Unspliced X-box-binding protein 1 (XBP1) protects endothelial cells from oxidative stress through interaction with histone deacetylase 3.
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.
Affiliation: From the Cardiovascular Division, King's College London, London SE5 9NU, United Kingdom.Show MeSH
Related in: MedlinePlus
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.
Affiliation: From the Cardiovascular Division, King's College London, London SE5 9NU, United Kingdom.