FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2108455760> ?p ?o ?g. }

• W2108455760 endingPage "30634" @default.
• W2108455760 startingPage "30625" @default.
• W2108455760 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. 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. It is widely accepted that atherosclerosis occurs geographically at branch points where disturbed flow can trigger endothelial cell (EC) 4The abbreviations used are: ECendothelial cellHO-1heme oxygenase 1XBP1X-box-binding protein 1HDAC3histone deacetylase 3XBP1uunspliced XBP1mTORC2mammalian target of rapamycin complex 2IRE1αinositol-requiring enzyme 1 αERendoplasmic reticulumXBP1sspliced XBP1DMSOdimethyl sulfoxideHUVEChuman umbilical vein ECMOImultiplicity of infectionLucluciferaseAREantioxidant response elementAdAdenovirus. dysfunction via oxidative stress and inflammation (1Nigro P. Abe J. Berk B.C. Flow shear stress and atherosclerosis: a matter of site specificity.Antioxid. Redox Signal. 2011; 15: 1405-1414Crossref PubMed Scopus (170) Google Scholar). Under physiological conditions, cells can naturally establish homeostasis in response to different stimuli. Theoretically, the activation of oxidative stress under disturbed flow should be accompanied by the up-regulation of anti-oxidative mechanisms to maintain homeostasis within ECs. This may be a mechanism through which vascular integrity is maintained. The development of atherosclerosis could be a consequence of the disruption of the homeostasis by the intervention of other risk factors such as hyperlipidemia, diabetes, hypertension, smoking, and etc. (2Davies P.F. Civelek M. Fang Y. Guerraty M.A. Passerini A.G. Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo.Semin. Thromb. Hemost. 2010; 36: 265-275Crossref PubMed Scopus (37) Google Scholar, 3Nitenberg A. Cosson E. Pham I. Postprandial endothelial dysfunction: role of glucose, lipids and insulin.Diabetes Metab. 2006; 32: 2S28-33Crossref PubMed Google Scholar). endothelial cell heme oxygenase 1 X-box-binding protein 1 histone deacetylase 3 unspliced XBP1 mammalian target of rapamycin complex 2 inositol-requiring enzyme 1 α endoplasmic reticulum spliced XBP1 dimethyl sulfoxide human umbilical vein EC multiplicity of infection luciferase antioxidant response element Adenovirus. Heme oxygenase 1 (HO-1) is an inducible isoform of the heme oxygenase family, which catalyzes the degradation of heme, producing biliverdin, iron, and carbon monoxide (4Kikuchi G. Yoshida T. Noguchi M. Heme oxygenase and heme degradation.Biochem. Biophys. Res. Commun. 2005; 338: 558-567Crossref PubMed Scopus (339) Google Scholar). HO-1 was originally identified as a 32-kDa stress responsive protein to UV-irradiation, hydrogen peroxide, and sodium arsenite (5Keyse S.M. Tyrrell R.M. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 99-103Crossref PubMed Scopus (1108) Google Scholar). HO-1 is induced ubiquitously in cells in response to oxidative stress, hypoxia, heavy metal ions, cytokines, glutathione depletion, and etc. (6Siow R.C. Ishii T. Sato H. Taketani S. Leake D.S. Sweiry J.H. Pearson J.D. Bannai S. Mann G.E. Induction of the antioxidant stress proteins heme oxygenase-1 and MSP23 by stress agents and oxidised LDL in cultured vascular smooth muscle cells.FEBS Lett. 1995; 368: 239-242Crossref PubMed Scopus (79) Google Scholar). The induction of HO-1 and the derived carbon monoxide plays a protective role against cell apoptosis (7Brouard S. Berberat P.O. Tobiasch E. Seldon M.P. Bach F.H. Soares M.P. Heme oxygenase-1-derived carbon monoxide requires the activation of transcription factor NF-κB to protect endothelial cells from tumor necrosis factor-α-mediated apoptosis.J. Biol. Chem. 2002; 277: 17950-17961Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Disruption of the HO-1gene (HMOX-1) does not affect mouse survival but increases end-organ damage and mortality during endotoxemia due to increased oxidative stress (8Wiesel P. Patel A.P. DiFonzo N. Marria P.B. Sim C.U. Pellacani A. Maemura K. LeBlanc B.W. Marino K. Doerschuk C.M. Yet S.F. Lee M.E. Perrella M.A. Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice.Circulation. 2000; 102: 3015-3022Crossref PubMed Scopus (176) Google Scholar). In EC, HO-1 can be induced by both laminar flow and disturbed flow through oxidative stress and Nrf2 (NF-E2-related factor 2) activation (9Warabi E. Takabe W. Minami T. Inoue K. Itoh K. Yamamoto M. Ishii T. Kodama T. Noguchi N. Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species.Free Radic. Biol. Med. 2007; 42: 260-269Crossref PubMed Scopus (145) Google Scholar). However, the existence of a signaling pathway between the mechanosensor and Nrf2-mediated HO-1 expression remains unknown. The X-box binding protein 1 (XBP1) is also a stress responsive gene. In contrast to most stress responsive genes, XBP1 mRNA undergoes alternative splicing via inositol-requiring enzyme 1 α (IRE1α). This occurs in response to endoplasmic reticulum (ER) stress, resulting in an open reading frameshift (10Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2961) Google Scholar, 11Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response.Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (828) Google Scholar). XBP1 protein exists as 29-kDa unspliced (XBP1u) and 56-kDa spliced (XBP1s) isoforms. Both isoforms have an identical N-terminal dimerization domain and internal DNA binding domain but differ in the C terminus. XBP1s contains a transcriptional activation domain in the C terminus and functions as an intact transcription factor (10Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2961) Google Scholar). The majority of the previously described XBP1 functions are assigned to XBP1s. Our previous studies have demonstrated that XBP1s plays multiple roles in EC proliferation, autophagy response, and apoptosis (12Zeng L. Xiao Q. Chen M. Margariti A. Martin D. Ivetic A. Xu H. Mason J. Wang W. Cockerill G. Mori K. Li J.Y. Chien S. Hu Y. Xu Q. Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.Circulation. 2013; 127: 1712-1722Crossref PubMed Scopus (83) Google Scholar, 13Margariti A. Li H. Chen T. Martin D. Vizcay-Barrena G. Alam S. Karamariti E. Xiao Q. Zampetaki A. Zhang Z. Wang W. Jiang Z. Gao C. Ma B. Chen Y.G. Cockerill G. Hu Y. Xu Q. Zeng L. XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation.J. Biol. Chem. 2013; 288: 859-872Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar14Zeng L. Zampetaki A. Margariti A. Pepe A.E. Alam S. Martin D. Xiao Q. Wang W. Jin Z.G. Cockerill G. Mori K. Li Y.S. Hu Y. Chien S. Xu Q. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8326-8331Crossref PubMed Scopus (175) Google Scholar). The C terminus of XBP1u contains a signal for proteasome-mediated degradation, negatively regulating XBP1s function (15Yoshida H. Oku M. Suzuki M. Mori K. pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response.J. Cell Biol. 2006; 172: 565-575Crossref PubMed Scopus (315) Google Scholar). There remains very little investigation into the role of XBP1u compared with XBP1s. Histone deacetylase 3 (HDAC3) is a class I HDAC (16de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Histone deacetylases (HDACs): characterization of the classical HDAC family.Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2454) Google Scholar). Disruption of the HDAC3 gene is lethal at an early embryonic stage (17Bhaskara S. Chyla B.J. Amann J.M. Knutson S.K. Cortez D. Sun Z.W. Hiebert S.W. Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control.Mol. Cell. 2008; 30: 61-72Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). It is reported that cigarette smoke reduces HDAC3 activity via posttranslational modification (18Yang S.R. Chida A.S. Bauter M.R. Shafiq N. Seweryniak K. Maggirwar S.B. Kilty I. Rahman I. Cigarette smoke induces proinflammatory cytokine release by activation of NF-κB and posttranslational modifications of histone deacetylase in macrophages.Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: L46-57Crossref PubMed Scopus (396) Google Scholar), which is the first indirect evidence that HDAC3 is involved in response to oxidative stress. Our previous study provides direct evidence that up-regulation of HDAC3 by disturbed flow is essential for EC survival under oxidative stress via activation of Akt phosphorylation (19Zampetaki A. Zeng L. Margariti A. Xiao Q. Li H. Zhang Z. Pepe A.E. Wang G. Habi O. deFalco E. Cockerill G. Mason J.C. Hu Y. Xu Q. Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.Circulation. 2010; 121: 132-142Crossref PubMed Scopus (119) Google Scholar). HDAC3 deficiency in ECs accelerates vessel injury-induced neointima formation. Our studies have also demonstrated that HDAC3 homeostasis is essential for EC differentiation from stem/progenitor cells (20Zeng L. Xiao Q. Margariti A. Zhang Z. Zampetaki A. Patel S. Capogrossi M.C. Hu Y. Xu Q. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells.J. Cell Biol. 2006; 174: 1059-1069Crossref PubMed Scopus (211) Google Scholar, 21Xiao Q. Zeng L. Zhang Z. Margariti A. Ali Z.A. Channon K.M. Xu Q. Hu Y. Sca-1+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2244-2251Crossref PubMed Scopus (110) Google Scholar), inflammatory reactions (22Alam S. Li H. Margariti A. Martin D. Zampetaki A. Habi O. Cockerill G. Hu Y. Xu Q. Zeng L. Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3.J. Biol. Chem. 2011; 286: 44211-44217Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and endothelial-to-mesenchymal transition (23Zeng L. Wang G. Ummarino D. Margariti A. Xu Q. Xiao Q. Wang W. Zhang Z. Yin X. Mayr M. Cockerill G. Li J.Y. Chien S. Hu Y. Xu Q. Histone deacetylase 3 unconventional splicing mediates endothelial-to-mesenchymal transition through transforming growth factor β2.J. Biol. Chem. 2013; 288: 31853-31866Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this study, we found that HDAC3 cooperated with XBP1u to modulate HO-1 expression in response to disturbed flow. To scrutinize the molecular mechanisms of this process, the present study aims to clarify the role of XBP1 interaction with the partners in maintaining endothelial functions. We demonstrated that an interaction between XBP1 and HADC3 resulted in PI3K/Akt1 activation and HO-1 expression. This process is crucial for endothelial survival in response to oxidative stress. All cell culture medium and serum were purchased from Invitrogen, whereas cell culture supplements were purchased from Sigma. Antibodies against XBP1 (sc-7160), HDAC3 (sc-136290) phospho-Akt (sc-7985R), Akt1 (sc-1619), Nrf2 (sc-722), mTOR (sc-1549), histone H3 (sc-10809), IRE1α (sc-20790), and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology; antibodies against FLAG (F2426, F1804, and F7425), HA (H6908) and tubulin (T8203) were from Sigma; antibody against HO-1 (ab13248) was purchased from Abcam. Antibodies against XBP1u and XBP1s were raised in rabbits with peptides CRSSQRSTQKDPVPY and DSGGIDSSDSESDIC, respectively, by GenScript Corp. All secondary antibodies were from DakoCytomation. Inhibitors LY294002, PD98059, SU5416, actinomycin D, cycloheximide, and Tin protoporphyrin IX were purchased from Sigma. AZD2014 was purchased from Selleckchem. All inhibitors were dissolved in DMSO. All other chemicals were also from Sigma. Human umbilical vein ECs (HUVECs) were cultured on 0.04% gelatin-coated flasks in M199 medium supplemented with 1 ng/ml β-EC growth factor, 3 µg/ml EC growth supplement from bovine neural tissue, 10µ/ml heparin, 1.25 µg/ml thymidine, 10% fetal bovine serum (FBS), 100 µ/ml penicillin, and streptomycin in a humidified incubator supplemented with 5% CO2. The cells were split every 3 days at a ratio of 1:3. Cells up to passage 10 were used in this study. HEK293 cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin and were split every 3 days at a ratio of 1:4. Mouse embryonic fibroblasts were isolated from XBP1+/− cross-bred embryonic day 8.5 embryos (12Zeng L. Xiao Q. Chen M. Margariti A. Martin D. Ivetic A. Xu H. Mason J. Wang W. Cockerill G. Mori K. Li J.Y. Chien S. Hu Y. Xu Q. Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.Circulation. 2013; 127: 1712-1722Crossref PubMed Scopus (83) Google Scholar), cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. The genotype of the cells was verified by PCR with primer triplet of P1 (5′-atcctgtcttgaaatggcaagtgttgg-3′), P2 (5′-tggcaaggctgagcctgatcg-3′), and P3 (5′-ggaactagagataccactgag-3′), giving rise to a 265-bp band for wild type and a 365-bp band for XBP1−/− homozygous and double bands for XBP1+/− heterozygous. Flow experiments were performed exactly as described previously (14Zeng L. Zampetaki A. Margariti A. Pepe A.E. Alam S. Martin D. Xiao Q. Wang W. Jin Z.G. Cockerill G. Mori K. Li Y.S. Hu Y. Chien S. Xu Q. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8326-8331Crossref PubMed Scopus (175) Google Scholar). Briefly, the disturbed flow was created by placing the flask on a platform shaker (Labnet, model Rocker 25) with parameters of 2.0-mm culture medium depth, 10-cm length flask, ± 7° rotating angle and frequency of 0.5 Hz (i.e. 2 s per cycle), respectively. Unshaken cells were kept for same duration as static control. For inhibitor assays, the inhibitors were included in culture medium for 1 h prior to flow and maintained during the flow process. HUVECs were challenged with 50 µmol/liter H2O2 for 24 h after 24 h post-infection with Ad-null or Ad-XBP1u virus at 10 MOI or with 20 µmol/liter H2O2 for 24 h after 72 h post-infection with non-target shRNA or XBP1 shRNA lentivirus at 10 MOI, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay with the CellTiter 96 Aqueous One Solution Cell Proliferation assay kit according to the protocol provided (Promega). Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was diluted at 1:4 with M199 containing 2% FBS, applied to HUVECs at 300 µl/well in 24-well plate and incubated at 37 °C for 30 min to 2 h. 300 µl/well of 0.2% SDS was then added to stop the reaction. The absorbance at A490 nm was measured with a laminator luminometer. The relative cell survival was defined as the ratio of A490 nm of the test group to that of control group with that of control group set as 1.0. Mouse embryonic fibroblasts were challenged with 50 µmol/liter H2O2 for 24 h, followed by apoptosis analysis using the Apo-Direct flow cytometry kit (Chemicon) with protocol provided. PBS was included as control. Briefly, the treated cells were detached with trypsin and fixed in solution with 1% paraformaldehyde on ice for 1 h and washed three times with PBS. The cell pellet was resuspended in 50 µl of staining solution (TdT reaction buffer, TdT enzyme, fluorescein-dUTP) and incubated for 60 min at 37 °C, with shaking every 15 min. The reaction was stopped through the addition of 1 ml of rinse buffer and washed three times with the rinse buffer. The cell pellet was resuspended in 500 µl of propidium iodide/RNase A solution and incubated in the dark at room temperature for 30 min, followed by flow cytometry analysis of fluorescence at 520 nm with a 488-nm Argon laser. The artery ex vivo survival experiments were performed as described previously (14Zeng L. Zampetaki A. Margariti A. Pepe A.E. Alam S. Martin D. Xiao Q. Wang W. Jin Z.G. Cockerill G. Mori K. Li Y.S. Hu Y. Chien S. Xu Q. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8326-8331Crossref PubMed Scopus (175) Google Scholar). Briefly, arteries were isolated from Tie2-LacZ/ApoE−/− mice and cut into an ∼2-mm2 segment. The segments were incubated with 0.5 ml of M199 medium plus 15% FBS containing no virus (control), empty virus (Ad-null), or Ad-XBP1u virus at 1 × 106 plaque-forming unit/ml in 24-well plate for 6 h. Three segments were included in each group. The virus solutions were then removed, and 1 ml of fresh medium was added to each well. Twenty-four hour post infection, the segments were treated with PBS or 50 µmol/liter H2O2 for 24 h. The segments were fixed with 4% formaldehyde and 1% glutaraldehyde in PBS for 5 min, followed by X-gal staining overnight (24Hu Y. Davison F. Zhang Z. Xu Q. Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells.Circulation. 2003; 108: 3122-3127Crossref PubMed Scopus (200) Google Scholar). The segments were mounted on slide with vessel lumen face up. Images were assessed by Zeiss Axioplan 2 Imaging microscope with Plan-NEOPLUAR 20×/0.5 objective lenses, AxioCam camera and Axiovision software at room temperature and were processed by Adobe Photoshop software. Cell numbers were counted under the microscope. The relative cell number was defined as the ratio of cell numbers/mm2 of virus-infected group to that of uninfected control group with that of control set as 1.0. A 700-bp mouse HDAC3 promoter fragment was amplified by PCR using a primer set of 5′-gacactctcgagaatgcctactcgcgttgc-3′ and 5′-gtgaccaagcttcgagcctcagctgcc-3′, cloned into the XhoI/HindIII sites of pGL3-Luc (Promega), and verified by DNA sequencing. The resulting plasmid was designated as HDAC3-Luc. The XBP1u cDNA sequence was amplified by RT-PCR with a primer set of 5′-ggagctggtaccctggtggtggtggcagcc-3′ and 5′-tctgagaagcttacagtattggatcattcc-3′, cloned into the KpnI/HindIII site of pCMV5-HA vector, and verified by DNA sequencing. The XBP1u open reading frame is fused to HA tag at the N terminus, designated as pCMV5-HA-XBP1u. The cloning of other plasmids, pShuttle2-FLAG-XBP1s, pShuttle2-FLAG-XBP1u, pShuttle2-FLAG-HDAC3, and the HDAC3 mutant variants has been described in previous reports (14Zeng L. Zampetaki A. Margariti A. Pepe A.E. Alam S. Martin D. Xiao Q. Wang W. Jin Z.G. Cockerill G. Mori K. Li Y.S. Hu Y. Chien S. Xu Q. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8326-8331Crossref PubMed Scopus (175) Google Scholar, 19Zampetaki A. Zeng L. Margariti A. Xiao Q. Li H. Zhang Z. Pepe A.E. Wang G. Habi O. deFalco E. Cockerill G. Mason J.C. Hu Y. Xu Q. Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.Circulation. 2010; 121: 132-142Crossref PubMed Scopus (119) Google Scholar). HUVECs were seeded in 12-well plates at 5 × 104 cells/well 24 h prior to transfection. HDAC3-Luc (0.1 µg/well) vector was co-transfected with 0.1 µg/well pShuttle2-FLAG-XBP1s or -XBP1u expression vector into HUVECs with FuGENE 6 (Roche Applied Science). pGL3-Luc basic vector and grp78-luc vector were included as negative and positive luciferase vector control, respectively. pShuttle2-lacZ vector was used as an empty vector control. Renilla-Luc (0.05 µg/well) was included as an internal control. Forty-eight hours later, firefly and Renilla luciferase activity was assessed with respective assay kit (Promega). The relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for Renilla luciferase with that of control group set as 1.0. Total cellular RNA was extracted using Qiagen RNeasy kit according to the protocol provided. Two microgram RNA was transcribed into cDNA using Improm-II reverse transcription system (Promega). Twenty nanogram cDNA (relative to RNA amount) was amplified by PCR with cyber green solution (Applied Biosystems) in a Sequence Detection System 7000 (Applied Biosystems). The primers were as follows: XBP1, 5′-agcactcagactacgtgcacct-3′ and 5′-tgcccaacag gatatcagactc-3′; HDAC3, 5′-catctctgctggtagaagagg-3′ and 5′-catcatagaactcattgggtg-3′; HMOX-1, 5′-gccagcaacaaagtgcaagatt-3′ and 5′-tgagtgtaaggacccatcggag-3′; Nrf2, 5′-cagtggatctgccaactactc-3′ and 5′-tggagaggatgctgctgaagg-3′; and 18s, 5′-cccagtaagtgcgggtcataa-3′ and 5′-ccgagggcctcactaaacc-3′. For adenoviral infection, HUVECs were incubated with Ad-null, Ad-HDAC3, or Ad-XBP1u virus at 10 MOI for 6 h and then cultured in fresh complete growth medium for time duration indicated in figure legends. For shRNA lentiviral infection, HUVECs were incubated with 100 transduction unit/cell of non-target shRNA or XBP1 shRNA or IRE1α shRNA or HDAC3 shRNA lentiviruses in the presence of 10 mg/ml polybrene for 16 h, followed by culture in fresh complete growth medium for 72 h, and subjected to further treatments. Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly. For siRNA transfection assay, HUVECs in 75-ml flasks were transfected with 50 µl of 10 µmol/liter control siRNA or Nrf2 siRNA together with 50 µl of Lipofectamine RNAimax (Invitrogen) according to protocol provided. For virus infection assays, 48 h post-transfection, the cells were infected with 10 MOI Ad-null, Ad-XBP1u or Ad-HDAC3 viruses and incubated for 24 h, followed by Western blot analysis. For shear stress assays, 72 h post-transfection, the cells were subjected to disturbed flow for 4 h, followed by Western blot analysis. Immunoprecipitation and immunoblotting were performed according to standard procedures described elsewhere. One milligram lysate was used for immunoprecipitation, whereas 25 µg was used for input or direct immunoblotting. For unconjugated antibody, 2 µg of antibody and 10 µl of protein G beads (Sigma) were used for one immunoprecipitation assay. For agarose-conjugated antibody, 10 µl of such beads were directly used for each immunoprecipitation assay. Immunofluorescence staining was performed using standard procedures. Briefly, adenovirus-infected or uninfected HUVECs were seeded on 0.04% gelatin-coated glass slides with or without flow treatment. The cells were fixed with methanol and permeabilized with 0.1% Triton X-100, blocked with 5% normal swine serum, incubated with primary antibodies, followed by incubation with Alexa Fluor 488- or 594-labeled secondary antibodies and counterstaining with DAPI. Images were taken by using SP5 confocal microscope (Leica) and were processed by Adobe Photoshop software. Magnification was indicated in figures. HUVECs were infected with Ad-null, Ad-XBP1u, or Ad-HDAC3 at 10 MOI for 6 h and incubated for another 18 h. Fresh medium containing DMSO or 5 µmol/liter AZD2014 were added and incubated for 24 h. The cytosol and nuclear extracts were harvested with procedures described previously (12Zeng L. Xiao Q. Chen M. Margariti A. Martin D. Ivetic A. Xu H. Mason J. Wang W. Cockerill G. Mori K. Li J.Y. Chien S. Hu Y. Xu Q. Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.Circulation. 2013; 127: 1712-1722Crossref PubMed Scopus (83) Google Scholar). Chromatin immunoprecipitation was performed with ChIP assay kit (17-295, Millipore) according to the protocol provided. Briefly, HUVECs were subjected to disturbed flow for 2 h, followed by the ChIP assay. Rabbit anti-XBP1u and anti-XBP1s were used, and normal rabbit IgG was included as negative control. Six sets of primer pairs were used to cover the 1.5-kb HDAC3 promoter region (GenBankTM accession no. AB457579.1). The sequences include the following: position +1 ∼ −276, 5′-ccacggtcttggccatggtgc-3′ versus 5′-tcggcttcccgaggatctgac-3′; position −276 ∼ −520, 5′-tcagatcctcgggaagccgag-3′ versus 5′-gtttgggtccgggtaggggac-3′; position −520 ∼ −720, 5′-cccctacccggacccaaactc-3′ versus 5′-gctgagagcggtggcaggctc-3′; position −720 ∼ −960, 5′-gagcctgccaccgctctcagc-3′ versus 5′-ttctcccaccctgaccacctg-3′; position −960 ∼ −1195, 5′-gccaggtggtcagggtgggag-3′ versus 5′-agctctctaccacgaccatgg-3′; position −1195 ∼ −1467, 5′-accatggtcgtggtagagagc-3′ versus 5′-aagagcatatatagcccatgttgg-3′. Routine PCR was performed using these primer sets to amplified XBP1u- or XBP1s-bound DNA fragment. Data expressed as the mean ± S.E. were analyzed using GraphPad Prism software (version 5) with t test for pair-wise comparisons or analysis of variance, when t test was inappropriate, followed by Dunnett's multiple comparison tests, and significance was depicted by asterisks (*, p < 0.05). Our previous studies have demonstrated that disturbed flow sustainably activates XBP1 expression and splicing (14Zeng L. Zampetaki A. Margariti A. Pepe A.E. Alam S. Martin D. Xiao Q. Wang W. Jin Z.G. Cockerill G. Mori K. Li Y.S. Hu Y. Chien S. Xu Q. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8326-8331" @default.
• W2108455760 created "2016-06-24" @default.
• W2108455760 creator A5004429744 @default.
• W2108455760 creator A5012509728 @default.
• W2108455760 creator A5022101190 @default.
• W2108455760 creator A5030921116 @default.
• W2108455760 creator A5036831600 @default.
• W2108455760 creator A5044907152 @default.
• W2108455760 creator A5064842058 @default.
• W2108455760 creator A5072167506 @default.
• W2108455760 creator A5075240741 @default.
• W2108455760 creator A5087780082 @default.
• W2108455760 creator A5088028330 @default.
• W2108455760 date "2014-10-01" @default.
• W2108455760 modified "2023-10-14" @default.
• W2108455760 title "Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3" @default.
• W2108455760 cites W153428855 @default.
• W2108455760 cites W1967765358 @default.
• W2108455760 cites W1968472971 @default.
• W2108455760 cites W1968809860 @default.
• W2108455760 cites W1970090817 @default.
• W2108455760 cites W1970198745 @default.
• W2108455760 cites W1976382640 @default.
• W2108455760 cites W1976448006 @default.
• W2108455760 cites W1982669767 @default.
• W2108455760 cites W1983307716 @default.
• W2108455760 cites W1985096908 @default.
• W2108455760 cites W1990488806 @default.
• W2108455760 cites W1997507706 @default.
• W2108455760 cites W1997671473 @default.
• W2108455760 cites W2003943211 @default.
• W2108455760 cites W2008209363 @default.
• W2108455760 cites W2024883268 @default.
• W2108455760 cites W2026873752 @default.
• W2108455760 cites W2036504407 @default.
• W2108455760 cites W2037686094 @default.
• W2108455760 cites W2041553103 @default.
• W2108455760 cites W2042404313 @default.
• W2108455760 cites W2051690606 @default.
• W2108455760 cites W2062145087 @default.
• W2108455760 cites W2085225882 @default.
• W2108455760 cites W2085707502 @default.
• W2108455760 cites W2088510522 @default.
• W2108455760 cites W2091802988 @default.
• W2108455760 cites W2094320086 @default.
• W2108455760 cites W2104836104 @default.
• W2108455760 cites W2106314483 @default.
• W2108455760 cites W2107733280 @default.
• W2108455760 cites W2117028091 @default.
• W2108455760 cites W2118814154 @default.
• W2108455760 cites W2119511051 @default.
• W2108455760 cites W2119621267 @default.
• W2108455760 cites W2121193141 @default.
• W2108455760 cites W2129227644 @default.
• W2108455760 cites W2132382458 @default.
• W2108455760 cites W2133869227 @default.
• W2108455760 cites W2138018577 @default.
• W2108455760 cites W2145091080 @default.
• W2108455760 cites W2148563903 @default.
• W2108455760 cites W2158281895 @default.
• W2108455760 cites W2169633992 @default.
• W2108455760 cites W2170191859 @default.
• W2108455760 cites W2192080449 @default.
• W2108455760 cites W2326832882 @default.
• W2108455760 cites W4238567307 @default.
• W2108455760 doi "https://doi.org/10.1074/jbc.m114.571984" @default.
• W2108455760 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4215241" @default.
• W2108455760 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25190803" @default.
• W2108455760 hasPublicationYear "2014" @default.
• W2108455760 type Work @default.
• W2108455760 sameAs 2108455760 @default.
• W2108455760 citedByCount "72" @default.
• W2108455760 countsByYear W21084557602015 @default.
• W2108455760 countsByYear W21084557602016 @default.
• W2108455760 countsByYear W21084557602017 @default.
• W2108455760 countsByYear W21084557602018 @default.
• W2108455760 countsByYear W21084557602019 @default.
• W2108455760 countsByYear W21084557602020 @default.
• W2108455760 countsByYear W21084557602021 @default.
• W2108455760 countsByYear W21084557602022 @default.
• W2108455760 countsByYear W21084557602023 @default.
• W2108455760 crossrefType "journal-article" @default.
• W2108455760 hasAuthorship W2108455760A5004429744 @default.
• W2108455760 hasAuthorship W2108455760A5012509728 @default.
• W2108455760 hasAuthorship W2108455760A5022101190 @default.
• W2108455760 hasAuthorship W2108455760A5030921116 @default.
• W2108455760 hasAuthorship W2108455760A5036831600 @default.
• W2108455760 hasAuthorship W2108455760A5044907152 @default.
• W2108455760 hasAuthorship W2108455760A5064842058 @default.
• W2108455760 hasAuthorship W2108455760A5072167506 @default.
• W2108455760 hasAuthorship W2108455760A5075240741 @default.
• W2108455760 hasAuthorship W2108455760A5087780082 @default.
• W2108455760 hasAuthorship W2108455760A5088028330 @default.
• W2108455760 hasBestOaLocation W21084557601 @default.
• W2108455760 hasConcept C104317684 @default.
• W2108455760 hasConcept C185592680 @default.
• W2108455760 hasConcept C27253355 @default.
• W2108455760 hasConcept C2776151105 @default.

• next