Kravchenko V V, Pan Z, Han J, Herbert J M, Ulevitch R J, Ye R D
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037, USA.
J Biol Chem. 1995 Jun 23;270(25):14928-34. doi: 10.1074/jbc.270.25.14928.
The capability of platelet-activating factor (PAF) to induce transcription factor activation was examined. In stably transfected Chinese hamster ovary cells expressing the PAF receptor (CHO-PAFR), PAF stimulation resulted in the nuclear expression of a DNA binding activity with specificity to the kappa B sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor kappa B (NF-kappa B), were identified as components of the DNA protein complexes by antipeptide antibodies in gel supershift as well as UV cross-linking experiments. PAF induced an initial decrease and subsequent increase of cytoplasmic I kappa B alpha levels, accompanied by up-regulation of the I kappa B alpha messenger RNA, a feature of NF-kappa B activation. PAF-induced kappa B binding activity was detected within 15 min after agonist stimulation, peaked at 30-40 min, and remained detectable by 2.5 h. SR 27417, a PAF receptor antagonist, blocked PAF-induced kappa B binding activity but not that induced by tumor necrosis factor-alpha (TNF alpha). Cholera toxin treatment markedly reduced PAF-induced kappa B binding activity, whereas pertussis toxin had no significant inhibitory effect. Neither of the two toxins affected the kappa B binding activity induced by TNF alpha in the same cells. In addition to the CHO-PAFR cells, PAF stimulated kappa B binding activity in the murine P388D1 macrophage and the human ASK.0 B cell lines that express endogenous PAF receptors. These results imply a potential role of PAF in the regulation of gene expression through a G protein-coupled transcription factor activation pathway.
研究了血小板活化因子（PAF）诱导转录因子激活的能力。在稳定转染表达PAF受体的中国仓鼠卵巢细胞（CHO-PAFR）中，PAF刺激导致出现一种对κB序列具有特异性的DNA结合活性的核表达。通过凝胶超迁移以及紫外线交联实验中的抗肽抗体，鉴定出原型核因子κB（NF-κB）的组成成分p50和p65蛋白是DNA-蛋白质复合物的组成部分。PAF诱导细胞质IκBα水平先降低后升高，同时伴有IκBα信使核糖核酸的上调，这是NF-κB激活的一个特征。在激动剂刺激后15分钟内检测到PAF诱导的κB结合活性，在30 - 40分钟达到峰值，并在2.5小时内仍可检测到。PAF受体拮抗剂SR 27417可阻断PAF诱导的κB结合活性，但不能阻断肿瘤坏死因子-α（TNFα）诱导的该活性。霍乱毒素处理显著降低了PAF诱导的κB结合活性，而百日咳毒素没有明显的抑制作用。这两种毒素均不影响同一细胞中TNFα诱导的κB结合活性。除了CHO-PAFR细胞外，PAF还刺激了表达内源性PAF受体的小鼠P388D1巨噬细胞和人ASK.0 B细胞系中的κB结合活性。这些结果表明PAF可能通过G蛋白偶联转录因子激活途径在基因表达调控中发挥作用。
