The Effect And Mechanism Of TIPE On Macrophage Function

BackgroundTumor necrosis factor alpha induced protein 8(TIPE) family has four members so far, which includs TIPE, TIPE1, TIPE2 and TIPE3. Their gene sequences are highly homology. They play important roles in cell proliferation, apoptosis, inflammation, oncogenesis as well as in maintaining immune homeostasis. The present study shows that TIPE is a cytoplasmic protein with a molecular weight of 21 kD, and it plays important roles in apoptosis, signal transduction, tumor growth, tumor invasion and metastasis. TIPE is not only expressed constitutively in many tumor tissues and cells, but also expressed in immune tissues and cells. The current research mainly focused on TIPE as a oncogene which inhibited tumor apoptosis and promoted tumor growth, invasion and metastasis. But it is not clear about the roles of TIPE in immune cells activation and function. As we know, Innate immunity is the first line of immune system to protect the body from infection. And macrophages as major effector cells play an important role in innate immunity. Therefore, it is necessary to make it clear whether TIPE is involved in macrophage proliferation and activation and its molecular mechanism. This study is essential in theory development and clinical application. ObjectiveTo study the effect of TIPE on activation and function of mouse macrophages by knocking down TIPE expression in RAW264.7 cells, and explore the molecular mechanism of TIPE in macrophage activation. MethodsTIPE gene silencing stable cell line was obtained by knocking down TIPE expression in RAW264.7 cells. Cell morphology was observed by microscope and cell adhesive ability was assessed by flat dish adhesion experiment. Cell proliferation was measured by clone formation assay and MTT asssy. Flow cytometry was performed to detect cell phagocytosis. Migration of macrophages was tested by woundhealing assay as well as transwell assay. ELISA kits were purchased and undertaken to detect inflammatory cytokines expression in cell culture supernatant. The molecules phosphorylation of MAPK signal pathway were mesured by western blot. ResultsTipe gene silence in RAW264.7 cells significantly impaired cell adhesive ability with 15 min, 30 min or 2 hours culture. Compared with the control cells, TNFAIP8 sliencing RAW264.7 cells looked round, with a smaller number of cellular extensions. MTT and clone formation assay data showed that knocking down TIPE expression decreased macrophages proliferation. The flow data indicated that TIPE knockdown inhibited macrophages phagocytosis. Both Wound-healing assay and transwell assay data showed that TIPE knockdown inhibited macrophages migration. According to ELISA data, the expression level of inflammatory cytokines in TIPE knockdown RAW264.7 cells were decreased significantly. After being induced by LPS, the increase of c-Raf, Erk1/2 and p38 phosphorylation were suppressed significantly by TIPE knocking down in RAW264.7 cells, which suggests that MAPK signal pathway may be regulated by TIPE protein. Conclusion1. TIPE knocking down inhibited macrophages adhesion, proliferation, phagocytosis and inflammatory cytokines secretion.2. c-Raf, Erk1/2 and p38 phosphorylation of MAPK signal pathway were downregulated in TIPE knocking down RAW264.7 cells.