Trevillyan J M, Chiou X G, Chen Y W, Ballaron S J, Sheets M P, Smith M L, Wiedeman P E, Warrior U, Wilkins J, Gubbins E J, Gagne G D, Fagerland J, Carter G W, Luly J R, Mollison K W, Djuric S W
Global Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
J Biol Chem. 2001 Dec 21;276(51):48118-26. doi: 10.1074/jbc.M107919200. Epub 2001 Oct 9.
NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.
活化T细胞核因子（NFAT）蛋白在大多数免疫系统细胞中表达，并调节对免疫反应至关重要的细胞因子基因的转录。NFAT蛋白的活性受到Ca(2+)/钙调蛋白依赖性蛋白磷酸酶2B/钙调神经磷酸酶（CaN）的严格调控。CaN使NFAT去磷酸化是NFAT核定位所必需的。目前的免疫抑制药物，如环孢素A和FK506，可阻断CaN活性，从而抑制NFAT的核转位以及随之而来的细胞因子基因转录。在免疫系统以外的细胞中抑制CaN可能会导致与环孢素A治疗相关的毒性。为了寻找更安全的免疫抑制药物，我们鉴定了一系列3,5-双三氟甲基吡唑（BTP）衍生物，它们可阻断Th1和Th2细胞因子基因的转录。通过电泳迁移率变动分析确定，BTP化合物可阻断NFAT的活化依赖性核定位。对表达荧光标记NFAT的细胞进行共聚焦显微镜检查证实，BTP化合物可阻断钙诱导的NFAT从细胞质向细胞核的移动。对NFAT的抑制具有选择性，因为BTP化合物不影响NF-κB和AP-1转录因子的活化。在用钙离子载体诱导CaN活化之前，用BTP化合物处理完整的T细胞会使NFAT保持高度磷酸化状态。然而，BTP化合物在体外并不直接抑制CaN对NFAT的去磷酸化作用，这些药物也不阻断其他CaN底物的去磷酸化作用，包括蛋白激酶A的II型调节亚基和转录因子Elk-1。数据表明，BTP化合物使NFAT以磷酸化状态维持在细胞质中，并通过直接抑制CaN磷酸酶活性以外的机制阻断NFAT的核输入，从而阻断NFAT依赖性细胞因子基因转录。本文所述的新型抑制剂将有助于更好地确定NFAT活化的细胞调控机制，并可能有助于识别治疗自身免疫性疾病和移植排斥反应的新治疗靶点。
