Adiponectin Promote TPO Response for Treating ITP By Relating Rab6A-myosin9 Trafficking Vehicle Carrying c-Mpl to Cell Surface

Abstract

Impaired megakaryocyte maturation is one of the pathogenic mechanisms of primary immune thrombocytopenia (ITP). Exploring the regulatory mechanisms of megakaryocyte maturation is important for the clinical treatment of ITP. The thrombopoietin (TPO) receptor c-Mpl on the membrane surface of megakaryocytes plays a critical role in the cellular reception of TPO signals and megakaryocyte maturation. But how the distribution of c-Mpl on the membrane surface of megakaryocytes influences the pathogenesis of ITP and the specific regulatory mechanisms are still unclear. In this project, we found that the distribution of c-Mpl on the membrane surface of megakaryocytes of ITP patients was significantly reduced, confirming that the abnormal distribution of c-Mpl is associated with the pathogenesis of ITP. Further, we applied immunoprecipitation and protein profiling to find the intracellular c-Mpl transport-related protein molecules. Motor protein myosin 9 and G protein-associated protein Rab6A were screened out. Next, we down-regulated the expression of myosin 9 and Rab6A, proved that aberrant expression of both leads to reduced transportation of the c-Mpl to membranes and impaired megakaryocyte maturation, identifying the regulatory complex for c-Mpl transport to membranes was composed of myosin 9 and Rab6A. Moreover, we confirmed that the formation of the above complex is dependent on GTP activation of Rab6A by transient overexpression of wild-type Rab6A (wt), dominant-negative (T27N) and GTPase-deficient (Q72L) Rab6A, clarified the working mechanism of how the Rab6A-myosin9 trafficking vehicle regulate c-Mpl transport to membranes. At the same time, we demonstrated that the abnormally low expression of Adiponectin in the plasma of ITP patients affects the transportation of the c-Mpl to membranes by inhibiting the interaction between Rab6A and myosin 9, thereby reducing the responsiveness of megakaryocytes to TPO and inhibiting the activation of its corresponding intracellular signaling pathways. Finally, using a mouse model of ITP disease, we demonstrated that Adiponectin could mediate the transportation of the c-Mpl to membranes by regulating the Rab6A-myosin9 complex and can improve the efficacy of TPO in the treatment of ITP. In conclusion, the present study identified a novel trafficking complex mediating the transportation of the c-Mpl to membranes, clarified the abnormal mechanism of this complex in ITP, and confirms that Adiponectin can regulate this complex to improve the efficacy of TPO, which provides a new idea for the clinical treatment of ITP.