Studies On Molecular Mechanisms Of HsBAFF-inhibited Autophagy Contributing To Proliferation And Survival In B Cells

The present study, using cellular and molecular biological techniques of cell culture, fluorescence staining, RNA interference, MTS assay, Western blotting, etc., and employing B lymphoma cell line Raji cells as experimental objects, investigated the relationship of hsBAFF-stimulated proliferation and survival to autophagy in B cells, and deeply explored the role and molecular mechanisms of hsBAFF in promoting B-cell proliferation and survival via affecting autophagy by Ca2+/CaMKII-Akt/mTOR signaling pathway. The detailed results were summarized as follows:1 hsBAFF inhibits autophagy contributing to B-cell proliferation and survivalRaji cells, or Raji cells infected with shRNA LC3-Ⅰ/Ⅱ, shRNA GFP or Ad-GFP-LC3, respectively, were stimulated with different concentrations of hsBAFF (0,0.5,1,1.5,2.5,5μg/ml) for 12 h, or with/without 2.5μg/ml hsBAFF for different time (0,4,8,12,24,48 h), or with hsBAFF (0,1,2.5μg/ml) for 12,24 and/or 48 h. After that, the manifestation of cell autophagy was detected using MDC staining and GFP-LC3 staining, cell proliferation and survival were evaluated using MTS assay and cell counting, and expression of LC3Ⅰ/Ⅱ, p62 and Beclinl proteins was determined by Western blotting. The results showed that hsBAFF inhibited autophagy and caused decrease of LC3Ⅱ expression concentration-and time-dependently in B cells, which was consistent with hsBAFF-stimulated B-cell proliferation and survival. Down-regulation of LC3Ⅰ/Ⅱ attenuated hsBAFF-promoted proliferation and survival in B cells. The findings suggest that hsBAFF inhibits autophagy contributing to proliferation and survival in B cells.2 hsBAFF prevents autophagy contributing to B-cell proliferation and survival via activation of Akt/mTOR signaling pathwayRaji cells, Raji cells infected with Ad-dn-Akt or Ad-GFP, or Raji cells with shRNA LC3Ⅰ/Ⅱ、shRNA mTOR or shRNA GFP, respectively, were pretreated with/without rapamycin (100 ng/ml) or PP242 (1μM) for 2 h, or with/without Akt inhibitor X (10μM) or CQ (5μM) for 1 h, then stimulated with/without 2.5μg/ml hsBAFF for 12 h or 48 h. After that, cell proliferation and survival were evaluated using MTS assay and cell counting, and expression of LC3Ⅰ/Ⅱ, p62, Beclinl,S6K1 and Akt proteins was determined by Western blotting. The results exhibited that Akt inhibitior X, rapamycin or PP242 caused p62 decrease and LC3Ⅱ increase, and meanwhile blocked hsBAFF-induced phosphorylation of Akt and S6K.1. Down-regulation of LC3Ⅰ/Ⅱ enhanced Akt inhibitor X or rapamycin suppression of hsBAFF-promoted proliferation and survival in B cells. Expression of dn-Akt potentiated rapamycin or PP242 blockage of hsBAFF-inhibited autophagy contributing to B-cell proliferation and survival. Silencing mTOR strengthened Akt inhibitor X or PP242 prevention of hsBAFF-inhibited autophagy contributing to proliferation and survival in the cells. The findings reveal that hsBAFF prevents autophagy contributing to proliferation and survival via activation of Akt/mTOR signaling pathway in B cells.3 hsBAFF blocks autophagy contributing to B-cell proliferation and survival through Ca2+-CaMKⅡ activation of Akt/mTOR signaling pathwayRaji cells, or Raji cells infected with shRNA CaMKⅡ or shRNA GFP, respectively, were pretreated with/without PP242 (1μM) for 2 h, or with/without BAPTA/AM (20μM), EGTA (100μM),2-APB (100μM) or KN93 (10μM) for 1 h, then treated with 2.5μg/ml hsBAFF for 12 h or 48 h. After that, cell proliferation and survival were evaluated using MTS assay and cell counting, and expression of LC3Ⅰ/Ⅱ, p62, Beclinl, S6K1, Akt and CaMKII proteins was determined by Western blotting. The results showed that chelating [Ca2+]i with BAPTA or reducing [Ca2+]i with EGTA or 2-APB increased expression of LC3Ⅱ, and meanwhile prevented hsBAFF-induced phosphorylation of Akt and S6K1. However, these effects are more significant in the cells pretreated with co-combination of BAPTA, EGTA or 2-APB with PP242, respectively, indicating that hsBAFF-inhibited autophagy contributing to proliferation and survival by activation of Akt/mTOR signaling pathway was Ca2+-dependent in B cells. BAPTA/AM, EGTA or 2-APB inhibited hsBAFF-induced phosphorylation of CaMKⅡ, Inhibition of CaMKⅡ by KN93 significantly increased expression of LC3Ⅱ, and decreased hsBAFF-induced phosphorylation of Akt, S6K1 and CaMKⅡ, yet these events were more potent in the cells pretreated with co-combination of KN93 with PP242. Silencing CaMKⅡ enhanced PP242 blockage of hsBAFF-inhibited autophagy contributing to B-cell proliferation and survival. The data imply that hsBAFF blocks autophagy contributing to proliferation and survival through Ca2+-CaMKⅡ activation of Akt/mTOR signaling pathway in B cells.