Tsuzaka Kensei, Itami Yuka, Kumazawa Chika, Suzuki Miyuki, Setoyama Yumiko, Yoshimoto Keiko, Suzuki Katsuya, Abe Tohru, Takeuchi Tsutomu
Division of Rheumatology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe, Saitama 350-8550, Japan.
Biochem Biophys Res Commun. 2008 Mar 7;367(2):311-7. doi: 10.1016/j.bbrc.2007.12.145. Epub 2008 Jan 3.
We have demonstrated that T-cell receptor zeta (zeta) mRNA with a 562-bp deleted alternatively spliced 3'-untranslated region (3'UTR) observed in T cells of patients with systemic lupus erythematosus (SLE) can lead to a reduction in zeta and TCR/CD3 (J. Immunol., 2003 & 2005). To determine the region in zeta mRNA 3'UTR for the regulation of zeta, zeta mRNA with 3'UTR truncations ligated into pDON-AI was used to infect murine T-cell hybridoma MA5.8 cells, which do not contain zeta. As a Western blot analysis demonstrated the importance of the regions from +871 to +950, containing conservative sequence 1 (CS1), and +1070 to +1136, containing CS2, for the production of zeta, we constructed MA5.8 mutants carrying zeta mRNA 3'UTR with deletions of these regions (DeltaCS1 and DeltaCS2 mutants). Western blot and FACS analyses showed significant reduction in the cell surface zeta and TCR/CD3 in both these mutants, and IL-2 production was decreased, compared with MA5.8 cells transfected with wild-type zeta mRNA. Furthermore, real-time PCR demonstrated the instability of zeta mRNA with 3'UTR deletions in these MA5.8 mutants. In conclusion, CS1 and CS2 may be responsible for the regulation of zeta and TCR/CD3 through the stability of zeta mRNA in SLE T cells.
我们已经证明，在系统性红斑狼疮（SLE）患者的T细胞中观察到的具有562 bp缺失的选择性剪接3'非翻译区（3'UTR）的T细胞受体ζ（ζ）mRNA可导致ζ和TCR/CD3减少（《免疫学杂志》，2003年和2005年）。为了确定ζ mRNA 3'UTR中调控ζ的区域，将具有3'UTR截短的ζ mRNA连接到pDON-AI中，用于感染不含ζ的小鼠T细胞杂交瘤MA5.8细胞。由于蛋白质免疫印迹分析表明，包含保守序列1（CS1）的+871至+950区域和包含CS2的+1070至+1136区域对于ζ的产生很重要，我们构建了携带缺失这些区域的ζ mRNA 3'UTR的MA5.8突变体（DeltaCS1和DeltaCS2突变体）。蛋白质免疫印迹和荧光激活细胞分选分析显示，与转染野生型ζ mRNA的MA5.8细胞相比，这两种突变体的细胞表面ζ和TCR/CD3均显著减少，白细胞介素-2的产生也减少。此外，实时聚合酶链反应证明这些MA5.8突变体中具有3'UTR缺失的ζ mRNA不稳定。总之，CS1和CS2可能通过SLE T细胞中ζ mRNA的稳定性来调控ζ和TCR/CD3。
