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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 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- 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.
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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- 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.

Mentions: Our previous studies have demonstrated that disturbed flow sustainably activates XBP1 expression and splicing (14) and that disturbed flow activates HDAC3 in a KDR/PI3K-Akt pathway-dependent manner (19). In this study, we found that disturbed flow-induced up-regulation of XBP1u was ablated by the presence of KDR inhibitor SU5416 and PI3K/Akt inhibitor LY294002, whereas XBP1 splicing was only ablated by SU5416 (Fig. 1A). This suggests that XBP1u was regulated by a similar mechanism to HDAC3 (19). As disturbed flow concomitantly up-regulated HDAC3, XBP1u, and XBP1s, we wondered whether there was cross-talk between HDAC3 and both XBP1 isoforms. XBP1s is produced by IRE1α activation (11) and down-regulation of XBP1s can be achieved by knockdown of IRE1α. Knockdown of XBP1 or IRE1α abolished disturbed flow-induced HDAC3 up-regulation (Fig. 1B), indicating that there is relationship between both XBP1 isoforms and HDAC3 under disturbed flow. To further examine the involvement of XBP1s in flow-induced HDAC3 up-regulation, exogenous overexpression of XBP1s was introduced into HUVECs via adenoviral gene transfer. As shown in Fig. 1C, overexpression of XBP1s actually decreased HDAC3 protein due to transcriptional repression as revealed by the HDAC3-Luc reporter analysis (Fig. 1D). Overexpression of XBP1u had no effect on HDAC3 transcription (Fig. 1D) but antagonized the effect of XBP1s and protected HDAC3 protein levels (Fig. 1E). A ChIP assay revealed that both XBP1u and XBP1s could bind to the −960 ∼ −1195 region of HDAC3 promoter (Fig. 1F). Under static condition, more XBP1u bound to this region, whereas during disturbed flow, more XBP1s bound to this region. These results suggest that there may be a cross-talk between HDAC3 and XBP1u.


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 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- 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.
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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- 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.
Mentions: Our previous studies have demonstrated that disturbed flow sustainably activates XBP1 expression and splicing (14) and that disturbed flow activates HDAC3 in a KDR/PI3K-Akt pathway-dependent manner (19). In this study, we found that disturbed flow-induced up-regulation of XBP1u was ablated by the presence of KDR inhibitor SU5416 and PI3K/Akt inhibitor LY294002, whereas XBP1 splicing was only ablated by SU5416 (Fig. 1A). This suggests that XBP1u was regulated by a similar mechanism to HDAC3 (19). As disturbed flow concomitantly up-regulated HDAC3, XBP1u, and XBP1s, we wondered whether there was cross-talk between HDAC3 and both XBP1 isoforms. XBP1s is produced by IRE1α activation (11) and down-regulation of XBP1s can be achieved by knockdown of IRE1α. Knockdown of XBP1 or IRE1α abolished disturbed flow-induced HDAC3 up-regulation (Fig. 1B), indicating that there is relationship between both XBP1 isoforms and HDAC3 under disturbed flow. To further examine the involvement of XBP1s in flow-induced HDAC3 up-regulation, exogenous overexpression of XBP1s was introduced into HUVECs via adenoviral gene transfer. As shown in Fig. 1C, overexpression of XBP1s actually decreased HDAC3 protein due to transcriptional repression as revealed by the HDAC3-Luc reporter analysis (Fig. 1D). Overexpression of XBP1u had no effect on HDAC3 transcription (Fig. 1D) but antagonized the effect of XBP1s and protected HDAC3 protein levels (Fig. 1E). A ChIP assay revealed that both XBP1u and XBP1s could bind to the −960 ∼ −1195 region of HDAC3 promoter (Fig. 1F). Under static condition, more XBP1u bound to this region, whereas during disturbed flow, more XBP1s bound to this region. These results suggest that there may be a cross-talk between HDAC3 and XBP1u.

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