Activation of calcineurin signal transduction by Clostridium perfingens beta-toxin in HL-60 cell

产气荚膜梭菌β毒素是坏死性肠炎的重要病原体。在10个细胞系中，只有HL-60细胞系对β -毒素敏感。毒素引起细胞肿胀和溶解。毒素通过激活磷脂酶C （PLC）诱导二酰基甘油和肌醇-3-磷酸（IP3）在37℃与HL-60细胞孵育时呈剂量依赖性和时间依赖性，毒素诱导的细胞肿胀被Ca^<2+>拮抗剂（TMB-8、BAPTA-AM）和钙调磷酸酶（CN）抑制剂（环孢素a）抑制，而Ca^<2+>进入阻断剂（维拉帕米）和PKC抑制剂没有抑制作用。当fura -2- am处理的HL-60细胞在细胞外Ca^<2+>缺失的情况下与毒素孵育时，细胞内Ca^<2+>呈剂量和时间依赖性增加。结果表明，毒素诱导的细胞裂解与细胞内Ca^<2+>的动员密切相关。用β -毒素处理HL-60细胞可在5分钟内激活CN活性。环孢素A抑制毒素诱导的CN活性升高。CN的一个重要细胞内底物是NFTA（活化t细胞的核因子）。CN的激活诱导NFTA向细胞核的易位。毒素刺激HL-60细胞NFAT的核易位。数据表明，β -毒素诱导的细胞肿胀依赖于Ca^<2+>/ cam依赖的CN，通过PLC的活性。

Clostridium perfringens beta-toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only HL-60 cell line was susceptible to beta-toxin. The toxin induced swelling and lysis of the cells. The toxin induced production of diacylglycerol and inositol-3-phosphate (IP3) in a dose-and a time-dependent manner through the activation of phospholipase C (PLC) when incubated with HL-60 cells at 37 ℃, the toxin induced cell swelling were suppressed by Ca^<2+> antagonists (TMB-8,BAPTA-AM) and calcineurin (CN) inhibitor (cyclosporin A) but not by Ca^<2+> entry blocker (verapamil) and PKC inhibitor. When Fura-2-AM-treated HL-60 cells were incubated with the toxin in the absence of extracelluar Ca^<2+>, the cells increased intracellular Ca^<2+> in a dose-and a time-dependent manner. The results suggested that the toxin induced lysis of cells is closely related the intracelluar Ca^<2+> mobilization. Treatment of HL-60 cells with beta-toxin resulted in the activation of CN activity within 5 min. Cyclosporin A inhibited the toxin-induced increase of CN activity. An important intracelluar substrate for CN is NFTA (nuclear factor of activated T-cells). Activation of CN induced the translocation of NFTA to nucleus. The toxin stimulated the nuclear translocation of NFAT in HL-60 cells. The data indicated that beta-toxin-induced cell swelling is dependent on the Ca^<2+>/CaM-dependent CN through activity of PLC.