GPI-anchored protein-mediated cell signaling system in B-precursor cells

我们先前报道，分化簇(CD)24的交联会诱导Burkitt淋巴瘤细胞和B-preendsor ALL细胞的凋亡。CD24是一种GPI锚定蛋白，通过RAFT微域介导的细胞信号转导系统诱导细胞凋亡。为了研究GPI锚定蛋白在正常B前体细胞中的细胞信号转导作用，我们建立了CD34+细胞分化为Pro-B细胞的体外培养体系。利用该培养体系，我们发现CD24的交联可诱导正常人Pro-B细胞的凋亡。B细胞连接蛋白(BLNK)是B细胞受体(BCR)信号通路的组成部分，RAFT微区参与其中。我们研究了人Pre-B-ALL细胞系中BLNK的表达，发现四个被测试的细胞系中的一个，HPB-Null细胞，被发现缺乏BLNK的表达，我们使用这些表达和不表达BLNK的人Pre-B-ALL细胞系来研究Pre-BCR交联后的细胞内信号事件。当Pre-Bcr与抗μ重链抗体交联时，观察到细胞内分子包括Syk、Shc、ERK MAPK和AKT的显著磷酸化，以及RAS的激活，而不考虑BLNK表达的缺失，这表明BLNK不是Pre-BCR介导的MAP激酶和磷脂酰肌醇3(Pi3)激酶信号转导所必需的。相反，只有在表达BLNK的细胞中才观察到磷脂酶C-Gamma2(PLC-Gamma2)的磷酸化和细胞内钙离子浓度的升高，这表明BLNK在PLC-Gamma2诱导的Ca(2+)内流中是必不可少的。

We previously reported that the cross-linking of cluster of differentiation (CD)24 induces apoptosis in Burkitt's lymphoma cells as well as B-predursor ALL cells. CD24 is a GPI-anchored protein and this apoptosis induction is mediated by raft microdomain-mediated cell signaling system. To investigate the cell signaling mediated by GPI-anchored protein in normal B precursor cells, we developed an in vitro culture system to differentiate CD34+ cells into pro-B cells. Using this culture system, we found that the cross-linking of CD24 induced apoptosis in normal human pro-B cells.B-cell linker protein (BLNK) is a component of the B-cell receptor (BCR) signalling pathway, in which raft microdomain is deeply involved. We investigated BLNK expression in human pre-B ALL cell lines and found that one of the four cell lines tested, HPB-NULL cells, was found to lack BLNK expression, and we used these human pre-B ALL cell lines that express and do not express BLNK to investigate the intracellular signalling events following pre-BCR cross-linking. When pre-BCR was cross-linked with anti-μ heavy-chain antibodies, significant phosphorylation of intracellular molecules, including Syk, Shc, ERK MAP kinase, and AKT, and an activation of Ras were observed without regard to deficiency of BLNK expression, suggesting that BLNK is not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signalling. By contrast, phospholipase C-gamma2 (PLC-gamma2) phosphorylation and an increase in intracellular Ca(2+) level mediated by pre-BCR cross-linking were observed only in the BLNK-expressing cells, indicating that BLNK is essential for PLC-gamma2-induced Ca(2+) influx.