| The present study, using techniques and methods of cell culture, flow cytometry, Western blot analysis, etc., and chosing BAPTA/AM (an intracellular Ca2+chelator), EGTA (an extracellular Ca2+chelator), and2-APB (a membrane-permeable inhibitor of IP3receptors on the endoplasmic reticulum), KN93(a specific inhibitor for CaMKII) and rapamycin (a specific inhibitor for mTOR), under in vitro and/or in vivo experiment conditions, synthetically investigated the relation of hsBAFF-stimulated B lymphocyte proliferation with [Ca2+]i elevation. The role and molecular mechanisms of hsBAFF in promoting B lymphocyte proliferation through Ca2+-CaMKII signaling activation of mTOR pathway were discussed to provide new thread and theory for hsBAFF exertion of effective immune regulation. The results were summarized as follows:1Studies on mechanisms of hsBAFF activation of mTOR pathway and cell proliferation through elevated [Ca2+]i in B lymphocytesThe purified mouse splenic B lymphocytes were isolated using anti-CD19magnetic fluorobeads. Then, cell suspensions were seeded in96-well (2×10cells/well) or6-well (2×106cells/well) flat-bottomed plates and cultured at37℃,5%CO2. The next day, cells were treated with hsBAFF (2.5and5μg/ml) in the presence or absence of anti-IgM (2.5μg/ml) for12h or0-24h. In addition, B cells were treated with/without hsBAFF (5μg/ml) for12h following pre-incubation with/without BAPTA/AM, EGTA or2-APB for1h. Afterwards, cells were loaded with Fluo-3/AM and [Ca2+]i fluorescent intensity was detected using Flow Cytometry and fluorescent colorimetry, cell proliferation was evaluated using an MTT assay, and Western blotting was performed to test the phosphorylation of CaMKII, Akt, S6K1, S6and4E-BP1proteins. We showed that Treatment of B lymphocytes with hsBAFF or hsBAFF+anti-IgM resulted in significant elevation of [Ca2+]i in a time-or dose-dependent manner. We conformed hsBAFF-elevated [Ca2+]i activation Akt/mTOR pathway, leading to the increase of B lymphoctye proliferation using BAPTA/AM for chelating intracellular Ca2+. Reduction of [Ca2+]i with EGTA or2-APB revealed that hsBAFF-induced extracellular Ca2+influx and endoplasmic reticulum (ER) Ca2+release elevates [Ca2+]i contributing to B cell proliferation via activation of mTOR pathway. These results suggest that hsBAFF may induce extracellular Ca2+influx and ER Ca2+release, which is involved in [Ca2+]i elevation, thereby leading to mTOR signaling activation and proliferation increase of B lymphocytes. 2Studies on mechanisms of hsBAFF activation of mTOR pathway and cell proliferation via CaMKII phosphorylation in B lymphocytesThe purified mouse splenic B lymphocytes was isolated using anti-CD19magnetic fluorobeads. Then, cell suspensions were seeded in96-well (2×104cells/well) or6-well (2×106cells/well) flat-bottomed plates and cultured at37℃,5%CO2. The next day, cells were treated with hsBAFF (1-10μg/ml, or5μg/ml) in the presence or absence of anti-IgM (2.5μg/ml) for12h or0-24h. In addition, B cells were treated with/without hsBAFF (5μg/ml) for12h following pre-incubation with/without BAPTA/AM, EGTA,2-APB or KN93for1h. Afterwards, cell proliferation was evaluated using an MTT assay, and Western blotting was performed to test the phosphorylation of CaMKII, Akt, S6K1, S6and4E-BP1proteins. The results showed that hsBAFF induced CaMKII phosphorylation in a time-and concentration-dependent manner in B lymphocytes. Chelating intracellular Ca2+with BAPTA/AM or preventing [Ca2+]i elevation using EGTA or2-APB obviously inhibited hsBAFF-or anti-IgM plus hsBAFF-stimulated phosphor-CaMKII increase in B lymphocytes. We observed that KN93significantly suppressed hsBAFF-or anti-IgM plus hsBAFF-stimulated phosphorylation of CaMKII, Akt, mTOR-mediated S6K1and4E-BP1, as well as cell proliferation in B lymphocytes. The findings suggest that hsBAFF-induced CaMKII phosphorylation activates mTOR signaling pathway and cell proliferation via elevation of [Ca2+]i. in B lymphocytes.3Studies on in vivo hsBAFF activation of mTOR pathway and cell proliferation via calcium signaling in B lymphocytes as well as rapamycin regulationForty ICR mice, half males-half females were chosen and randomly divided into a normal control group (n=8); four hsBAFF treatment groups (n=8,8,8,8). The mice in four hsBAFF treatment groups were given abdominal cavity injection of hsBAFF solution which was diluted with phosphate buffered saline at dosage of0.1,0.5,1,2mg/kg body weight once each day for over eight days, respectively. The mice in control group were received abdominal injection of PBS at the same dose and frequency. On the9th day post injection, animals from each group were sacrificed via cervical dislocation to collect spleens under sterile conditions. Spleen B cells were isolated from splenic cell suspensions using anti-CD19magnetic fluorobeads, seeded at a density of2×106cells/well in6-well plates or2×104cells/well in96-well plates and cultured at37℃,5%CO2. Meanwhile, the purified normal mouse splenic B lymphocytes was isolated and seeded in6-well plates, then treated with5μg/ml hsBAFF with/without2.5μg/ml anti-IgM for12h following pretreatment with0.2rapamycin for2h. Cells were loaded with Fluo-3/AM and then [Ca2+]i fluorescent intensity was detected using fluorescent colorimetry, cell proliferation was evaluated using an MTT assay, and Western blotting was performed to test the phosphorylation of CaMKII, Akt, S6K1, S6and4E-BP1proteins. We observed that in vivo administration of hsBAFF dramatically stimulated B cell proliferation with a concomitant [Ca2+]i elevation in concentration-dependent fashion, and the activation of Akt and mTOR-mediated S6K1,S6and4E-BP1with a concomitant increase of CaMKII phosphorylaiton in B cells of hsBAFF-administered mice also existed. We found that pretreatment with rapamycin significantly inhibited hsBAFF and/or anti-IgM stimulation of [Ca2+]i elevation and of phosphorylated CaMKII increase in B lymphoctyes. These results indicates that B cell proliferation in hsBAFF-administered mice was related to Ca2+-CaMKII signaling activation of mTOR pathway; Rapamycin may inhibit hsBAFF-induced [Ca2+]i elevation and CaMKII phosphorylatio. |
