Studies On Mechanisms Of HsBAFF-activated Erk1/2Leading To Proliferation Via Ca²⁺-caMKII-dependent Inhibition Of PP2A In B Cells

The present study, using RNA interference, Western blotting, cell culture, flow cytometry, etc., via in vivo and in vitro experiments, investigated the role of PP2A activity and Erkl/2signaling in hsBAFF-sitmulated B cell proliferation, and further discussed relation of hsBAFF-upregulated intracellular free calcium ions ([Ca2+]i) to PP2A activity and Erkl/2activation through chosing Ca2+chelator or CaMKII inhibitor. This study is aimed at providing theoretical foundation and scientific basis for understanding mechanisms of BAFF-regulated humoral immunity and preventive treatment of related diseases. The results were summarized as follows:1. hsBAFF activates Erkl/2contributing to cell proliferation via inhibition of PP2A in B cellsFor in vivo experiment, four ICR healthy 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. After day8of injection, purified spleen B lymphocytes were isolated from splenic cell suspensions using anti-CD19magnetic fluorobeads. For in vitro experiment, Raji cells and purified normal mouse splenic B lymphocytes was seeded in6-,24-or96-well plates, and treated with0-5μg/ml hsBAFF for12or48h, or treated with2.5μg/ml hsBAFF for different time (0-24h). In above experiments, the changes of cell proliferation, PP2A activity, Erkl/2protein expression were assayed. To clarify the role of PP2A-Erkl/2signaling cascades in promoting B cell proliferation, Raji cells were infected by Ad-MKK1-R4F and Ad-MKK-K97M to constitutively active or inactivate MKK1activity, or by Ad-PP2A-wt to induce overexpression of PP2A. We found that after day8of in vivo administration of hsBAFF, purified spleen B lymphocyte proliferation increased in a concentration-dependent fashion, and dose-dependent increases of demethylated-PP2A, phosphor-PP2A and phosphor-Erkl/2in B lymphocytes from hsBAFF-administered mice also existed. The results observed by in vitro experiments showed that hsBAFF-stimulated B cell proliferation was associated with its inhibiting PP2A leading to Erkl/2phosphorylaiton. This is supported by the findings that constitutively active or inactivate MKK1, respectively, enhanced or attenuated hsBAFF-induced Erkl/2phosphorylation and proliferation in B cells, and overexpression of PP2A partially prevented hsBAFF-activated Erkl/2and B cell proliferation. The findings reveal that hsBAFF activates Erkl/2contributing to cell proliferation via inhibition of PP2A in B cells.2. hsBAFF induces Erkl/2activation and cell proliferation via Ca2+-CaMKII-dependent inhibition of PP2A in B cellsRaji cells were seeded in6-or24-well flat-bottomed plates, cells were treated with0,1and2.5μg/ml hsBAFF for12or48h following treatment with BAPTA/AM (20μM), EGTA (100μM),2-APB (100μM) or KN93(10μM) for1h. Western blotting was performed to test t PP2A activity and activation of Erkl/2, and cell proliferation was evaluated using cell counting. The results showed that BAPTA/AM, EGTA and2-APB attenuated hsBAFF-inhibited PP2A activity, thereby reducing Erkl/2phosphorylation and B cell proliferation, indicating hsBAFF-inhibited PP2A activation of Erkl/2and B cell proliferation is Ca2+-dependent. KN93, a CaMKII inhibitor, obviously prevent the phosphorylation of Erkl/2and cell proliferation via blocking hsBAFF-induced inhibition of PP2A activity in B cells, revealing that hsBAFF-stimulated CaMKII inhibition of PP2A activates Erkl/2and B cell proliferation. Our data suggest that hsBAFF induces Erkl/2activation and cell proliferation via Ca2+-CaMKII-dependent inhibition of PP2A in B cells.