Functional Research Of Daintain In RAW264.7 Macrophages

Daintain was identified as a novel cytokine derived from macrophages in the mid of 1990s. It is a IFN-γ-induced, 17 kDa, cytoplasmic, calcium-binding and inflammation-responsive scaffold protein. The Daintain gene maps to the major histocompatibility complex class III region on chromosome 6p21.3, which is known for clusters of genes involved in the immune response, suggesting its important role in immune response. Although the detailed physiological functions of Daintain in vivo remain unclear, there is substantial evidence that its remarkable expression contributes to the pathogenesis of many inflammatory diseases, including organ-graft rejection, central lesion and autoimmunity. Therefore, Daintain is thought to play a fundamental role in monocyte and macrophage effector functions that contribute to inflammation and autoimmune reactions.It is well documented that estrogen, especially 17(3-estradiol (E2), is an immunomodulator and modulates cytokine gene expression. However, no data are available regarding the regulation of E2 on Daintain expression. In first part of the present study, we investigated the effects and molecular mechanisms of E2 on the mRNA and protein levels of Daintain using RAW264.7 cells, a commonly used mouse macrophage cell line. The main study is including:1) We investigated the effect of E2 on the expression of Daintain. E2 upregulated the mRNA and protein levels of Daintain in similar manners under physiological concentrations of 10-10 M to 10-7 M. Especially, under the concentrations of 10-10 M to 10-8 M, increasing the concentration of the incubated E2 resulted in a dose-dependent increase in Daintain production. Though the effect at 10-7 M was lower than that at 10-8 M, it was higher as compared to the control.2) It has been found that functional estrogen receptor a (ERa), but not estrogen receptorβ(ERβ), is constitutively expressed in RAW264.7 cells. The application of ICI 182,780, an estrogen receptor (ER) antagonist, attenuated E2-induced Daintain production, suggesting the involvement of ERa in this process. 3) Neither LPS alone nor LPS synergied with E2 had an effect on Daintain expression in RAW264.7 cells, suggesting that expression of Daintain is subject to direct hormonal control, which is different from other inflammatory factors.4) The mouse genome DNA was extracted from the macrophage cells RAW264.7, and from which 1.6 kb DNA sequence of Daintain gene 5'end UTR was amplified by PCR. The PCR product was directly inserted into pGL3-Basic vector by homologous recombination, and the luciferase report gene vector of mouse Daintain gene promoter (pGL3-Basic-DT(-1572_+59)) was well constructed. Additional constructs were generated by digestion of this plasmid with restriction enzymes to yield pGL3-Basic-DT(-1286_+59), pGL3-Basic-DT(-794_+59) and pGL3-Basic-DT(-595_+59). The construction of pGL3-Basic-DT vectors lay a foundation for further research on Daintain gene transcription regulation.5) The effect of E2 on Daintain gene promoter was detected by luciferase activity. E2 had a stimulated activity on pGL3-Basic-DT(-1572_+59), deletion from the 5'end revealed increasing promoter activity, with the greatest activity found in the pGL3-Basic-DT(-1286_+59). However, further 5'end deletion constructs, pGL3-Basic-DT(-794_+59) and pGL3-Basic-DT(-595_+59), lacked detectable promoter activity. The results suggest that sequences between-1286 bp and +59 bp contain the active region for E2.Daintain is constitutively expressed in monocytes and macrophages and is involved in macrophage activation. However, the detail functions of Daintain in macrophages remain unknown. In second part of the present study, we investigated the effect of Daintain on RAW264.7 proliferation and cell cycle. We also investigated the influence of Daintain on LPS-induced pinocytosis ability and the phenotype changes for antigen presentation in RAW264.7 cells. The main study is including:1) The stable over-expression of Daintain for RAW264.7 cells was well established.2) Daintain promoted the proliferation of RAW264.7 cells, probably via promoting more cells into S phage.3) Daintain enhanced LPS-induced pinocytosis ability in RAW264.7 cells, and facilitated the macrophages to differentiate into dendritic cells in phenotype. Moreover, Daintain upregulated the expression of surface antigen, including MHCⅡand CD86. Those effects of Daintain on RAW264.7 cells may due to it has stimulate effect on NF-κB activity.In conclusion, E2 upregulates Daintain expression via stimulating the Daintain gene promoter. Moreover, Daintain promotes the proliferation, pinocytosis ability and differentiation into dendritic cells in phenotype for RAW264.7 cells.