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

我们最近报道，系统性红斑狼疮患者T细胞受体ζ链(TCRζ)的表达显著降低或缺失，这是由于TCRζ与短3‘非编码区(TCRζ/AS-3’UTR)的选择性剪接(Int.免疫学1998，J.自身免疫学1998，关节炎大黄，1999)。在此，我们探讨了SLE T细胞TcRζ表达降低的机制。1)半定量聚合酶链式反应检测到SLE患者外周血中TCR-ζ基因/AS-3‘非编码区的表达明显高于正常对照组。2)体外翻译实验结果显示，TCRζm RNA/w-3‘UTR16-kD TCRζ单体和同源二聚体的编码量明显高于TCR-ζm RNA/AS-3’UtR。3)分别扩增TcRζ/w-3‘UtR和TcR-ζcDNAs/AS-3’UtR，并连接到pDON-AI载体上。通过转移重组逆转录病毒构建MA5.8突变体。用抗TcRζ抗体进行的Western印迹分析显示，TcRζ单体和同源二聚体在AS3UTR细胞中的表达降低。TIA-2和抗CD_3ε抗体的细胞表面蛋白免疫印迹和流式细胞仪分析显示，AS3‘UTR细胞表面TCRζ和TCR/CD_3复合体表达下调。抗CD3抗体刺激后，AS3‘UTR细胞IL-2和CD28的表达上调。AS3‘非编码区细胞的TCRmR NA稳定性明显低于WT细胞。这些结果表明，T细胞中主要表达TCRζ/AS-3‘ζ是由于TCRζ的稳定性降低而导致其表达降低的原因。

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.