The Effects And Mechanisms OfNMAAP1 Binding To IP3R And RegulatingM1 Macrophage Polarization

Regulation of adaptive and innate immune responses by tumor cells affects tumor development and progression. As an important part of innate immune system,macrophage is an abundant population of infiltration cells in tumors. Therefore,macrophage plays an important role in tumor development and progression.Monocytes recruited from the circulation differentiate into mature macrophages and then inhabit most tissues of the body(e.g., liver, brain, bone, lung, spleen) and exert an important function in the physiological and pathological processes. In 1988, Elie Metchnikoff initially described that macrophage as phagocytic cells responsible for pathogen elimination and tissue homeostasis. In Today’s Era of immunology,macrophage is mediators that shape the innate immune response and adaptive immune response, and play an indispensable role in the immune system. Recent studies have shown that heterogeneity and plasticity were key features of mononuclear phagocytes and their activation states. The capability of macrophages to express distinct functional phenotypes is typically manifested in microenvironmental conditions, and polarized macrophages can be broadly classified in two main groups:classically activated macrophages(or M1) and alternatively activated macrophages(or M2). M1 exhibit potent microbicidal and tumoricidal properties, elevated phagocytosis and promote strong IL-12-mediated Th1 responses. M2 polarized macrophages exert robustly clearance of debris, promote trophic factor synthesis, tissue remodeling and repair, promote cancer cell growth and spread, angiogenesis and immunosuppression,accompanied by reduced pro-inflammatory cytokine secretion. This paradoxical role of macrophages in cancer offers an explanation in their functional plasticity, which may result in the polarized expression of either pro- or anti-tumoural functions.BCG–activated macrophage(BAM) obtains surprisingly high tumoricidal activities.Bacilli Calmette-Guérin(BCG) is a strong stimulus that is used for the treatment of superficial bladder cancer in clinical treatment.BCG-activated macrophage acts ascutostatic/tytotoxic effector cells, which exerttumoricidal activity through highly expressed adhesion molecular, cell-cell contact and/or release of soluble cytotoxic effector molecules.In previous study, we found BCG-activated macrophage exerts tumoricidal activity to MCA207 sarcoma tumor cells through cell-cell dependent cytotoxicity, and this process is closely related with membrane proteins upon BCG-activated. So, we compared membrane proteins from BAM with thioglycolate-elicited macrophage(TEM) using capillary liquid chromatography tandem mass spectrometry(LC-MS/MS), there are 454 proteins specifically expressed in BAM, and then we selected a novel protein NMAAP1(Novel macrophage activated associated protein 1)through protein structure prediction, protein sequence analysis and bioinformatics analysis to illustrate the key effector molecular of macrophage during anti-tumor process.NMAAP1 is a novel Ca2+ dependence allosteric regulator of IP3 R binding protein, which is a member of DANGER superfamily. DANGER superfamily members are closely related with evolutionary developmental biology, and regulate cell and tissue differentiation and outgrowth processes. NMAAP1 specifically expressed in BCG-activated macrophage, whether it can regulates macrophage differentiation and biological activity. But it is not clear that whether NMAAP1 can exert this function by regulating intracellular Ca2+ concentration.IP3R is the most ubiquitous intracellular Ca2+-release channel predominantly located on the endoplasmic reticulum(ER) and responsible for a controlled release of Ca2+ ions in the cytoplasm, which is crucial for maintaining intracellular Ca2+dynamic balance. Calcium(Ca2+) acts as a versatile signal and second messenger in all cells. Its plays an important roles in the regulation of such physiological and pathological processes as cell differentiation, proliferation, growth, death, secretion,gene expression, transcription, biomembrane fusion, embryonic development and morphogenesis. In macrophages, intracellular Ca2+ was shown to regulate several cell functions such as the production of TNF-α and NO, improve macrophage-mediatedtumoricidal activity, regulatephagosome maturation, and improve macrophage phagocytosis. In DCs, Ca2+ was reported to promote activation and maturation and to play a role in DC responses to TLR ligands or bacteria.It is not clear whether NMAAP1 bindingto IP3 R regulates intracellular Ca2+ concentrationand modulatesmacrophage biologicalactivity.To identify the physiological role of NMAAP1 in macrophage, our research includes the following three parts:Part I: There is correlation between NMAAP1 expression and phenotype in BCG-activated macrophage.To delineate the correlation between NMAAP1 expression and phenotype of macrophage, the mice were challenged i.p with 2 mg BCG, and after 4 days, 8 days and 12 days we collected BCG-activated peritoneal macrophage and serum. We detected CD16/32 and CD23 markers expression of M1/M2 cells respectively using flow cytometers, detected genes expression of NMAAP1 and IL-10 by quantitative RT-PCR,detected IL-10 and TNF-α release by ELISA in the serum.Part II: The effects of NMAAP1 on biological functions of macrophageTo illustrate whether NMAAP1 could regulate macrophage biological functions,we constructed ON/RAW264.7 cell line which stably expressing full-length murine NMAAP1 and OV/RAW264.7 cell line which stably expressing pIRES2-EGFP vector.Then we evaluatedGFP expression through fluorescence microscope, identified NMAAP1 gene and protein expression using RT-PCR and western blotting. We assessed the effects of NMAAP1 to macrophage phenotype using FACS, RT-PCR and ELISA, the results revealed that up-regulated NMAAP1 expression in RAW264.7cells increased the expression of M1 markers, e.g., Inducible nitric oxide synthase(iNOS), tumor necrosis factor alpha(TNF-α), Interleukin 6(IL-6),Interleukin 12(IL-12), Monocyte chemoattractant protein-1(MCP-1) and Interleukin-1 beta(IL-1β); and decreased or did not affect M2 markers, e.g.,Kruppel-like factor 4(KLF4) and Suppressor of cytokine signaling 1(SOCS1),Arginase-1(Arg-1), Interleukin(IL-10), Transforming growth factor beta(TGF-β)and found in inflammatory zone 1(Fizz1), the Phagocytosis of fluorescent microspheres of OV/RAW264.7 and ON/RAW264.7 cells were measured using fluorescence microscope and flow cytometry; We next evaluated the influence of NMAAP1 on the tumoricidal activity of macrophages using MTT, 7AAD/Annexin-V dual staining and Hoechst 33342 staining. The results revealed that ON/RAW264.7cells presented a more prominent cytotoxicity effect for the inhibition of MCA207 tumor cell viability than OV/RAW264.7 cells, and the main source of cytotoxicity activity is the supernatants of OV/RAW264.7 and ON/RAW264.7 rather than cell-cell contact dependent tumoricidal function. These data suggest that NMAAP1 promote macrophage M1 differentiation, and enhance phagocytosis and tumoricidal activity through the secretion of inducible mediators.PartⅢ: The mechanism of NMAAP1 regulates macrophage polarization and functionsWe examined the interaction between NMAAP1 and IP3 R using co-localization and Co-Immunoprecipitation. And we determined the intracellular Ca2+concentration using flow cytometric analysis of cells that was stained with the fluorescent calcium probe Fluo-3/AM.We found that NMAAP1 can interact with IP3 R to lead the increase for the intracellular Ca2+levels. We next tested the effect of increased Ca2+flux on Ca2+ related signaling cascade involving PKC, ERK1/2, CaM and p65 in OV/RAW264.7 and ON/RAW264.7 cells using western blot. These results demonstrate that NMAAP1 to be a novel IP3R-interacting and co-localization membrane protein enhanced the Ca2+release activity of IP3 Rs in RAW264.7 cells, and increased intracellular Ca2+activated Ca2+/CaM/CaMKII and Ca2+/PKC/MAPK signaling pathway, which polarized macrophage to M1.The study is the first ever to prove that NMAAP1 can regulate macrophage M1 polarization, and exerts robustly phagocytosis and soluble cytokines dependent tumoricidal activity, which may also be important in clearing invading pathogens and enhancing tumor cells apoptosis. In the discovery process of regulatory mechanism of NMAAP1 in macrophage, we demonstrated NMAAP1 co-localized with IP3 R anddirectly bond to full-length IP3 R in vitro by co-immunoprecipitation and increased the intracellular Ca2+ release, induced a series of Ca2+ related signaling cascade, which cause a large amounts of pro-inflammatory cytokines secretion. These studies illustrated NMAAP1 as a promising protein to modulate macrophage phenotype and physiological functions and provide further evidences and understand for the mechanism of macrophage anti-tumor activity.