Deletional analysis of the murine IL-12 p35 promoter comparing IFN-γ and lipopolysaccharide stimulation
Deletional analysis of the murine IL-12 p35 promoter comparing IFN-γ and lipopolysaccharide stimulation
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DOI：
10.4049/jimmunol.167.10.5653
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发表时间：
2001-11-15
影响因子：
4.4
通讯作者：
Petro, TM
中科院分区：
文献类型：
--
作者：
Kollet, J;Witek, C;Petro, TM
IL-12是Th 1细胞发育的关键，由p35和p40亚基联合形成，由巨噬细胞和巨噬细胞系RAW 264.7产生。在这项研究中，p35的启动子克隆和分析。鼠IL-12 p35基因在前两个外显子中的每一个的上游具有启动子。外显子1和外显子2启动子，克隆到一个报告载体，响应LPS或IFN-γ/CD 40连接在转染的RAW 264.7细胞。外显子2启动子含有bp -809至+1，与人p35启动子具有显著的同源性。因此，进行缺失分析以确定对LPS、CD 40和/或IFN-γ的应答性所需的区域。碱基对-809到-740影响对LPS的反应性。相反，bp -740至-444和bp -122至-100是对IFN-γ、IFN-γ/LPS或IFN-γ/CD 40连接的应答所需的。去除bp-444至-392增加了外显子2启动子对每种刺激物的反应。IFN调节因子（IRF）-1参与该启动子在bp -108至-103处的活性，因为核IRF-1水平与响应IFN-γ和IRF-1过表达刺激和增强的外显子2启动子活性的外显子2启动子活性相关。此外，IRF-1元件在bp -108至-103处的位点或缺失突变降低了启动子和IRF-1与含有bp -108至-103的寡核苷酸结合的响应性。这些数据表明，p35启动子对IFN-γ的应答需要在bp -108至-103处的独特的IRF-1正调控元件。
IL-12, pivotal to the development of Th1 cells and formed by association of p35 and p40 subunits, is made by macrophages and the macrophage cell line RAW264.7. In this study, the promoter for p35 was cloned and analyzed. The murine IL-12 p35 gene has promoters upstream from each of the first two exons. The exon 1 and exon 2 promoters, cloned into a reporter vector, were responsive to LPS or IFN-gamma /CD40 ligation in transfected RAW264.7 cells. The exon 2 promoter containing bp -809 to +1 has significant homology to the human p35 promoter. Thus, deletion analysis was performed to determine the regions required for responsiveness to LPS, CD40, and/or IFN-gamma. Base pairs -809 to -740 influenced responsiveness to LPS. In contrast, bp -740 to -444 and bp -122 to -100 were required for responses to IFN-gamma, IFN-gamma /LPS, or IFN-gamma /CD40 ligation. Removal of bp -444 to -392 increased the response of the exon 2 promoter to each stimulant. IFN regulatory factor (IRF)-1 is involved in the activity of this promoter at bp -108 to -103 because levels of nuclear IRF-1 correlated with exon 2 promoter activity in response to IFN-gamma and IRF-1 overexpression stimulated and enhanced exon 2 promoter activity. Also, site or deletion mutation of the IRF-1 element at bp -108 to -103 reduced the responsiveness of the promoter and IRF-1 bound to an oligonucleotide containing bp -108 to -103. The data suggest that the response of the p35 promoter to IFN-gamma requires a distinct IRF-1 positive regulatory element at bp -108 to -103.