Shimokawa N, Nishiyama C, Hirota T, Tamari M, Hara M, Ikeda S, Okumura K, Ogawa H
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
Clin Exp Allergy. 2009 Feb;39(2):228-35. doi: 10.1111/j.1365-2222.2008.03165.x. Epub 2008 Dec 23.
Human IL-12B gene on chromosome 5q31 encodes the common p40 subunit of IL-12 and IL-23. IL-12 is known to play critical roles in the generation of T-helper type 1 (TH(1)) cells, whereas IL-23 is involved in maintenance and/or population expansion of TH(17) cells. Although several reports suggested an association between a polymorphism (-6415CTCTAA/GC) in IL-12B and asthma, the molecular mechanism how this polymorphism is involved in allergic inflammation is still unclear.
The transcription activity was analysed by reporter assay. A transcription factor binding to -6415 polymorphic site was identified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The amount of cytokines produced from peripheral monocytes were determined by ELISA.
Reporter assay showed that the transcription activity of the GC allele was higher than that of the CTCTAA allele. A transcription factor Sp1 bound to the region including the GC allele with a higher affinity than that of the CTCTAA allele in EMSA. In vivo binding of Sp1 to IL-12B gene carrying -6415GC was confirmed by ChIP assay. Overexpression of Sp1 up-regulated transcription activity of promoter carrying GC allele sequence, whereas the CTCTAA promoter was not affected by Sp1. We examined the correlation between -6415CTCTA/GC polymorphism and production of cytokine IL-12/23p40, IL-12p70, and IL-23 on peripheral blood monocytes, and monocytes with the GC/GC allele exhibited significantly higher expression of IL-12p70 protein than those with the CTCTAA/CTCTAA allele (P=0.009).
The -6415 polymorphism is involved in cytokine production potential by affecting Sp1-mediated transcription activity.
位于5号染色体q31区域的人类白细胞介素12B（IL-12B）基因编码白细胞介素12（IL-12）和白细胞介素23（IL-23）的共同p40亚基。已知IL-12在1型辅助性T细胞（TH(1)）的产生中起关键作用，而IL-23参与TH(17)细胞的维持和/或数量扩增。尽管有几份报告提示IL-12B基因中的一个多态性位点（-6415CTCTAA/GC）与哮喘有关，但该多态性如何参与过敏性炎症的分子机制仍不清楚。
通过报告基因检测分析转录活性。通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）检测确定与-6415多态性位点结合的转录因子。采用酶联免疫吸附测定（ELISA）法测定外周血单核细胞产生的细胞因子量。
报告基因检测显示，GC等位基因的转录活性高于CTCTAA等位基因。在EMSA中，转录因子Sp1与包含GC等位基因的区域结合，其亲和力高于与CTCTAA等位基因结合的亲和力。ChIP检测证实Sp1在体内与携带-6415GC的IL-12B基因结合。Sp1的过表达上调了携带GC等位基因序列启动子的转录活性，而CTCTAA启动子不受Sp1影响。我们检测了-6415CTCTA/GC多态性与外周血单核细胞中细胞因子IL-12/23p40、IL-12p70和IL-23产生之间的相关性，携带GC/GC等位基因的单核细胞中IL-12p70蛋白的表达明显高于携带CTCTAA/CTCTAA等位基因的单核细胞（P=0.009）。
-6415多态性通过影响Sp1介导的转录活性参与细胞因子产生潜能。
