OR14 HLA class I antibodies increased WNT/β-catenin pathway component protein lymphoid-binding factor 1 (LEF1) in endothelial cells in foxo1 protein dependent fashion

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Introduction and aim Forkhead box (FOX) proteins are a family of important transcription factors that plays important role in maintaining endothelial cells functions. The protective role of FOXO1 in multiple cellular functions is supported by several studies. In many cellular systems, the forkhead transcription factors such as FOXO1 activity is regulated by post-translational modification through phosphorylation and acetylation. The purpose of the current study was to investigate the effect of HLA class I on the phosphorylation/inactivation of the FOXO1 protein. We also investigated the impact of the HLA antibodies on the mRNA levels of the fibrosis protein LEF1. Methods Western blot analysis was used to examine the phosphorylation status of FOXO1 in human umbilical cord endothelial cells (HUVECs) after treatment with monoclonal antibodies directed against HLA class I. Real-time PCR assay was used to measure the mRNA levels of LEF1 in endothelial cells. Results Coupling of endothelial cells with monoclonal antibodies directed to HLA class I but not class II resulted in increased phosphorylation (inactivation) of the FOXO1 protein at Ser256 and Th24. In addition, the mRNA levels of FOXO1 down-stream target LEF1 were significantly up-regulated in endothelial cells treated with HLA class I antibodies compared to control treated cells. shRNAi mediated knock-down of FOXO1 protein in endothelial cells resulted in increased expression of LEF1 in endothelial cells. Conclusion This data identifies a new role of HLA antibodies in regulating endothelial cells functions by the inactivation of FOXO1 and increased its down-stream target LEF1. This data in addition to the previously published data indicating the involvement of the LEF1 in fibrosis might suggest a novel mechanism for allograft fibrosis mediated by HLA antibodies. Further studies to evaluate the in vitro findings in human allograft tissues are required.