Abstract

Polyclonal CD19+ B cells isolated from Bcl2-overexpressing Eμ-Bcl2 transgenic mice (Eμ-Bcl2tg+) overexpress SOCS3 protein. We previously reported that enforced expression of Bcl2 in BaF3 pro-B cells induces SOCS3 protein via p44/42 MAP Kinase activation. We also found that SOCS3 protein overexpression is an independent poor prognostic factor in patients with Bcl2-positive follicular lymphomas. We hypothesized that Bcl2 mediates induction of SOCS3 indirectly via cellular intermediaries that may alter cell signaling and/or apoptotic pathways in B cells. In the current study, magnetic bead B cell negative selection was used to isolate polyclonal CD19+ B cells from 24 week old Eμ-Bcl2tg+ mice (n=6). Bcl2-interacting proteins were then isolated from Eμ-Bcl2tg+ B cells by immunoprecipitation using a polyclonal anti-Bcl2 antibody. Bcl2 protein complexes separated by SDS-PAGE revealed several bands, with the most prominent bands noted at 70kDa and 90kDa, respectively. These bands were analyzed by mass spectrometry (LC MS/MS) and protein database analysis identified them as Heat Shock Proteins (HSP) 70 and 90β. LC MS/MS results were verified by co-immunoprecipitation of both HSP70 and HSP90β with Bcl2 in Eμ-Bcl2tg+ B cells. Co-immunoprecipitation assays with SOCS3 and Bax revealed no interaction with this protein complex. 5 × 106 Eμ-Bcl2tg+ B cells from individual mice were then cultured in microtiter plates using Supplented Iscove’s Modified Dulbecco’s Medium (SIMDM) with 10% FBS and were treated over 12 hours with escalating doses of the HSP90 inhibitor 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG). Immunoprecipitation with Bcl2 revealed that 17-AAG blocked the interaction between Bcl2 and HSP90β in a dose-dependent manner. Furthermore, Western analysis of Eμ-Bcl2tg+ B cells treated with 17-AAG revealed a marked decrease in SOCS3 protein expression compared with untreated cells. In addition, Eμ-Bcl2tg+ B cells exposed to increasing concentrations of 17-AAG showed a concentration-dependent increase in activity of caspase 3 and increased cell death compared with untreated cells. Next, to determine whether Bcl2-mediated SOCS3 overexpression in B cells involves STAT3-dependent or -independent mechanisms, we measured phospho-STAT3 levels relative to STAT3 in Eμ-Bcl2tg+ B cells cultured in the presence of escalating doses of IL-6. When probed with phospho-STAT3 and STAT3 antisera, Eμ-Bcl2tg+ B cells did not reveal STAT3 protein phosphorylation but revealed equivalent STAT3 protein levels in response to IL-6. Eμ-Bcl2tg+ B cells were then cultured as above in the presence or absence of a specific p44/42MAPK pathway inhibitor (MEK 1/2 inhibitor U0126), and whole cell lysates were analyzed for SOCS3 expression by Western analysis. When probed with SOCS3 antisera, B cells selectively grown in the presence of U0126 revealed complete abrogation of SOCS3 protein expression while those grown in the absence of inhibitor continued to harbor SOCS3 protein. Bcl2 protein levels remained unchanged in the presence or absence of U0126. Inhibitors of p38MAPK (SB203580), AKT (Triciribine), and PI3K (LY294002) did not affect SOCS3 expression. These results indicate that Bcl2-associated SOCS3 induction in murine B cells is dependent upon a HSP70/90 complex and involves activation of p44/42 MAPK. These studies illustrate a novel signaling pathway in B cells important for the regulation of caspase-mediated apoptosis.

Author notes

Disclosure: No relevant conflicts of interest to declare.