The Molecular Mechanism Of TP Inhibiting Macrophage Activation Mediated By Tab1

Triptolide (TP) is a biologically active diterpene triepoxide from a Chinese herb Tripterygium wilfordii Hook F, commonly known as thunder god vine. Extracts from this plant have been historically used in traditional Chinese medicine to treat inflammatory and autoimmune diseases such as rheumatoid arthritis, systemic lupus, psoriatic arthritis and Behcet’s disease. The studies of TP’s potent antiproliferative and immunosuppressive properties have uncovered some of its specific molecular targets.However, the identification of TP targets so far may not explained well that TP can inhibit the activation of immunocyte, such as macrophages, in the low nanomolar range whitout affecting the proliferation of them. Our previous works have shown that Takl-binding protein1(Tabl) was the target of TP in macrophages. In this study, we focused on the molecular mechanism of TP inhibiting macrophage activation mediated by Tabl. Our work can be categorized into items as following:1.TP inhibits Takl kinase activity through attenuation of Takl-Tabl Complex formation in ana-1cells The immunoprecipitation and immunoblotting analyses revealed that TP decreased LPS-induced Takl-Tab1complex formation both when Takl and Tabl were present at endogenous levels in cell line. Furthermore, TP also decreased the phosphorylation of Takl at Thr187which is necessary for Takl activation.Then, we examined whether TP affects Tak1-induced phosphorylation of MKK4and MKK3/6since these MKKs are the direct substrate of Takl kinase. The results indicated that the phosphorylation level of both MKK4and MKK3/6were reduced by TP pretreatment in LPS-induced ana-1cell line.We next examined whether TP inhibits LPS-induced JNK and p38activation. We pretreated ana-1cells with TP and stimulated the cells with LPS. The activated JNK and p38were detected with anti-phospho-JNK and anti-phospho-p38antibodies. The result shows that LPS-dependent JNK and p38activation was abrogated with treatment of TP. The above results suggest that TP inhibits the Tak1signaling pathway at Tak1or downstream of Tak1, e.g. MKKs and MAPKs.2. TP inhibits Takl kinase activity through attenuation of Takl-Tabl Complex formation in vivoTo verify in vivo the relevance of our ex vivo results demonstrating the effects of TP, we used a well established shock model involving mice sensitized to LPS. a single i.g. dose of triptolide (100μg/kg of body weight) was administered at18hours before subsequent coinjection with LPS(4mg/kg of body weight). The data shows that TP has the same effects on Takl-Tabl complex and the downstream of Takl both in vivo and ex vivo.In conclusion, We discovered that TP inhibits Tak1kinase activity by interfering with the formation of the Takl-Tabl complex, and the binding affinity of TP to Tabl correlated highly with the inhibitory activity of TP against MAPK pathway activation in macrophages.