Decreased expression of TCRζ and signal transduction due to the aberrantexpression of the TCR mRNA in SLE patient T cells

我们最近报道，在系统性红斑狼疮患者中，T细胞受体ζ链(ζ)的表达显著降低或缺失，这是由于ζ基因剪接变异体与交替剪接的短3‘非编码区(ζ)或外显子7缺失(ζ/EX7-)所致。在此，我们利用逆转录病毒系统研究了包括ζ在内的TcR/CD3的表达。因此，包括ζ在内的T细胞TcR/CD3mRNA/EX7-的表达和IL-2的产生由于ζmRNAEX7-的不稳定性而显著降低。为了验证包括ζ在内的TcR/CD3复合体的减少或缺失是否表现出提示ζ的差异转录模式，我们通过基因芯片分析比较了基因表达谱。结果表明，在ζ基因剪接变异体的转染体中，Syndecan-1、颗粒酶A、硒结合蛋白和溶质载体家族4基因普遍上调。另一方面，几种细胞因子(IL-2…)的表达IL-15、IL-18、转化生长因子-β)降低。因此，在T细胞中发现这些应答基因，并伴随包括ζ在内的TcR/CD3复合体的下调，可能有助于更好地了解T细胞功能障碍和系统性红斑狼疮的发病机制。因此，Sdc1、Sdc2和Sdc3在SLE患者T细胞和健康对照组之间的表达模式没有观察到差异。然而，在11例SLE患者T细胞中，有10例在抗SDC4抗体检测到的35kD和40kD蛋白条带缺失。10例系统性红斑狼疮患者经抗Ζ抗体免疫印迹法检测，ζ基因缺失。该SDC4相关的35-kD蛋白在抗HS抗体处理后被吸收。FACS分析显示，12例SLE患者中有9例细胞表面HS和SDC4减少或缺失。有趣的是，在这9例SLE患者T细胞中，有报道将N-乙酰氨基葡萄糖(GlcNAc)转移到SDC家族核心蛋白的公共连接物四糖的Exostosin-like 2(EXTL2)酶的产生显著减少或缺失。较少

We recently reported that expression of the T-cell receptor ζ chain (ζ) was significantly decreased or absent in systemic lupus erythematosus (SLE) patients due to the ζ mRNA splice variants with alternatively spliced short 3'UTR (ζ mRNA/as-3'UTR) or with deletion of exon7 (ζ mRNA/ex7-). Here we investigated the expression of TCR/CD3 including ζ by using retrovirus systems. As a result, the expression of TCR/CD3 including ζ and IL-2 production from ζ mRNA/ex7- was significantly decreased due to the instability of ζ mRNA/ex7-.To examine whether these T cells with decreased or absent of TCR/CD3 complex including ζ exhibit differential transcription patterns that are indicative of SLE, we compared the gene expression profile by DNA microarray analysis. As a result, genes of syndecan-1 (Sdc-1), granzyme A, selenium binding protein, and solute carrier family 4 were up-regulated commonly in the transfectant with ζ mRNA splice variant froms. On the other hand, those of several cytokines (IL-2 … More , IL-15, IL-18, TGF- β) and were decreased. From these observations, identification of these responsive genes in T-cells accompanied by the down-regulation of TCR/CD3 complex including ζ may help to better understand the mechanism underlying T-cell dysfunction and pathogenesis of SLE.We investigated the expression of Sdc families (Sdc-1 through Sdc-4) in SLE T cells by using Western blot (WB). As a result, there observed no differences in the expression patterns of Sdc1, Sdc2, and Sdc3 between in SLE patient T cells and in healthy normal controls. However, the 35- and 40-kD protein bands detected with anti-Sdc4 antibody were missing in 10 out of 11 SLE patient T cells. Ζ was also missing in these 10 SLE patients by WB using anti-ζ antibody. This Sdc4-related 35-kD protein was absorbed after the treatment of anti-HS antibody. In FACS analysis, cell surface HS and Sdc4 was decreased or missing in 9 out 12 SLE patients. Interestingly, the production of exostosin-like 2 (EXTL2) enzyme, which has been reported to transfer N-acetylglucosamine (GlcNAc) to the common linker tetrasaccharide of a core protein of Sdc families, was significantly reduced or missing in these 9 SLE patient T cells. Less