Mechanism Of RIP2Promoting B-NHL Anti-apoptosis By Regulating NF-κB Signal Pathway

B-cell non-Hodgkin lymphoma (B-NHL) represents the most common malignancy derived from B lymphocyte. Although the updated scheme of combined therapy has significantly improved the treatment efficiency, the effect on aggressive B-NHL is not significant and40%of the patients with B-NHL still died of the recurrence and resistance to treatment. Studies indicated that the activation of non-canonical and canonical NF-κB pathway mediated by BAFF-R on B-NHL cell membrane is closely related to B-NHL cell apoptosis, contributing to the process, prognosis and therapy of B-NHL. Therefore, the exploration of the mechanism for B-NHL anti-apoptosis mediated by non-canonical and canonical NF-κB activation might provide important information for the studies on molecular diagnosis and specific targeted drugs of B-NHL.Receptor-interacting protein2(RIP2) is a serine/threonine protein kinase belonging to the RIP kinase family. Previous study showed that RIP2was extensively expressed in a variety of human tissues and highly in Raji cell, a B-NHL cell line, analyzed by RNA blot of human tissue and cell line, suggesting that RIP2might play an important role in the survival of B-NHL cell. Our previous study also found that the high expression of RIP2existed in peripheral blood leukocycte from B-NHL patients and specimens from B-NHL patients with resistance, respectively, further suggesting that RIP2might be closely associated with B-NHL anti-apoptosis. Additionally, studies have found that RIP1, a member of RIP kinase family, is a negative regulator of the non-canonical NF-κB pathway in TNFR1signaling. The activation of non-canonical NF-κB results from the accumulated NIK, whereas dominant negative mutation of NIK inhibits RIP2-induced NF-κB activation. The data implied that RIP2might participate in the regulation of non-canonical pathway of NF-κB. The non-canonical NF-κB pathway is triggered by signaling from a subset of TNFR members, including LTβR, CD40, BAFF-R and RANK. Transmembrane-expressed BAFF-R only has a TRAF binding site in its intracellular domain, by which it specifically binds with TRAF3. Evidence suggests that TRAF3 plays an important role in negative regulation of NIK by serving as an only intracellular adaptive molecule of BAFF-R. Moreover, our previous studies found that RIP2and TRAF3can interact, further suggesting that RIP2might participate in the regulation of BAFF-NF-κB pathway through TRAF3. However, the effects of RIP2on the non-canonical NF-κB pathway mediated by BAFF-R and the mechanism for regulating B-NHL anti-apoptosis in B-NHL are not yet clear at present.We firstly obtained RIP2fusion protein with high purity and directly observed the effects of RIP2on proliferation, survival and apoptosis of tumor cells in vitro. Previous study reported that over-expression of RIP2induced apoptosis and inhibited proliferation in human MCF-7breast carcinoma cells. Our study showed that the purified rhRIP2inhibited the growth of MCF-7cells, suggesting that the rhRIP2produced in this study possesses its biological activity. However, our results also showed that rhRIP2could stimulate the growth of Ramos cells and U87MG cells, suggesting that RIP2is characteristic of the cell specificity. Further study displayed that the cell specificity of RIP2in vitro was associated with the expression of anti-apoptotic protein Bcl-xL regulated by RIP2. Subsequently, we investigated the mechanism by which RIP2promotes B-NHL cell anti-apoptosis.On the basis of our previous work, we further validated the interaction between RIP2and TRAF3, and determined the TRAF3-interacting region on RIP2. Co-immunoprecipitation(CoP) and GST pull-down assay demonstrated that the specific interaction in vivo and in vitro exists between RIP2and TRAF3. Previous study demonstrated that over-expression of RIP2can induce the transcription activity of NF-κB reporter gene. Therefore, we analyzed the influence of TRAF3on RIP2-induced NF-κB transcription activation activity. The result showed that TRAF3possessed significant inhibitory effect on the RIP2-induced the activation of NF-κB reporter gene transcription and in a dose dependent manner. Besides, the mammalian cell two-hybridization method was used to screen for the TRAF3-interacting site on RIP2. The result indicated that RIP2could interact with TRAF3-C through its intermediate region (390-435aa residues). TRAF3is involved in the regulation of non-canonical NF-κB pathway by serving as an only intracellular adaptive molecule of BAFF-R and negative regulator of NIK. In addition, our results confirmed that RIP2can specifically interact with TRAF3. These data suggest that RIP2may be involved in the regulation of non-canonical NF-κB and canonical NF-κB pathway through TRAF3. We further explored the regulation mechanism of RIP2on BAFF-R-mediated non-canonical NF-κB pathway. Results displayed that knockdown of endogenous RIP2expression could down-regulate the expression of NIK kinase and suppressed the activation of p100in Ramos cells. These results suggest that RIP2was involved in the activation of BAFF-R-mediated non-canonical NF-κB pathway and might play an important role by serving as a positive regulator in non-canonical NF-κB pathway.Recent studies demonstrated that the accumulation of NIK enhances IKK complex activity, leading to augmented activation of canonical NF-κB pathway. Therefore, we observed whether the suppression of NIK kinase expression by RIP2could affect the activation of NIK-mediated canonical NF-κB pathway or not. Our results showed that knockdown NIK could suppress the phosphorylation level of IκBα and p65in the Ramos cells, whereas RIP2could not. Additionally, results showed that knockdown RIP2, on the basis of knockdown NIK, further suppressed phosphorylation level of IκBα (S32) and p65(S536),compared with that of knockdown NIK. These results indicated that RIP2and NIK might have synergistic action on the BAFF-R mediated canonical NF-κB pathway.Both canonical NF-κB pathway and non-canonical NF-κB pathway mediate cell inflammatory reaction and affect the expression of proinflammatory cytokines. Moreover, the expression of inflammatory factors also plays an important role in the genesis and development of B-NHL cell. Therefore, we observed whether the positive regulation of RIP2in non-canonical NF-κB pathway could affect the expression of inflammation-related factors or not. Real time qRT-PCR results demonstrated that knockdown RIP2could down-regulate the expression of TNFα and ICAM-1at the mRNA level. ELISA results also showed that knockdown RIP2could down-regulate the secretion level of TNFα and soluble ICAM-1(sICAM-1) in the cell supernatant. The above-mentioned results suggested that RIP2might be involved in NF-κB mediated inflammatory reaction. Besides, the activation of NF-κB pathway mediates cell anti-apoptosis and promotes cell survival. In this study, we also observed the regulation effect of RIP2on cell anti-apoptosis. The results displayed that knockdown RIP2promoted the apoptosis of Ramos cell and significantly down-regulated the expression of Bcl-xL, an anti-apoptosis protein. Additionally, results showed that knockdown RIP2, on the basis of knockdown NIK, further promoted the apoptosis of Ramos cell and suppressed the expression of Bcl-xL, compared with that of knockdown NIK. The above-mentioned results indicated that RIP2plays an important role in B-NHL cell anti-apoptosis and the regulation effect of RIP2on non-canonical NF-κB pathway might be one of molecular mechanisms of B-NHL cell anti-apoptosis.Additionally, another investigator of our group found that the expression of RIP2in B-NHL specimens was closely related to the activation of non-canonical NF-κB pathway. Especially, the expression of RIP2in specimens from B-NHL patients with rituximab resistance was significantly elevated, which was remarkably different from that of B-NHL patients with non-resistance. Our research revealed that RIP2was involved in the activation of BAFF-R-mediated non-canonical NF-κB pathway and closely associated with B-NHL cell anti-apoptosis. We speculated that high expression of RIP2might play an important role in rituximab resistance. For this reason, we further observed the effect of RIP2in Rituximab-induced B-HNL cell apoptosis and investigated its potential mechanism. Primary results showed that in the basis of the knockdown RIP2, addition Rituximab could significantly suppress NIK kinase expression and p100activation. Based on the knockdown RIP2and NIK, the addition of Rituximab could affect the phosphorylation level of IκBα (S32) and p65(S536) as well as the expression of p65in BAFF-R mediated canonical NF-κB pathway in comparison with that of Rituximab alone in Ramos cells. It suggest that depletion of endogenous RIP2protein in Ramos cells can enhance the suppression effect of Rituximab on BAFF-R mediated non-canonical NF-κB pathway, and the combination with knockdown NIK can augment the suppression effect of Rituximab on canonical NF-κB pathway. Additionally, the expression of inflammatory factors and anti-apoptic protein also demonstrated that knockdown RIP2in Ramos cells could promote the antagonist effects of Rituximab against inflammatory factors, such as TNFa and ICAM-1, and significantly enhanced the suppression effect of Rituximab on Bcl-xL expression. Results of flow cytometry showed that knockdown RIP2together with Rituximab significantly induced cell apoptosis. Additionally, results showed that knockdown RIP2together with Rituximab, on the basis of knockdown NIK, further promoted the apoptosis of Ramos cell and suppressed the expression of Bcl-xL, compared with that of knockdown NIK together with Rituximab. The above-mentioned results suggest that RIP2is the potential therapy target of NF-κB activation, and RIP2depletion together with Rituximab can improve the therapeutic efficiency of Rituximab in B-NHL cell as well as increase the Rituximab-induced B-NHL cell apoptosis. In summary, this study confirmed that RIP2and TRAF3possess a specific interaction in vivo and in vitro, and also revealed that RIP2was involved in the activation of BAFF-R mediated non-canonical NK-κB pathway and regulated the canonical NK-κB pathway synergistic with NIK through TRAF3, thereby participating in NK-KB-mediated anti-apoptosis and inflammatory reaction. The primary study also showed that the effects of RIP2on regulation of canonical NK-κB and non-canonical NK-κB affected therapeutic efficiency of Rituximab on B-NHL cell. The above-mentioned results might be helpful to better understand the regulation mechanisms of B-NHL cell anti-apoptosis and provide critical clues for B-NHL clinical therapies.