Studies On Molecular Mechanisms Of HsBAFF Promoting B-cell Proliferation Through PTEN/Akt-Erk1/2 Signaling

The present study, using cellular and molecular biology techniques and methods including cell culture, MTS assay, RNA interference, Western blotting, etc., and employing Raji cells as experimental objects, investigated the role of PTEN activity and Akt signaling in hsBAFF-induced B-cell proliferation and survival, and further discussed the relation of PTEN/Akt function activity to activation of Erkl/2 pathway in the process of hsBAFF promoting B-cell proliferation and survival, by upregulating PTEN expression, or inhibiting Akt activity or dominant negative Akt. This study is aimed at providing theoretical foundation and scientific basis for understanding occurrence of BAFF-regulated B lymphadenoma and autoimmune diseases. The results were summarized as follows:1. hsBAFF-triggered B-cell proliferation and survival is associated with inhibition of PTEN and activation of AktRaji cells were treated with 0-0.25 μg/ml hsBAFF for 12 or 48 h, or treated with 0.25 μg/ml hsBAFF for 0-24 h, or treated with hsBAFF (0.1 and 0.25μg/ml) for 12 h or 48 h following pretreatment with/without Akt inhibitor X(10 μM) for 1 h. In some cases, Raji cells, infected with Ad-PTEN and Ad-GFP (as control), respectively, were treated with hsBAFF (0.1 and 0.25 μg/ml) for 12 h or 48 h. After that, cell proliferation and survival were evaluated by cell counting and MTS assay. Expression of PTEN, Akt and their related signaling molecules was determined by Western blotting. The results showed that hsBAFF promoted B-cell proliferation and survival in dose-and time-dependent manner, increased expression of survivin as well as phosphorylation of PTEN, Akt, S6K1,4E-BP1 and GSK3β. Inhibition of Akt activity blocked hsBAFF-induced increases of Bcl-2 and survivin, as well as phosphorylaiton of S6K1 and GSK3β, thereby attenuating cell proliferation and survival. Overexpression of PTEN partially inhibited hsBAFF-induced elevation of Bcl-2 and survivin, as well as phosphorylation of PTEN, Akt, S6K1,4E-BP1 and GSK3β, and thus reduced cell proliferation and survival. The findings reveal that hsBAFF inhibits PTEN and activates Akt contributing to B-cell proliferation and survival.2. hsBAFF promotes B-cell proliferation and survival via activation of Akt-Erkl/2 signaling pathwayRaji cells, Raji cells infected with Ad-dn-Akt、Ad-MKK1-K97M, were pretreated with Akt inhibitor X (10 μM) or PD98059 (10 μM) for 1 h, followed by adding hsBAFF (0.1 and 0.25 μg/ml) for treatment of 12 h or 48 h. After that, cell proliferation and survival were evaluated by cell counting and MTS assay. Expression of related signaling molecules was determined by Western blotting. The results showed that hsBAFF regulated Erkl/2 activity contributing to proliferation and survival by Akt pathway in Raji cells. This is supported by the findings that inhibition of Akt activity attenuated hsBAFF-induced phosphorylation of Erkl/2 as well as proliferation and survival in Raji cells, expression of dominant negative Akt partially blocked hsBAFF-induced phosphorylation of Erkl/2 as well as proliferation and survival in Raji cells, and expression of dominant negative MKK1 inhibited proliferation and survival in Raji cells. The findings reveal that hsBAFF promotes B-cell proliferation and survival via activation of Akt-Erkl/2 signaling pathway.3. hsBAFF promotes B-cell proliferation and survival via inhibiting PTEN activation of Erkl/2 pathwayRaji cells, infected with Ad-PTEN and Ad-GFP, were treated with hsBAFF (0.1 and 0.25 μg/ml) for 12 h, or pretreated with PD98059 (10 μM) for 1h, followed by adding hsBAFF (0.25 μg/ml) for treatment of 12 h or 48 h. After that, cell proliferation and survival were evaluated by cell counting and MTS assay. Expression of related signaling molecules was determined by Western blotting. The results showed that overexpression of PTEN attenuated hsBAFF-induced phosphorylation of Erkl/2, elevated PD98059-inhibited hsBAFF promoting proliferation and survival in Raji cells. The findings reveal that hsBAFF promotes B-cell proliferation and survival via inhibiting PTEN activation of Erkl/2 pathway.