TAB1-a New Target Of Triptolide In Macrophage

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 without affecting the proliferation of them. In this study, we took a ’comprehensive’ approach to identifying the molecular target of triptolide in macrophage, including in vivo, ex vivo and in vitro experiments. Our work can be categorized into items as following:1. TP binds to Tabl proteinTo isolate the binding protein of TP, the monoclonal antibody to TP (anti-TP) was prepared. Then the Co-Immunoprecipitation with anti-TP and protein A/G agarose was used to pull down the TP-binding protein. The Takl-binding protein 1(Tabl) protein was identified by Mass Specrometry. Co-Immunoprecipitation and Western-blot using anti-Tab1 antibody reconfirmed the Tab1 as the target of TP.We prepared fluorescence-tagged TP probe by modification of the hydroxyl group at position 14 of TP by using Coumarin derivatives (Br-mmc), which named TP-mmc. To identified the combination between TP and Tab1 in living cell, we treated cell line with TP-mmc and then stained endogenous Tab1 with immunofluorescence assay. The merged color showed the existence of TP-Tab1 complex.2. TP binds to recombinant Tabl-his in vitroTo investigate the characterization of TP-Tab1 complex in vitro, the his6-tagged Tabl was successfully expressed in bacterial and purified via metal-affinity columns. The interaction of TP with Tabl-his at physiological pH 7.43 was studied by molecular spectroscopy including UV-visible and synchronous fluorescence spectra. The alteration of characteristic absorption peak of TP suggests that interactions occur between TP and Tabl-his. The synchronous fluorescence spectroscopy reflects the fact that the microenvironment of the tryptophan residue was significantly affected by TP binding. The equilibrium dialysis and UV-vis spectra were used to investigate the interaction of TP and Tabl-his. The maximum binding amount (amax) is 3.53μM and the binding constant (Ka) is 0.116μM-1.3. TP inhibits Takl kinase activity through attenuation of Takl-Tabl Complex formation in ana-1 cellsThe immunoprecipitation and immunoblotting analyses revealed that TP decreased LPS-induced Takl-Tabl complex formation both when Takl and Tabl were present at endogenous levels in cell line. Furthermore, TP also decreased the phosphorylation of Takl at Thr 187 which is necessary for Takl activation. Then, we examined whether TP affects Takl-induced phosphorylation of MKK4 and MKK3/6 since these MKKs are the direct substrate of Takl kinase. The results indicated that the phosphorylation level of both MKK4 and MKK3/6 were reduced by TP pretreatment in LPS-induced ana-1 cell line.We next examined whether TP inhibits LPS-induced JNK and p38 activation. We pretreated ana-1 cells with TP and stimulated the cells with LPS. The activated JNK and p38 were detected with anti-phospho-JNK and anti-phospho-p38 antibodies. The result shows that LPS-dependent JNK and p38 activation was abrogated with treatment of TP. The above results suggest that TP inhibits the Takl signaling pathway at Takl or downstream of Takl, e.g. MKKs and MAPKs.4.TP inhibits Tak1 kinase activity through attenuation of Tak1-Tab1 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. The data shows that TP has the same effects on Tak1-Tab1 complex and the downstream of Takl both in vivo and ex vivo.In this study, we explored and identified that Tab1 was the target of TP in macrophages. To ensure the interactions between them, we investigate the characterization of TP-Tab1 complex in vitro. We also found that TP inhibits Tak1 kinase activity through attenuation of Tak1-Tab1 Complex formation both in cell line and in mice. In conclusion, elucidation of the mechanisms of action of TP offers not only new insights into the cellular functions of Tab1-Takl complex but also evidences for Tab1 as a novel therapeutic target in inflammatory disease.