Zitt Christof, Strauss Bettina, Schwarz Eva C, Spaeth Nicola, Rast Georg, Hatzelmann Armin, Hoth Markus
Department of Biochemistry (RDR/B2), ALTANA Pharma AG, 78467 Konstanz, Germany.
J Biol Chem. 2004 Mar 26;279(13):12427-37. doi: 10.1074/jbc.M309297200. Epub 2004 Jan 12.
Ca2+ entry through store-operated Ca2+release-activated Ca2+ (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole (BTP) derivatives are specific inhibitors of Ca2+-dependent transcriptional activity in T-cells (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., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118-48126). Whereas inhibition of Ca2+ signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441-4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134-142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC(50) of approximately 10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca2+-dependent gene expression (interleukins 2 and 5 and interferon gamma) and proliferation of T-lymphocytes with similar IC(50) values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC(50) values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.
通过储存操纵性Ca2+释放激活的Ca2+（CRAC）通道的Ca2+内流对于T细胞活化和增殖至关重要。最近，研究表明3,5-双三氟甲基吡唑（BTP）衍生物是T细胞中Ca2+依赖性转录活性的特异性抑制剂（特里维廉，J.M.，邱，X.G.，陈，Y.W.，巴拉伦，S.J.，希茨，M.P.，史密斯，M.L.，维德曼，P.E.，沃里尔，U.，威尔金斯，J.，古宾斯，E.J.，加涅，G.D.，法格兰德，J.，卡特，G.W.，卢利，J.R.，莫利森，K.W.，和朱里克，S.W.（2001年）《生物化学杂志》276，48118 - 48126）。虽然有报道称BTP2可抑制Ca2+信号（石川，J.，大贺，K.，吉野，T.，竹泽，R.，市川，A.，久保田，H.，和山田，T.（2003年）《免疫学杂志》170，4441 - 4449），但未发现BTP3有此作用（陈，Y.，史密斯，M.L.，邱，G.X.，巴拉伦，S.，希茨，M.P.，古宾斯，E.，沃里尔，U.，威尔金斯，J.，苏罗维，C.，中根，M.，卡特，G.W.，特里维廉，J.M.，莫利森，K.，和朱里克，S.W.（2002年）《细胞免疫学》220，134 - 142）。我们发现BTP2能特异性抑制T细胞中的CRAC通道，半数抑制浓度（IC50）约为10纳米。它不会干扰T细胞中对Ca2+信号重要的其他机制，包括Ca2+泵、线粒体Ca2+信号传导、内质网Ca2+释放和K+通道。BTP2可抑制外周血T淋巴细胞（特别是CD4+ T细胞）和人Jurkat T细胞中的Ca2+信号。Ca2+信号的抑制与刺激方法无关，因为在用激活T细胞受体的抗CD3刺激后，以及在用毒胡萝卜素或肌醇1,4,5 - 三磷酸刺激后，Ca2+内流均被阻断。BTP2还以相似的IC50值抑制T淋巴细胞中Ca2+依赖性基因表达（白细胞介素2和5以及干扰素γ）和增殖。BTP2是原代T淋巴细胞中首个强效且特异性的CRAC通道抑制剂。在T淋巴细胞中，以相似的IC50值抑制CRAC通道以及Ca2+依赖性信号转导，强调了CRAC通道活性在T细胞活化过程中的重要性。此外，BTP2可能最终成为揭示CRAC通道分子身份的工具。
