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, 289 (44), 30625-34

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells From Oxidative Stress Through Interaction With Histone Deacetylase 3

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Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells From Oxidative Stress Through Interaction With Histone Deacetylase 3

Daniel Martin et al. J Biol Chem.

Abstract

It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. 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. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. 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.

Keywords: Cell Signaling; Endothelial Cell; Histone Deacetylase (HDAC); Oxidative Stress; Shear Stress.

Figures

FIGURE 1.
FIGURE 1.
XBP1u protein was essential for disturbed flow-induced HDAC3 up-regulation. A, VEGF-PI3K/Akt pathway was involved in disturbed flow (4 h)-induced up-regulation of XBP1 expression and splicing. DM, DMSO (vehicle control); SU, SU5416 (1 μmol/liter, VEGF receptor inhibitor); PD, PD98059 (5 μmol/liter, ERK inhibitor); LY, LY294002 (5 μmol/liter, PI3K/Akt inhibitor). B, knockdown of XBP1 or IRE1α abolished disturbed flow (4 h)-induced HDAC3 up-regulation. UT, untransfected; NT, non-target shRNA transfected; Xsh, XBP1 shRNA transfected; Ish, IRE1α shRNA transfected. Disturbed flow was applied to HUVECs 72 h post transfection for 4 h. C, Overexpression of spliced XBP1 down-regulated HDAC3 protein. FLAG indicates the exogenous XBP1s protein. D, spliced XBP1 suppressed HDAC3 gene transcription. RLA, relative luciferase activity. pGL3-luc basic vector was included as negative control, whereas grp78-Luc vector was used as positive control. Mock, pShuttle-LacZ plasmid; XBP1s, pShuttle-FLAG-XBP1s plasmid; XBP1u, pShuttle-FLAG-XBP1u plasmid. E, XBP1u antagonized XBP1s on the regulation of HDAC3 protein. HUVECs were co-infected with Ad-XBP1s and Ad-XBP1u at 10 MOI each for 48 h. Ad-null was included as control and to compensate the MOI. FLAG indicates the exogenous XBP1s and XBP1u proteins. F, XBP1u and XBP1s differentially bound to HDAC3 promoter in response to disturbed flow. ChIP assay was performed to analyze the binding of XBP1u and XBP1s to the HDAC3 promoter in static and disturbed flow-treated HUVECs (4 h). Six sets of primer pairs covered the +1 ∼ −1467 region (upper panel), and PCR showed that XBP1u and XBP1s differentially bound to −960 ∼ −1195 region in response to disturbed flow (lower panel). SS, shear stress. Data presented are representative or average of three independent experiments. *, p < 0.05.
FIGURE 2.
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-null (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.
FIGURE 3.
FIGURE 3.
XBP1u-mediated cell survival was through regulation of HO-1 expression. A, HO-1 inhibitor SnPPIX abolished the protective effect of XBP1u overexpression on cell survival under H2O2 challenging. B, quantitative RT-PCR revealed that over-expression of XBP1u or HDAC3 up-regulated HMOX-1 mRNA level without effect on Nrf2 mRNA level. C, Western blot analysis showed that overexpression of XBP1u or HDAC3 up-regulated both Nrf2 and HO-1 protein levels. D, knockdown of Nrf2 abolished XBP1u or HDAC3-induced HO-1 expression. CTLsi, control siRNA. E, overexpression of XBP1u or HDAC3 increased Nrf2 nuclear translocation and HO-1 expression in the infected and adjacent cells. Double immunofluorescence staining was performed with anti-Akt1 (red) and anti-Nrf2 (green) antibodies or with anti-FLAG (red) for exogenous XBP1u or HDAC3 and anti-HO-1 (green) antibodies. Data presented are representatives or average from three independent experiments. *, p < 0.05.
FIGURE 4.
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-null 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-null 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-null 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-null 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.
FIGURE 5.
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-null 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.
FIGURE 6.
FIGURE 6.
A schematic illustration of flow-induced HO-1 expression. Disturbed flow (D-Flow) may activate VEGF receptor (KDR) in a ligand-independent manner, which in turn induces the complex formation among mTOR, Akt1, XBP1u, and HDAC3. The complex formation stabilizes both XBP1u and HDAC3 and activates Akt1 phosphorylation (p), leading to Nrf2 stabilization. Nrf2 translocates into nucleus and binds to the ARE in the HMOX-1 gene promoter and recruit co-activators (ca), promoting the HMOX-1 transcription. HO-1 catalyzes the heme degradation, which produces antioxidant biliverdin and carbon monoxide (CO), antagonizing disturbed flow-induced reactive oxygen species (ROS), leading to the maintenance of the redox homeostasis.

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