Gorelik Gabriela J, Yarlagadda Sushma, Patel Dipak R, Richardson Bruce C
University of Michigan, Ann Arbor, MI 48109-2200, USA.
Arthritis Rheum. 2012 Sep;64(9):2964-74. doi: 10.1002/art.34503.
CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells.
CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation.
Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T(505) phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells.
Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.
活动性狼疮患者的CD4+ T细胞存在细胞外调节蛋白激酶（ERK）信号通路受损的情况，这会降低DNA甲基转移酶的表达，导致DNA去甲基化、免疫基因过度表达及自身免疫。ERK信号通路缺陷是由于蛋白激酶Cδ（PKCδ）激活环中苏氨酸（T）505位点的磷酸化受损。然而，狼疮T细胞中阻止PKCδ T505位点磷酸化的机制尚不清楚。其他人报道过，T细胞以及血清蛋白的氧化修饰，尤其是硝化作用，与狼疮疾病活动相关。我们开展这项研究以检验我们的假设，即硝化作用使PKCδ失活，导致狼疮T细胞中ERK信号通路受损。
从狼疮患者和对照中纯化CD4+ T细胞，然后用佛波酯（PMA）刺激。通过对T细胞裂解物进行免疫沉淀和免疫印迹来定量信号蛋白水平、硝化作用及磷酸化。通过电穿孔进行转染。
用过氧亚硝酸盐处理CD4+ T细胞会使PKCδ硝化，阻止PKCδ T505位点磷酸化，并抑制ERK信号通路，这与在狼疮T细胞中观察到的情况相似。活动性狼疮患者的硝化T细胞PKCδ水平高于对照，且与疾病活动直接相关，抗硝基酪氨酸免疫沉淀表明，硝化的PKCδ而非未修饰的PKCδ对PMA刺激的T505位点磷酸化具有抗性。这与过氧亚硝酸盐处理细胞中的PKCδ相似。
氧化应激导致PKCδ硝化，阻止其磷酸化，并导致狼疮T细胞中ERK信号减少。这些结果确定PKCδ是氧化应激与狼疮中T细胞表观遗传修饰之间的一个联系。
