The Regulation Of Yap Protein On NF-?B Signaling Pathway And Its Molecular Mechanism

Adult T-cell leukemia(ATL)is a malignant lymphatic disease caused by human T-cell leukemia virus type 1(HTLV-1).HTLV-1 encoded viral protein Tax regulates multiple signaling pathways that are related with diseases.Studies have shown that Tax promotes the malignant proliferation of ATL cells by activating the NF-?B signaling pathway,which results in the occurrence of ATL.However,in the later stages of ATL,the expression of Tax was lost because of epigenetic and epigenetic modifications,but the NF-?B signaling pathway is still activated.It suggests that in the later stage of ATL exist other molecular mechanisms that keep the activation of the NF-?B signaling pathway.Accumulating evidences have showed that YAP,the key protein of the Hippo signaling pathway,is highly expressed in many tumors.Hippo signaling pathway not only regulates organ size and cell regeneration,but also influences the growth of cells by regulating other signaling pathways.Therefore,we speculated whether YAP can promote the growth of tumor cells by regulating NF-?B signaling pathway in ATL cells? In order to prove this and elucidate its molecular mechanism,we carried on these researches as the following aspects:Part ?: High expression of YAP in ATLWe firstly detected the expression of YAP mRNA in ATL cell lines(ATL-T,ATL-2,MT-1,MT-2,MT-4,TL-Om1),control cell lines(MOTL-4,Hut78,Jurkat),and normal peripheral blood monocytes stimulated by PHA.The results showed that in ATL cell lines the YAP mRNA was highly expressed.Later,we verified that the YAP protein was highly expressed in ATL cell lines by Western blot.Finally,we confirmed the high expression of YAP mRNA in ATL clinical patient samples by q RT-PCR.Part ?: YAP regulates NF-?B signaling pathwayIn order to explore the relationship between YAP and NF-?B signaling pathway,we performed dual-luciferase reporter assay to detect the effect of YAP on NF-?B signaling pathway mediated by Tax.The results showed that YAP alone could not activate NF-?B signaling pathway,but could promote Tax-mediated NF-?B signaling pathway.Previous studies have shown that Tax activated classical and the alternative NF-?B signaling pathway in ATL cells.In order to further study the effect of YAP on these two pathways,we studied the effects of YAP on the p65-mediated classical pathways and p52-mediated alternative pathways.The results showed that YAP had no effect on the p52-mediated alternative pathways,but it can promote the classical signaling pathway mediated by p65.Subsequently,we constructed the ATL-T YAP knockdown cell line and the ATL-T YAP overexpressed cell line through lentivirus infection.and verified the effects of YAP on classical signaling pathway in T cells.The results showed that,in the YAP knockdown cells,the activation of NF-?B signaling pathway was down-regulated.Besides,the activation of NF-?B signaling pathway was significantly enhanced in the YAP overexpressed ATL-T cells.Those experiments further proved that NF-?B signaling pathway is positively regulated by YAP in ATL-T cells.Part ?: Molecular mechanisms of YAP regulating NF-?B signaling pathwayIn order to study the molecular mechanism of YAP reg ulating NF-?B signaling pathway,we firstly used Western blot experiment to detect whether YAP has an effect on the stability of p65,a key protein of classical NF-?B signaling pathway.The results showed that YAP had no effect on p65 protein expression.Subsequently,we found that YAP can bind to p65 protein by immunoprecipitation assay.Immunofluorescence assay showed that YAP and p65 protein co-located in the nucleus.We also conducted immunoprecipitation assays on mutants of YAP and p65.We found that the TAD domain of YAP protein is involved in binding with p65,and YAP protein mainly associated with the RHD homologous domain of p65 protein.Then we found that YAP itself could not bind to p50,but it could promote the binding between p65 and p50.In addition,we found that YAP could induce the nucleus localization of p65.Part ?: YAP regulates the expression of NF-?B downstream target genes in ATL cellsTo investigate whether YAP can influence the expression of NF-?B signaling pathway downstream target genes,we firstly treated ATL-T cells with YAP inhibitor Verteporfin(VP).In a RT-PCR experiment,we found that the expression of TNF?,ICAM-1,CCR7,IL2 RA,IRF4,IRF1 and VEGF gene were significantly down-regulated when the function of YAP was inhibited.The result collectively indicated that YAP can regulate the downstream target genes of NF-?B signaling pathway and thus affect the development of ATL.Taken together,we found in this study that YAP was highly expressed in ATL,and it activated the classical NF-?B signaling pathway.Mechanism studies have shown that YAP can bind to p65,and YAP can enhance the binding of p65 and p50 and promote the entry of p65 into nucleus.Finally,it was proved that YAP could regulate the downstream target genes of NF-?B signaling pathway.This study provides a new idea to elucidate the molecular mechanism of the continuously activation NF-?B signaling pathway in the late stage of ATL.It also provides a new target and strategy for the clinical diagnosis and treatment of ATL.