[Toxoplasma gondii Rhoptry Protein 17 Inhibits the Apoptosis of Mouse Macrophages via Activation of Activator Protein 1 Signaling].

Abstract

Objective: To investigate the effect of Toxoplasma gondii rhoptry protein 17（ROP17） on γ-interferon (IFN-γ)-induced apoptosis of mouse J774A.1 monocyte macrophages.

Methods: The J774A.1 cells were transfected with recombinant plasmid p3×Flag-CMV-14/TgROP17 or empty plasmid p3×Flag-CMV-14. After addition of IFN-γ, flow cytometry and Western blotting were performed to detect apoptosis and the protein levels of phosphorylated c-Jun and apoptosis-related proteins cleaved Caspase-3, Bcl-2, Bcl-xL and Bcl-3. The p3×Flag-CMV-14/TgROP17 plasmid and c-Jun shRNA were co-transfected into J774A.1 cells, after which IFN-γ was added to induce cell apoptosis. The levels of cleaved Caspase-3 and Bcl-3 were analyzed using Western blotting.

Results: Flow cytometry showed that the apoptosis rate of cells overexpressing ROP17［（3.73±0.51）%］ was significantly lower than that of the control cells［（7.78±1.10）%, P<0.05］. Western blotting showed significant differences in protein levels of phosphorylated c-Jun（0.196±0.028 vs. 0.075±0.010）, Bcl-3（0.461±0.063 vs. 0.108±0.013） and cleaved Caspase 3（0.015±0.004 vs. 0.174±0.026） between the cells overexpressing ROP17 and control cells （all P<0.05）. However, the levels of Bcl-2 and Bcl-xL were not significantly different between the cells overexpressing ROP17 and the control. When the expression of c-Jun and phosphorylation of c-Jun were inhibited by c-Jun shRNA, the relative level of cleaved Caspase 3 in the RNA interferenced cells and control cells was 0.147±0.024 and 0.087±0.010, respectively (P<0.05), and the relative level of Bcl-3 was 0.085±0.010 and 0.162±0.011, respectively (P<0.05).

Conclusion: The anti-apoptosis effect of ROP17 is dependent on the phosphorylation of c-Jun and the expression of Bcl-3.