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。在所测试的10个细胞系中，仅HL-60细胞系对β-毒素敏感。毒素引起细胞肿胀和溶解。在37 ℃下，毒素通过激活磷脂酶C（PLC）诱导HL-60细胞产生甘油二酯和肌醇-3-磷酸（IP 3），并呈剂量和时间依赖性，Ca^<2+>拮抗剂可抑制毒素诱导的细胞肿胀（TMB-8，BAPTA-AM）和钙调磷酸酶（CN）抑制剂（环孢菌素A），但不被Ca^2+进入阻断剂（维拉帕米）和PKC抑制剂所抑制。当Fura-2-AM处理过的HL-60细胞在细胞外Ca^<2+>缺乏的情况下与毒素一起孵育时，细胞内Ca^<2+>以剂量和时间依赖的方式增加。结果提示，毒素诱导的细胞溶解与细胞内Ca^2+动员密切相关。用β-毒素处理HL-60细胞后，CN活性在5 min内被激活，环孢菌素A可抑制毒素诱导的CN活性的增加。CN的一种重要的细胞内底物是NFTA（活化T细胞的核因子）。激活CN可诱导NFTA向细胞核转位。该毒素刺激HL-60细胞NFAT核转位。这些数据表明，β-毒素诱导的细胞肿胀依赖于通过PLC活性的Ca^<2+>/CaM依赖性CN。

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.