PAX5 Regulates Nf-kB Signal Pathway Through Binding To RIP2 In Multiple Myeloma

Multiple Myeloma (MM) is a kind of malignant blood cancer which is formed by the transformation from normal B cells to cancerous plasma cells. The high monoclonal immunoglobulin level in the serum often appears in Multiple Myeloma with the companion of bone destruction, kidney failure and anemia. Lots of causes have the ability to induce Multiple myeloma, including ionizing radiation, chemical carcinogen and viral infection. These inducers could make the B cell genome unstable through affecting chromosome translocation, epigenetic alterations and gene mutation and deregulate the causative genes associated with multiple myeloma. It has been identified that a large part of Multiple Myeloma samples possess the high PAX5 expression after the analysis of gene expression profiles in numbers of multiple myeloma patients. As a B cell specific transcription factor, PAX5 promotes the expression of B cell-lineage genes (CD 19, BLNK et al), meanwhile blocking the non-B cell lineage and plasma cell genes transcription (Notchl, XBP-1 et al). A procedural expression of PAX5 exists in B cell differentiation, emerging in the pro-B cell phase and shutting down during the formation of plasma cells which is essential for the B cell terminal differentiation. Considering that The role of PAX5 in the pathogenesis of multiple myeloma still remains unknown, we focus on this point to initiate our project.We first silenced PAX5 expression using PAX5 shRNA knockdown technology in IM9 multiple myeloma cell line contains high level of PAX5, establishing IM9/Control and IM9/PAX5-KD cell with PAX5 stable knockdown, In drugs-induced apoptosis assay, the increasing sensitivity of IM9 to Bortezomib, Doxorubicin and Vinblastine was observed after knocking down PAX5. We also found the tumor formation capacity of IM9/PAX5-KD cells was attenuated and bortezomib facilitated IM9/PAX5-KD tumor shrink compared with IM9/Control tumor in tumor-bearing nude mice. Next, the western blot data showed the TNF-a and LPS induced-NF-κB activation was strongly enhanced by PAX5 which performed the effect through binding to RIP2 kinase. The interaction domains of PAX5 and RIP2, which involved intermediate domain of PAX5 and kinase domain of RIP2 respectively, were identified by Pull down assay and CoIP detection. The association between PAX5 and RIP2 was responsible for the RIP2 ser176 phosphorylation located in RIP2 kinase domain. RIP2 has been widely known as a crucial protein in NOD-NF-κB pathway, but our results suggested that RIP2 participated in TNF-α-induced NF-κB activation through RIP2 ser176 phosphorylation-regulated K209 ubiquitination. In a word, PAX5 binding to RIP2 facilitates RIP2 ser176 phosphorylation following with the activation of inducible NF-κB pathway. Similarly, Bortezomib, Doxorubicin and Vinblastine could activate RIP2 ser176 and NF-κB signal which was dependent on PAX5 expression in IM9. When we specifically inhibited NF-κB activation, the difference of bortezomib and vinblastine-induced apoptosis between IM9/Control and IM9/PAX5-KD was vanished. It indicated that PAX5-regulated NF-κB activation acted as a pivotal role in bortezomib and vinblastine-induced drug resistence.With the unclear function of PAX5 in multiple myeloma pathogenesis and treatment, our work first suggests that PAX5 induces the fall of drug sensitivity of multiple myeloma containing high PAX5 level. The phenomenon which is meaningful and beneficial for the following research and clinical treatment, has been well explained by our study on the PAX5-regulated NF-κB pathway. We also find out PAX5 is able to bind to RIP2, affecting RIP2 and NF-κB activity. The finding elevates the understanding of PAX5 function, especially in the part of B cell differentiation and B cell carcinoma. Beside the novel outcomes above, we present a new regulation model about RIP2 ser176 phosphorylation and RIP2 ubiquitination, which could be helpful to understand the relationship between the two distinctive RIP2 posttranslational modifications. With the observation on RIP2 involved in TNF-a-induced NF-κB pathway, the hypothesis proposed by the former researchers acquires our experimental support, which considers that RIP2 possesses the similar function as RIP 1.