Nambiar M P, Enyedy E J, Warke V G, Krishnan S, Dennis G, Kammer G M, Tsokos G C
Department of Cellular Injury, Walter Reed Army Institute of Research, Building 503, Robert Grant Avenue, Silver Spring, MD 20910-7500, USA.
J Autoimmun. 2001 Mar;16(2):133-42. doi: 10.1006/jaut.2000.0475.
A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR zeta-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR zeta-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3' untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P = 0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P = 0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a -76T insertion compared to the published sequence, in all of the SLE samples and controls. RT-PCR analysis of the TCR zeta-chain 3' untranslated region showed a 344 bp product in addition to the expected 906 bp product. Cloning and sequence analysis of the 344 bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672-1233 of TCR zeta-chain mRNA. Unlike the nomal TCR zeta-chain, the expression of TCR zeta-chain with the alternatively spliced 344 bp 3' untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906 bp TCR zeta-chain 3' untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P = 0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3' untranslated region may affect the stability and/or transport of TCR zeta-chain mRNA, leading to its downregulation in SLE T cells.
绝大多数系统性红斑狼疮（SLE）患者的TCRζ链mRNA表达降低，TCRζ链是一种关键的信号分子，参与TCR库的选择及自身免疫的预防。为了确定SLE T细胞中TCRζ链转录本下调所涉及的分子机制，我们研究了启动子和3'非翻译区中多态性/突变的可能性。对基因组DNA中启动子区域进行PCR、克隆和序列分析，结果显示与非SLE对照受试者相比，SLE T细胞中的多态性数量显著更高（P = 0.044）。还从粒细胞中分析启动子序列，以确定活化的SLE T细胞中发生体细胞突变的可能性。与非SLE对照相比，SLE患者粒细胞中的启动子多态性显著更高（P = 0.048），表明这些多态性源自基因组。启动子序列的核苷酸分析显示，与已发表序列相比，所有SLE样本和对照中均存在-76T插入。对TCRζ链3'非翻译区进行RT-PCR分析，除了预期的906 bp产物外，还出现了一个344 bp的产物。对344 bp产物进行克隆和序列分析表明，它是一种具有剪接供体和受体位点的可变剪接形式，导致TCRζ链mRNA的核苷酸672-1233缺失。与正常TCRζ链不同，具有可变剪接的344 bp 3'非翻译区的TCRζ链在SLE T细胞中的表达高于非SLE对照。与非SLE受试者相比，SLE T细胞中906 bp TCRζ链3'非翻译区的突变/多态性数量显著更高（P = 0.032）。下游3'非翻译区频繁的突变/多态性和异常剪接可能会影响TCRζ链mRNA的稳定性和/或转运，导致其在SLE T细胞中下调。
