Molecular characterization of the constitutive expression of the plasma platelet-activating factor acetylhydrolase gene in macrophages

Abstract

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.