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

在这项研究中，我们成功地产生了RasGRP 3缺陷小鼠。我们发现在RasGRP 3缺陷的B cAls中，BCR介导的Ras活化受损，并且在B细胞中Ras的活化主要是由于RasGRP 3的活化，这与以前的一些报道中认为Ras的活化是由SOS途径介导的相反。血清抗DNA抗体滴度显著高于对照组，提示自身反应性B细胞的选择可能受损。为了证实这个问题，我们将小鼠与抗BEL IG转基因小鼠杂交，并将B细胞转移到sBEL表达受体小鼠中。结果我们发现转移的B细胞没有完全缺失，这表明RasGRP 3缺陷的B细胞中的凋亡受损。为了进一步分析这个问题，在表面BCR的强连接后，检测了线粒体膜电位，这对凋亡的调节至关重要，BCR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，线粒体膜电位失调。此外，我们还发现RasGRP 3缺陷的B细胞中bc 1 -2的激活受到了抑制，这可能是RasGRP 3缺陷的B细胞中凋亡受到抑制的原因之一。

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.