule. The left panel indicates the schematic illustration of HDAC3 truncated mutants. The right panel shows the interaction of XBP1u and truncated HDAC3 as revealed by immunoprecipitation assays. E, disturbed flow increased XBP1u association with HDAC3/Akt1. Co-immunoprecipitation with anti-XBP1u antibody was performed on static and disturbed flow (4 h)-treated cells, followed by Western blot with anti-HDAC3 or Akt1 and anti-XBP1u antibodies. F, disturbed flow induced mTOR/Akt1/HDAC3/XBP1u complex formation in the cytoplasm. Double immunofluorescence staining was performed on static and disturbed flow (4 h)-treated cells. Antibodies are indicated with red or green letters reflecting the color in the images. Data presented are representatives of three independent experiments.
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Figure 5: XBP1 physically interacted with HDAC3.A, XBP1u and HDAC3 synergistically activated HO-1 expression. HUVECs were co-infected with Ad-XBP1u and Ad-HDAC3 at 10 MOI each for 24 h, followed by Western blot analysis. Ad- virus was included as control and to compensate the MOI. FLAG antibody was used to detect exogenous XBP1u and HDAC3. B, knockdown of HDAC3 via shRNA lentivirus (HDAC3sh) attenuated Ad-XBP1u-induced HO-1 expression. Non-target shRNA lentivirus (NTsh) was included as control. C, XBP1u physically interacted with HDAC3. HEK293 cells were co-transfected with HA-XBP1u and FLAG-HDAC3 plasmids, followed by immunoprecipitation with anti-HA antibody and Western blot analysis with anti-FLAG and anti-HA antibodies. D, XBP1u bound to amino acid 201–323 region in HDAC3 molecule. The left panel indicates the schematic illustration of HDAC3 truncated mutants. The right panel shows the interaction of XBP1u and truncated HDAC3 as revealed by immunoprecipitation assays. E, disturbed flow increased XBP1u association with HDAC3/Akt1. Co-immunoprecipitation with anti-XBP1u antibody was performed on static and disturbed flow (4 h)-treated cells, followed by Western blot with anti-HDAC3 or Akt1 and anti-XBP1u antibodies. F, disturbed flow induced mTOR/Akt1/HDAC3/XBP1u complex formation in the cytoplasm. Double immunofluorescence staining was performed on static and disturbed flow (4 h)-treated cells. Antibodies are indicated with red or green letters reflecting the color in the images. Data presented are representatives of three independent experiments.
Mentions: As described above, both XBP1u and HDAC3 up-regulate HO-1 expression, whereas flow-induced HDAC3 is XBP1-dependent. Therefore, we hypothesized that there was cross-talk between XBP1u and HDAC3 during the regulation of HO-1. To test this, co-expression of HDAC3 and XBP1u was first introduced into HUVECs by co-infection with two viruses. As shown in Fig. 5A, overexpression of either XBP1u or HDAC3 alone up-regulated Akt1 phosphorylation, Nrf2 and HO-1, whereas co-expression of XBP1u and HDAC3 had a synergistic effect. Further experiments revealed that knockdown of HDAC3 attenuated XBP1u-induced Akt1 phosphorylation and HO-1 expression (Fig. 5B). Co-immunoprecipitation assays revealed that XBP1u physically bound to HDAC3 in transfected cells (Fig. 5C). Using truncated HDAC3 mutants, the binding domain in HDAC3 molecule could be defined to the amino acid 201∼323 region (Fig. 5D). Immunoprecipitation with antibody against endogenous XBP1u revealed that XBP1u bound to HDAC3 and Akt1 under disturbed flow (Fig. 5E). Double immunofluorescence staining showed that mTOR/Akt1, Akt1/HDAC3, Akt1/XBP1u, and HDAC3/XBP1u co-localized in the cytoplasm (Fig. 5F). These results suggest XBP1u/HDAC3/Akt1/mTOR may form a complex to regulate Akt1 phosphorylation, leading to Nrf2 stabilization and HO-1 expression.

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