Wilson B E, Mochon E, Boxer L M
Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center, California, USA.
Mol Cell Biol. 1996 Oct;16(10):5546-56. doi: 10.1128/MCB.16.10.5546.
Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.
成熟B细胞表面免疫球蛋白的结合可使其免于凋亡并促进增殖。随着表面免疫球蛋白的交联，bcl-2 mRNA和蛋白水平会升高。我们已在5'侧翼区域定位到B细胞中控制bcl-2表达的主要正调控区域。该正调控区域可分为上游和下游调控区域。下游调控区域包含一个环磷酸腺苷反应元件（CRE）。我们通过抗体超迁移实验以及紫外线交联后变性聚丙烯酰胺凝胶电泳表明，CREB和ATF家族成员在体外均能结合该区域。导致CREB结合丧失的CRE位点突变也会导致在瞬时转染实验中bcl-2启动子功能活性的丧失。bcl-2启动子中存在活性CRE位点意味着bcl-2表达的调控与B细胞中的信号转导途径相关。用抗免疫球蛋白M或佛波醇12-肉豆蔻酸酯13-乙酸酯处理成熟B细胞系BAL-17会导致由CRE位点介导的bcl-2表达增加。用佛波醇酯处理更不成熟的B细胞系Ramos可使细胞免于钙依赖性凋亡。佛波醇酯处理后bcl-2表达增加，且增加的表达依赖于CRE位点。这些刺激导致CREB在丝氨酸133处磷酸化。导致激活的CREB磷酸化是由蛋白激酶C而非蛋白激酶A介导的。尽管CRE位点是必需的，但bcl-2表达的最佳诱导需要上游调控元件的参与，这表明CREB的磷酸化改变了其与上游调控元件的相互作用。bcl-2启动子中的CRE位点似乎在成熟B细胞激活过程中以及未成熟B细胞免于凋亡过程中bcl-2表达的诱导中起主要作用。CRE位点有可能负责在其他细胞类型中诱导bcl-2表达，特别是那些涉及蛋白激酶C的细胞类型。
