Studies On Molecular Mechanisms Of Rapamycin Inhibits Hsbaff-stimulated B-cell Proliferation And Survival Through Ca²⁺-CaMKⅡ-mediated MTORC1/2 And PP2A-PTEN/Erk1/2 Signaling

The present study, using cellular and molecular biology techniques and methods including cell culture, trypan blue exclusion,cell counting,MTS assay, flow cytometry, RNA interference and Western blotting, etc., and employing Raji cells,Daudi cells and mouse spleen B lymphocytes as experimental objects, systematically investigated the role of rapamycin in inhibition of hsBAFF-stimulated B cell proliferation and survival, the mechanisms of rapamycin inhibition of hsBAFF-activated Erkl/2-dependent B-cell proliferation and survival, the mechanisms of rapamycin inhibition of hsBAFF-stimulated B-cell proliferation and survival via PP2A-Erkl/2 signaling pathway, the mechanisms of rapamycin inhibition of hsBAFF-stimulated B-cell proliferation and survival via suppressing mTOR-mediated PP2A-Erkl/2 signaling pathway, the mechanisms of rapamycin inhibition of hsBAFF-stimulated B-cell proliferation and survival via targeting both mTORC1 and mTORC2 pathways. In addition, we also dissected the molecular mechanisms of rapamycin inhibition of hsBAFF-stimulated B-cell proliferation and survival through Ca2+-CaMKII-dependent PTEN/Akt-Erkl/2 signaling pathway. The detailed results were summarized as follows:1. Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survivalRaji cells, Daudi cells or/and purified mouse splenic B lymphocytes were stimulated with different concentrations of hsBAFF (0.5-5 μg/ml) for 48 h; or pretreated with 50-200 ng/ml rapamycin for 2 h and then treat with 2.5 μg/ml hsBAFF for 48 h. After that, analysis for MTS, cell counting, trypan blue exclusion and flow cytometry was conducted. The results showed that hsBAFF stimulated B-cell proliferation and survival in a dose-dependent manner. Pretreatment with rapamycin obviously attenuated hsBAFF-induced proliferation and survival in the cells dose-dependently. Our findings indicate that rapamycin may inhibit hsBAFF-stimulated B-cell proliferation and survival.2. Rapamycin blocks hsBAFF activation of Erkl/2-dependent B-cell proliferation and survivalRaji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with 50-200 ng/ml rapamycin for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h, or pretreated with 0.1-25 μM PD98059 for 1 h and then stimulated with 2.5μg/ml hsBAFF for 12 h, or co-pretreatment with 5 μM U0126, 10 μM PD98059 for 1 h and then stimulated with 1 and 2.5 μg/ml hsBAFF for 12 h or 48 h, or pretreated with 50 and 100 ng/ml rapamycin for 2 h, co-pretreatment with 1 and 10μM PD98059 for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. In some cases, Raji cells, infected with Ad-MKK1-R4F, Ad-MKK1-K97M or shRNA Erk1/2, were pretreated with 100 ng/ml rapamycin for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. After that, the cell proliferation and survival were evaluated by cell counting and MTS assay, the changes of protein exppression for the related pathway were determined by Western blotting. The results showed that rapamycin inhibited hsBAFF-activated Erkl/2 pathway in a dose-dependent manner,pretreatment with PD98059, down-regulation of Erkl/2, expression of dominant negative MKK1 potentiated rapamycin’s suppression of hsBAFF-induced activation of Erkl/2 and proliferation/viability in B cells, whereas expression of constitutively active MKK1 attenuated the inhibitory effects of rapamycin. The findings suggest that rapamycin blocks hsBAFF-activated Erkl/2-dependent proliferation and survival in B cells.3. Rapamycin attenuates hsBAFF-stimulated B-cell proliferation and survival by PP2A-Erkl/2 signaling pathwayRaji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with 50-200 ng/ml rapamycin for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h, or pretreatment with 100 ng/ml rapamycin for 2 h, co-pretreatment with 100 nM OA for 1h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. In some cases,Raji cells, infected with Ad-PP2A and Ad-dn-PP2A, were pretreated with 100 ng/ml rapamycin for 2 h, co-pretreatment with 10 μM PD98059 for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. After that, the cell proliferation and survival were evaluated by cell counting and MTS assay, the changes of protein expression for the related pathway were determined by Western blotting. The results showed that rapamycin down-regulated hsBAFF-induced expression of de-PP2A and p-PP2A.Overexpression of wild-type PP2A potentiated rapamycin’s suppression of hsBAFF-induced activation of Erkl/2 and proliferation/viability in B cells, whereas inhibition of PP2A by OA or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. The findings imply that rapamycin attenuates hsBAFF-stimulated B-cell proliferation and survival by targeting PP2A-Erkl/2 signaling pathway.4. Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival via suppressing mTOR-mediated PP2A-Erk1/2 signaling pathwayRaji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with 100 ng/ml rapamycin for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. In some cases, Raji cells, infected with rapamycin-resistant and kinase-active mTOR (Ad-mTOR-T), rapamycin-resistant and kinase-dead mTOR(Ad-mTOR-TE) or shRNA mTOR, were pretreated with 100 ng/ml rapamycin for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. After that, the cell proliferation and survival were evaluated by cell counting and MTS assay, the changes of protein expression for the related pathway were determined by Western blotting. The results showed that rapamycin inhibited hsBAFF-stimu lated phosphorylation of S6K1/4E-BP1 and Akt, as well as proliferation and survival in the cells,rapamycin inhibited hsBAFF-stimulated PP2A-Erk1/2 pathway in a mTOR kinase dependent manner, down-regulate mTOR inhibited hsBAFF-stimulated PP2A-Erkl/2 pathway and proliferation/viability in the cells. The data reveal that rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival via suppressing mTOR-mediated PP2A-Erkl/2 signaling pathway.5. Rapamycin inhibits hsBAFF-stimulated B cell proliferation and survival via targeting both mTORCl and mTORC2 pathwaysRaji cells and/or purified mouse splenic B lymphocytes were pretreated with 100 ng/ml rapamycin or 1 μM PP242 for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h; or pretreatment with 100 ng/ml rapamycin for 2 h, co- pretreatment with/without 20μM Akt inhibitor X for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. In some cases, Raji cells, infected with Ad-S6K1-wt,Ad-S6K1-ca, Ad-4EBP1-5A, Ad-myr-Akt, Ad-dn-Akt, shRNA Raptor, shRNA Rictor,shRNA Raptor/Rictor,shRNA S6K1 or shRNA 4E-BP1,were pretreated with 100 ng/ml rapamycin or 1 μM PP242 for 2 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. After that, the cell proliferation and survival were evaluated by cell counting and MTS assay, the changes of protein expression for the related pathway were determined by Western blotting. The results exhibited that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin. This is supported by the evidences that silencing Raptor, Rictor or Raptor/Rictor enhanced rapamycin’s inhibition of hsBAFF-induced B-cell proliferation and survival. PP242 also inhibit hsBAFF-stimulated survivin and cell proliferation/viability, and the inhibitory effect is more powerful than rapamycin. In addition, down-regulation of S6K1, ectopic expression of 4E-BP1-5A, dominant negative Akt, or co-treatment with Akt inhibitor X also potentiated rapamycin’s prevention against B-cell proliferation/viability, while Ad-S6K1-ca, down-regulation of 4E-BP1, Ad-myr-Akt attenuated inhibitory effect of rapamycin. Our findings uncover that rapamycin inhibits B-cell proliferation and survival via suppressing hsBAFF-induced activation of mTORC1-mediated S6K1 and 4E-BP1 pathways, as well as mTORC2-mediated Akt pathway.6. Rapamycin relieves hsBAFF-stimulated B-cell proliferation and survival through Ca2+-CaMKII-dependent PTEN/Akt-Erkl/2 signaling pathwayRaji cells and/or purified mouse splenic B lymphocytes were pretreated with 100 ng/ml rapamycin, co-pretreatment with/without 20 μM BAPTA/AM, 100 μM EGTA,100 μM 2-APB or 10 μM KN93 for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. In some cases, Raji cells, infected with Ad-PTEN, Ad-dn-Akt or shRNA CaMKII, were pretreated with 100 ng/ml rapamycin for 2 h, 20 μM Akt inhibitor X or 5 μM U0126 for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h. After that, the cell proliferation and survival was detected by cell counting and MTS assay, the changes of protein expression for the related pathway were determined by Western blotting. The results showed that overexpression of PTEN or dominant negative Akt potently strengthened rapamycin’s inhibition of hsBAFF-induced p-Erkl/2, Bcl-2 and survivin. BAPTA/AM, EGTA, 2-APB co-treatment with/without rapamycin inhibit hsBAFF-stimulated p-PTEN, p-Akt,p-Erkl/2, Bcl-2 and survivin, as well as cell proliferation and survival, of note, the inhibitory effect of co-treatment with rapamycin is more powerful. Inhibition of CaMKII by KN93 or down-regulation of CaMKII by RNA interference suppressed hsBAFF-induced p-CaMKII, p-PTEN, p-Akt, p-Erkl/2, Bcl-2 and survivin, as well as cell proliferation/viability, and significantly potentiated rapamycin’s inhibitory effect.Our data suggest that rapamycin relieves hsBAFF-stimulated B cell proliferation and survival through Ca2+-CaMKII-dependent PTEN/Akt-Erkl/2signaling pathway.