Roles Of BCA3in Regulating The Function Of Macrophages

BackgroundIn2003,Kitching et al.cloned and characterized the cDNA of BCA3, a novel human breast cancerassociated gene which is highly expresssed in breast tumor cells and prostate tumor cell lines. The BCA3gene encode a proline-rich protein and there are a number of putative functional domains in this proteinsuggesting that it may indeed have multiple cellular roles. Qin et al. reported this protein to be aKyoT2-binding protein (KBP1), related to signaling of RBP-J which mediates transcriptional activation bythe Notch intracellular domain (NICD). BCA3/KBP1compets with RING1for binding to the LIM domainsof KyoT2. Overexpression of KBP1can abrogate the suppression of the NICD-mediated transactivation ofRBP-J by RING1. BCA3has also been reported to be a PKA-interacting protein (AKIP1) whichparticipates in transcription regulation of NFκB by regulating the phosphorylation of p65. BCA3participates in the regulation of these important signaling pathways, suggesting that it may play aregulatory roles in a variety of biological activities. Thomas Ho-Yin Leung et al. reported that BCA3canenhance the sensitivity of cervical cancer cells in response to irradiation-induced apoptosis by interactionwith TAp73.Kuan-ping Yu et al. reported that BCA3is a novel Rac1-interacting protein and overexpressionof BCA3markedly attenuated the spreading response to CSF-1in osteoclasts. Mature osteoclasts are highlymotile, multinucleated, terminally differentiated cells of the monocyte/macrophage lineage that areresponsible for resorbing mineralized bone. Therefore BCA3may be involved in regulating the function ofmacrophages. Macrophages are one of important immune cells. Quantities of studies have shown that thedysfunction of macrophages is closely related to autoimmune diseases, cancer, and other diseases. Thus ithas great guiding significance to explore the biological functions of BCA3in macrophages and immunesystem on the mechanisms of immune response and the treatment of many diseases.ObjectiveTo investigate the roles of BCA3in regulating the function of macrophages and the potientialmechanism. MethodsWestern blot was undertaken to detect the expression of BCA3in many kinds of immune cells andimmune tissues.we also detected the expression of BCA3during the macrophage differentiation.Knockdown BCA3protein by infection with recombinant lentivirus encoding a BCA3shRNA inRAW264.7and U937cell lines, and then investigated the change of biological function of these cells,focused on the response to inflammatory stimulus, such as the changes in phagocytosis, surface molecules,cytokine secretion and so on.Then we examined the activity of NFκB pathway in normal and BCA3knockdown macrophages by Western Blot. We also established inflammatory model in Balb/c mice byintraperitoneal injection of LPS, and investigated the expression of BCA3in peritoneal macrophages indifferent stages of immune response.In order to further analyze the roles of BCA3in macrophages, weanalysised the signaling pathways related to those differentially expressed genes by DNA microarrays.ResultsBCA3is highly expressed in various immune cells and immune tissues; and the expression of BCA3protein upregulated significantly during the macrophage differentiation. We found that BCA3regulatingthe functions of macrophages. Knockdown of BCA3expression by RNA interference in RAW264.7andU937cell lines,increased phagocytosis and production of inflammatory cytokines and chemotacticfactor,and upregulated some surface molecules such as CD14、CD11b、CD86.We found that constitutiveexpression of p65is upregulated along with the knockdown of BCA3and the phosphorylation of p65isenhanced after LPS-stimulation. In animal models of inflammation,the expression of BCA3in peritonealmacrophages is downregulated in the early stage and then return to normal level with the progress ofinflammation.The DNA microarrays revealed that many pathways are involved in those differentiallyexpressed genes induced by the knockdown BCA3.Some of those pathways are related to the functions ofmacrophages, such as antigen processing and presentation and toll-like receptor signaling pathway.ConclutionsBCA3is highly expressed in various immune cells,and regulates the functions of macrophagesthrough the NFκB pathway.Under physiological conditions,BCA3inhibit NFκB transcriptional activity toavoid host-tissue damage caused by the abnormal activation of macrophages. In the early stage ofinflammation, BCA3is downregulated,so the NFκB transcriptional activity is enhanced, and macrophagesbecame activated for phagocytosis as well as secretion of inflammatory cytokines to against infection.With the progress of inflammation, BCA3expression recovery to prevent the excessive activation ofmacrophages.