Regulation of auto-reactive B cell activation by Ras signaling pathway

在本研究中，我们成功地产生了RasGRP3缺陷小鼠。我们发现在RasGRP3缺陷的B CAL中，BCR介导的RAS激活受到损害，B细胞中RAS的激活主要是由于RasGRP3的激活，这与以往的一些报道认为RAS的激活是通过SOS途径介导的形成相反。血清抗DNA抗体滴度显着高于对照组，提示自身反应性B细胞的选择可能受到损害。为了证实这一问题，我们用抗BEL Ig转基因小鼠与B细胞杂交，并将B细胞转移到表达sbel的受体小鼠中。结果我们发现，转移的B细胞并没有完全被删除，这表明RasGRP3缺陷的B细胞的凋亡受到了损害。为了进一步分析这一问题，我们检测了在细胞凋亡调控中至关重要的线粒体膜电位，并对表面BCR进行了强烈的连接，这是已知的诱导细胞凋亡的刺激之一。结果发现，在交叉结扎后的早期时间点，线粒体膜电位出现紊乱。此外，我们还发现RasGRP3缺陷的B细胞中bc1-2的活化受到抑制，这可能是RasGRP3缺陷的B细胞凋亡受损的原因之一。

In this study we are succeeded in the generation of RasGRP3-deficient mice. We found that BCR-mediated Ras activation is impaired in RasGRP3-deficient B cAls and that activation of Ras in B cells are mainly due to the activation of RasGRP3, which is in contrast with some of the previous reports which suggested activation of Ras is mediated by SOS pathway. Serum anti-DNAantilicdy titer was significantly higher than the control suggesting that selection of autoreactive B cells might be impaired. In order to confirm this issue, we crossed the mice with anti-BEL Ig transgenic mice and transferred the B cells into sBEL expressing recipient mice. As the result we found that transferred B cells are not completely deleted suggesting that apoptosis in RasGRP3-deficientB cells are impaired.To further analyze this issue, mitochondorial membrane potential, which is crucial for the regulation of apoptosis, was examined after strong ligation of surface BCR, which is one of the stimuli known to induce apopotosis. As the result, we found that mitochondorial membrane potential was disregulated at the early time point after the cross-ligation. Further, we found that activation of bc1-2 was impaired in RasGRP3-deficient B cells, which might be one of the reason which can explain the impaired apoptosis in RasGRP3-deficient B cells.