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Analysis of the aberrant TCRζ mRNA 3'UTR in SLE patient T cells

SLE 患者 T 细胞中异常 TCRz mRNA 3UTR 的分析

基本信息

批准号：
12670436
负责人：
TSUZAKA Kensei
金额：
$ 2.11万
依托单位：
Saitama Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670436/
关键词：
Systemic lupus erythematosus TCRζ mRNA3'UTR alternative splicing MA5.8 signal transduction mRNA3'UTR In vitro translation dimerization 半定量PCR法

项目摘要

We recently reported that expression of the T-cell receptor ζchain (TCRζ) was significantly decreased or absent in systemic lupus erythematosus (SLE) patients due to the TCRζ mRNA with alternatively spliced short 3'UTR (TCRζ mRNA/as-3'UTR) (Int. Immunol 1998, J.Autoimmunity 1998, Arthritis Rheum, 1999). Here we investigated the mechanisms of the decreased expression of TCRζ in SLE T cells. 1)By the semi-quantitative PCR, TCRζ mRNA/as-3'UTR was predominantly detected in the SLE patients compared with the normal controls. 2)By using the in vitro translation assay, TCRζ mRNA/w-3'UTR consistently coded much more 16-kD TCRζ monomer and homodimer than TCRζ mRNA/as-3'UTR. 3) TCRζ cDNA/w-3'UTR and TCRζ cDNA/as-3'UTR was amplified and ligated into pDON-AI vector. Then the MA5.8 mutants were constructed by transferring the recombinant retrovirus. Western blot analysis using an anti-TCRζ antibody showed decreased expression of the TCRζ monomer and homodimer in AS3'UTR cells. FACS analysis and the IP using the cell surface paroteins using TIA-2 and anti-CD3ε antibody demonstrated downregulation of the cell surface expression of TCRζ and TCR/CD3 complex on the AS3'UTR cells. The expression of IL-2 and CD28 in the AS3'UTR cells was up-regulated after stimulation with anti-CD3 antibody. Also the TCRζ mRNA stability of AS3'UTR cells was significantly lower than that in WT cells. From these observations, predominant expression of TCRζ mRNA/as-3'UTR in SLE T cells is responsible for the decreased expression of TCRζ because of the reduced stability of TCRζ mRNA.

我们最近报道，由于具有可变剪接的短3 'UTR的TCR γ mRNA（TCR γ mRNA/as-3' UTR），在系统性红斑狼疮（SLE）患者中T细胞受体γ链（TCR γ）的表达显著降低或缺失（Int.Immunol1998，J.Autoimmunity 1998，Arthritis Rheum，1999）。本文探讨了SLE T细胞TCR γ表达降低的机制。1）半定量PCR检测结果显示，SLE患者TCR α mRNA/as-3 'UTR阳性率明显高于正常对照组。2）体外翻译实验表明，TCR α mRNA/w-3 'UTR比TCR α mRNA/as-3' UTR编码更多的16-kD TCR α单体和同源二聚体。3)扩增TCR γ cDNA/w-3 'UTR和TCR γ cDNA/as-3' UTR，并连接到pDON-AI载体上。然后通过转移重组逆转录病毒构建MA5.8突变体。使用抗TCR γ抗体的蛋白质印迹分析显示在AS 3 'UTR细胞中TCR γ单体和同源二聚体的表达降低。流式细胞术分析和使用TIA-2和抗CD 3 ε抗体的细胞表面蛋白的IP证实了AS 3 'UTR细胞上的TCR γ和TCR/CD 3复合物的细胞表面表达下调。用抗CD 3抗体刺激后，AS 3 'UTR细胞中IL-2和CD 28的表达上调。AS 3 'UTR细胞的TCR γ mRNA稳定性也显著低于WT细胞。从这些观察结果来看，SLE T细胞中TCR γ mRNA/as-3 'UTR的主要表达是由于TCR γ mRNA稳定性降低而导致TCR γ表达降低的原因。