Studies On The Relationship Between Negative Costimulatory Molecule B7-H4and PI3K/AKT Signaling Pathway And Its Mechanisms

B7-H4, a member of B7family, is known to negatively regulate T cell immuneresponse. Early studies have shown that B7-H4is a transmembrane protein and is involvedin the regulation of immune response. Our previous study found that B7-H4contained anuclear localization sequence (NLS) and was capable of shuttling between cytoplasm andnucleus. B7-H4expressed on cell membrane has been shown to promote tumor growth byinhibiting T cell proliferation, but studies on nuclear B7-H4are rare. Individual studiesfound intracellular B7-H4is related to cell proliferation and apoptosis.Phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway is closelyrelated to cell growth, proliferation, tumor occurrence and development, and canparticipate in intracellular translocation of certain protein molecules. B7-H4can influenceT cell proliferation by inhibiting the activation of AKT signaling pathway in T cells,however, little known is the regulation of PI3K/AKT signaling pathway on B7-H4subcellular localization.This study aimed to construct wild type B7-H4and NLS mutant type B7-H4stablytransfected cell lines and characterize their biological functions primarily. Then explore therelationship between B7-H4and PI3K/AKT signaling pathway by controlling the activityof PI3K/AKT pathway in wild type B7-H4transfected cells.The paper includes the following two parts:Partâ…  Establishment of human B7-H4transfected cells and preliminary studyof its biological functionsObjective: To construct B7-H4WT/293, B7-H4H250Q MT/293and Mock/293stably transfected cell lines and characterize their biological functions.Methods: Recombinant plasmid (pIRES2-EGFP-B7-H4-WT, pIRES2-EGFP- H250Q-MT, pIRES2-EGFP) were transfected into HEK293cell line with Lipofectamine2000and the transfected cells were further selected with G418, whether the expressionvector was inserted into HEK293cell line was assayed by flow cytometry analysis.Observe B7-H4localization by immunofluorescence after treating these cell lines withLeptomycin B (LMB). The biological functions of transfected cells were researched byproliferation assay in vitro and in vivo.Results: Stable cell lines expressing human B7-H4WT and B7-H4H250Q MT wereestablished. Results of immunofluorescence further validate the existence of human B7-H4NLS. In vivo and in vitro studies on the biological function showed that nuclear B7-H4canpromote cell proliferation.Conclusion: The transgenic cell lines stably expressing B7-H4WT and B7-H4H250Q MT were obtained and afford effective tools for further study.Partâ…¡ Regulation of PI3K/AKT signaling pathway on the nuclear translocationof B7-H4Objective: To investigate the regulation of PI3K/AKT signaling pathway on thesubcellular distribution of negative co-stimulatory molecule B7-H4.Methods: The stably transfected B7-H4/HEK293cells were treated with the specificPI3K inhibitor LY294002, the subcellular localization of B7-H4was detected by ConfocalLaser Scanning Microscopy (CLSM). Then B7-H4WT/293cells were treated withLY294002, IGF-1, Wortmannin and Nutlin-3, changes of B7-H4subcellular localizationwere detected using Western blot. Then cell adhesion was detected before and afterLY294002treatment.Results: The result of CLSM demonstrated that LY294002could effectively inducethe nuclear translocation of B7-H4when compared with vehicle group. When the B7-H4WT/293cells were treated with LY294002, more B7-H4was translocated into nucleus.Results of Western blot showed that after the PI3K/AKT signal pathway was inhibited byLY294002for24h, the localization of B7-H4in cell membrane and cytoplasm wasdecreased significantly (p<0.05) while the localization in nucleus was increasedsignificantly (p<0.05). IGF-1treatment, on the contrary, reduced B7-H4nuclearlocalization. Rapamycin played a similar role to LY294002, while Nutlin-3had no significant effect on the localization of B7-H4. These results indicate that B7-H4subcellular localization may be regulated through PI3K-AKT-mTORC1pathway. Adhesionresults showed that nuclear B7-H4is associated with cell adhesion, and after LY294002treatment, adhesion of B7-H4WT/293was significantly enhanced.Conclusion: The PI3K/AKT signal pathway takes part in the regulation of B7-H4subcellular localization. And this regulation may be involved in PI3K-AKT-mTORC1pathway regulation.