HsBAFF-upregulated intracellular free Ca2+ homeostasis regulates ERK1/2 activity and cell proliferation in B cells in vitro

Abstract

We studied hsBAFF activity in in vitro mouse splenic B cells. hsBAFF effects on intracellular free Ca(2+) concentration ([Ca(2+)](i)) were assayed, using a laser scanning confocal microscope with fluorescent probe, Fluo-3/AM. We showed that treatment of B cells with 0.5-5 microg/ml hsBAFF resulted in significantly higher [Ca(2+)](i) levels in a dose-dependent fashion at 12 and 24 h, respectively (p<0.05 or p<0.01 vs. control). Furthermore, we noticed that 2.5 microg/ml hsBAFF-treated cells were significantly resistant to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor (p<0.05 hsBAFF plus Tg group vs. Tg group). Thus hsBAFF may promote B cell survival by direct upregulation of [Ca(2+)](i) physiological homeostasis contributing to prevention of [Ca(2+)](i) dysfunction. Using immunocytochemistry and Western blot analysis, we found that the activation of ERK1/2 due to hsBAFF was triggered by a [Ca(2+)](i) -dependent pathway, leading to elevation of B cell proliferation. This is supported by the findings that intracellular Ca(2+) chelator BAPTA/AM attenuated phosphorylated ERK1/2 expression and cell proliferation in hsBAFF-stimulated B cells. hsBAFF-stimulated B cell proliferation was obviously reduced by mitogen extracellular kinase 1/2 (MEK1/2, upstream of ERK1/2) inhibitor U0126. Taken together, the main finding of this study is that hsBAFF elicits higher but homeostatic [Ca(2+)](i) levels, which regulates ERK1/2 activity and cell proliferation in in vitro B cells.