Gabellini Chiara, Castellini Laura, Trisciuoglio Daniela, Kracht Michael, Zupi Gabriella, Del Bufalo Donatella
Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
J Neurochem. 2008 Nov;107(3):871-82. doi: 10.1111/j.1471-4159.2008.05661.x. Epub 2008 Sep 11.
We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.
我们最近报道，bcl-xL可调节胶质母细胞瘤细胞中白细胞介素8（CXCL8）的蛋白表达和启动子活性。在本文中，我们证明bcl-xL诱导CXCL8是通过核因子-κB（NF-κB）依赖性机制介导的。对CXCL8启动子的突变研究表明，NF-κB结合位点是bcl-xL诱导启动子活性所必需的，并且在bcl-xL过表达后观察到NF-κB亚基p65和p50的核表达增强。电泳迁移率变动分析表明，在bcl-xL过表达的细胞中NF-κB的DNA结合活性增加，使用特异性抗体证实p65和p50参与了NF-κB对CXCL8启动子序列的活性调节。bcl-xL对NF-κB活性的调节涉及IkBα和IKK复合物信号通路。事实上，bcl-xL过表达诱导IkBα蛋白的细胞质表达减少，同时相同的IkBα和IKKα/β的磷酸化增加。此外，通过RNA干扰下调异位或内源性bcl-xL表达证实了bcl-xL调节NF-κB途径的能力，并且在bcl-xL转染细胞中细胞质NF-κB抑制剂IkBα的抗降解形式的瞬时表达证实了该抑制剂参与bcl-xL诱导的CXCL8表达和启动子活性。总之，我们的结果证明了NF-κB作为bcl-xL诱导胶质母细胞瘤细胞中CXCL8上调的介质的作用。
