Wu Xiaoqing, McIntyre Thomas M, Zimmerman Guy A, Prescott Stephen M, Stafforini Diana M
Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112-5550, USA.
Biochem J. 2003 Oct 15;375(Pt 2):351-63. doi: 10.1042/BJ20030636.
Plasma platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase that inactivates platelet-activating factor (PAF) and PAF-like lipids to generate products with little or no biological activity. The levels of circulating PAF-AH correlate with several disease syndromes. We previously reported that mediators of inflammation regulate the expression of the human PAF-AH gene at the transcriptional level. In the present paper, we characterize the constitutive expression of plasma PAF-AH using the mouse gene as a model system, and we report comparative results obtained using human and mouse promoter constructs. We first cloned, sequenced and analysed the promoter region of the murine plasma PAF-AH (mPAF-AH) gene and found that this gene lacks a canonical TATA box. We demonstrated that the cis -elements required for basal transcription are localized within the -316 to -68 bp region. In vitro band-shift and supershift assays showed that Sp1 and Sp3 transcription factors from RAW264.7 and J774A.1 macrophage nuclear extracts bound strongly to a distal GC-rich site within -278/-243 [specificity protein (Sp-A)] and to a proximal TC-rich motif within -150/-114 (Sp-B). In addition, we observed weak binding to a GA-rich site within -110/-82 (Sp-C). The regions containing Sp-B and Sp-C are highly conserved between the human and mouse genes. Forced expression of Sp1 or Sp3 in Sp-lacking Drosophila SL2 cells induced markedly the activity of the exogenous mPAF-AH promoter in a dose-dependent manner, and this induction was dependent on the presence of intact Sp-A and Sp-B. Interestingly, we found that the Sp1- and Sp3-associated DNA-binding activities increased during the maturation of primary human monocytes into macrophages in cell culture. These results demonstrate that Sp1 and Sp3 are key factors that contribute to the basal, constitutive transcription of the plasma PAF-AH gene in macrophages.
血浆血小板活化因子乙酰水解酶（PAF-AH）是一种磷脂酶，可使血小板活化因子（PAF）和类PAF脂质失活，生成生物活性很小或无生物活性的产物。循环PAF-AH的水平与多种疾病综合征相关。我们之前报道过，炎症介质在转录水平调节人PAF-AH基因的表达。在本文中，我们以小鼠基因为模型系统，对血浆PAF-AH的组成型表达进行了表征，并报告了使用人和小鼠启动子构建体获得的比较结果。我们首先克隆、测序并分析了小鼠血浆PAF-AH（mPAF-AH）基因的启动子区域，发现该基因缺乏典型的TATA框。我们证明基础转录所需的顺式元件位于-316至-68 bp区域内。体外凝胶迁移和超迁移分析表明，RAW264.7和J774A.1巨噬细胞核提取物中的Sp1和Sp3转录因子与-278/-243内的一个富含GC的远端位点[特异性蛋白（Sp-A）]以及-150/-114内的一个富含TC的近端基序（Sp-B）强烈结合。此外，我们观察到与-110/-82内一个富含GA的位点（Sp-C）有弱结合。包含Sp-B和Sp-C的区域在人和小鼠基因之间高度保守。在缺乏Sp的果蝇SL2细胞中强制表达Sp1或Sp3以剂量依赖的方式显著诱导了外源mPAF-AH启动子的活性，并且这种诱导依赖于完整的Sp-A和Sp-B的存在。有趣的是，我们发现在细胞培养中，原代人单核细胞成熟为巨噬细胞的过程中，与Sp1和Sp3相关的DNA结合活性增加。这些结果表明，Sp1和Sp3是促成巨噬细胞中血浆PAF-AH基因基础组成型转录的关键因子。
