Abstract 662: X-box Binding Protein 1 Splicing Promotes Wound Healing via Modulating Endothelial Nitric Oxide Synthase

Abstract

Instruction: X-box binding protein 1 (XBP1) is a pivotal transcription factor in the pathogenesis of the immune system and in the endoplasmic reticulum stress response. Our previous studies have proved that XBP1 is involved in VEGF-mediated endothelial cell (EC) proliferation and angiogenesis. However, whether XBP1 can influence EC migration is unknown. Hypothesis: We assessed the hypothesis that XBP1 splicing is involved in endothelial migration contributing to wound healing and angiogenesis. Method and results: In this study, we used EC monolayer wound healing, tube formation and transwell migration models to study the involvement of XBP1 splicing in EC migration. We found that scratching on EC monolayer triggered XBP1 splicing, which was attenuated by the presence of LY294002, FAK-14 and L-NAME, suggesting that PI3K/Akt, FAK and eNOS may be involved in scratching-mediated upregulation of XBP1 splicing. Over-expression of spliced XBP1 (XBP1s) via Ad-XBP1s gene transfer increased EC migration, while knockdown of IRE1α or XBP1 by ShRNA lentiviruses suppressed EC migration. Over-expression of XBP1s upregulated eNOS at mRNA and protein levels leading to the increase of nitric oxide production. The eNOS promoter reporter analysis revealed that XBP1s down-regulated eNOS transcription. The pSI-Check2-eNOS-3’UTR reporter analysis indicated that XBP1s might upregulate eNOS mRNA via 3’UTR-mediated mRNA stabilization. The microRNA array assays found that XBP1s down-regulated microRNAs mir-24, mir-125 and mir-214. Transfection of EC with the miRNA mimics of those three miRNAs attenuated XBP1s-induced eNOS mRNA upregulation and EC migration. Conclusion: These results suggest that XBP1 splicing may increase eNOS mRNA stabilization and nitric oxide production via down-regulating miRNAs that target eNOS mRNA 3’-UTR, leading to EC migration and contributing to wound healing and angiogenesis. Significance: XBP1 splicing may provide a new therapeutic strategy to tackle endothelium injury-related cardiovascular disease.