Abstract 14155: Xbp1 Splicing is Crucial in Endothelial Cell Proliferation
The X-box binding protein 1 (XBP1) is an endoplasmic reticulum stress response transcription factor. Our previous study revealed that sustained activation of XBP1 splicing led to atherosclerosis development. However, the function of XBP1 expression and splicing in endothelial cells and angiogenesis remain unclear. To study this issue, we generated XBP1 knockout mice by deletion of the exons 1 and 2 of the gene. XBP1-null (XBP1−/−) embryos at E12.5 showed growth retardation and pale coloration phenotype. The average body weight of XBP1−/−embryos were 40% less than that of wild type (XBP1+/+) animals. The blood vessel density in XBP1−/− embryos was significantly reduced, due to a fewer number of CD31+ and Flk1+ cells. In vitro culture of whole embryonic cells, the XBP1−/− cells grew significantly slower and lost response to VEGF stimulation. To study the mechanism of XBP1-mediated cell growth, human umbilical vein endothelial cell were treated with VEGF that transiently activated IRE1α phosphorylation at Ser724. The mRNA movement inhibitor, cycloheximide, ablated VEGF-induced IRE1α phosphorylation and XBP1 splicing. Co-immunoprecipitation assay revealed that there was interaction among KDR, IRE1α and unspliced XBP1 (XBP1u), which could be increased by VEGF treatment. Further experiments demonstrated that the C-terminal region of KDR and the kinase domain of IRE1α are responsible for their interactions. MTT and BrdU incorporation assays indicate that transient activation of XBP1 splicing increased while long-term activation decreased endothelial survival and proliferation. Knockdown of XBP1 or IRE1α ablated VEGF-induced proliferation in endothelial cells. Immunofluorescent staining and TOP Flash reporter assay showed that over-expression of XBP1s increased beta-catenin translocation into nuclear. Furthermore, cell cycle analysis indicates that XBP1 splicing in HUVECs is cell cycle related, which peaks at G2 phase. Thus, this study demonstrated for the first time that XBP1 is crucial for endothelial growth and angiogenesis, in which VEGF-stimulated IRE1α/XBP1 splicing system and interaction with beta-catenin are key elements, indicating a potential target of XBP1 for protecting of endothelial integrity.
- © 2010 by American Heart Association, Inc.