Oda Akihisa, Ono Tomohiro, Yamamoto Mutsumi, Goitsuka Ryo, Kitamura Daisuke
Division of Molecular Biology, Tokyo University of Science, Noda, Chiba 278-0022, Japan.
Int Immunol. 2008 Nov;20(11):1417-26. doi: 10.1093/intimm/dxn101. Epub 2008 Sep 9.
Pre-B cell receptor (pre-BCR) signals promote pre-B cell differentiation, in which the adaptor protein B-cell linker (BLNK) plays a crucial role. However, the molecular pathways downstream of BLNK are currently unclear. Utilizing pre-B leukemia cell lines (BKO84 and others) derived from BLNK-deficient mice as in vitro models of the pre-B cell differentiation, we have demonstrated that reconstitution of BLNK as well as an active form of protein kinase C (PKC)eta induces the differentiation events, such as pre-BCR down-regulation and kappa gene rearrangement. Here we show that the same events are induced by cross-linking of pre-BCR with anti-mu antibody in these pre-B cell lines, as well as in ex vivo pre-B cells from BLNK-deficient mice, suggesting a function of BLNK as an internal cross-linker of pre-BCR. Anti-mu treatment of BKO84 cells up-regulated membrane recruitment of PKC eta and the expression of IRF-4, a transcription factor known to promote light chain gene rearrangements. Anti-mu induction of surface kappa chain on BKO84 cells was blocked by reagents that inhibit phospholipase C or PKC. Enforced expression of the active PKC eta in BKO84 cells resulted in up-regulation of IRF-4 expression. Conversely, siRNA-mediated silencing of PKC eta expression strikingly attenuated the anti-mu-induced IRF-4 expression and kappa gene rearrangement, which were restored by PKC eta reconstitution. Finally, enforced expression of IRF-4, but not of BLNK, in the PKC eta-silenced BKO84 cells resulted in kappa gene rearrangement. These results indicate that PKC eta directs the induction of IRF-4 expression downstream of BLNK in the pre-BCR signaling pathway promoting kappa gene rearrangement.
前B细胞受体（pre-BCR）信号促进前B细胞分化，其中衔接蛋白B细胞连接蛋白（BLNK）发挥关键作用。然而，目前尚不清楚BLNK下游的分子途径。利用源自BLNK缺陷小鼠的前B白血病细胞系（BKO84等）作为前B细胞分化的体外模型，我们已证明重建BLNK以及活性形式的蛋白激酶C（PKC）η可诱导分化事件，如前BCR下调和κ基因重排。在此我们表明，在这些前B细胞系以及来自BLNK缺陷小鼠的体外前B细胞中，用抗μ抗体交联前BCR可诱导相同的事件，这表明BLNK作为前BCR的内部交联剂发挥作用。用抗μ处理BKO84细胞可上调PKC η的膜募集以及IRF-4的表达，IRF-4是一种已知可促进轻链基因重排的转录因子。抑制磷脂酶C或PKC的试剂可阻断抗μ诱导的BKO84细胞表面κ链的表达。在BKO84细胞中强制表达活性PKC η可导致IRF-4表达上调。相反，siRNA介导的PKC η表达沉默显著减弱了抗μ诱导的IRF-4表达和κ基因重排，而PKC η重建可恢复这些现象。最后，在PKC η沉默的BKO84细胞中强制表达IRF-4而非BLNK可导致κ基因重排。这些结果表明，在促进κ基因重排的前BCR信号通路中，PKC η在BLNK下游指导IRF-4表达的诱导。
