PP2A B56α亚基介导镉诱导的细胞毒性

Role of PP2A B56 subunit in cadmium-induced cytotoxicity

目的：探讨蛋白磷酸酶2A（PP2A）B56亚基在调控Cd Cl诱导的细胞毒性中的作用及其机制。方法：利用病毒感染法在人胚肾2上皮细胞HEK上构建PP2A B56α表达降低的细胞株模型（HEK-SHB56α-1和HEK-SHB56α-2）,采用改良四甲基偶氮唑盐（MTT）法检测Cd Cl诱导的细胞毒性。用不同浓度（0、10、20、40和80μmol/L）Cd Cl联合或不联合c-Jun氨基末端激酶（JNK）抑制剂SP600125染毒细胞2 2不同时间（0、2、6、12和24 h）,采用免疫印迹检测B56α亚基、金属硫蛋白（MT）的表达和JNK的磷酸化水平。结果：免疫印迹结果证实细胞株HEK-SHB56α-1和HEK-SHB56α-2构建成功。与对照组比较,PP2A B 56α表达抑制导致镉诱导的细胞毒性减小,JNK的磷酸化水平和MT的表达水平均显著增加。用不同剂量Cd Cl处理细胞株12 h,发现B56α表达抑制导致JNK磷酸化（p-JNK）分别增加了2.78和21.26倍（P〈0.05）,MT的表达也分别增加了1.36和1.19倍（〈0.05）。用p-JNK抑制剂SP600125作用时,p-JNK的表达降低了35%~38%（P〈0.05）,MT的表达下降了13%~35%（P〈0.05）,Cd Cl诱导的细胞毒性显著增加（P〈0.05）。此外,用Cd Cl处理细胞不同时间2 2点,B56α的表达逐渐降低（P〈0.05）,而p-JNK和MT的表达水平呈逐渐增加趋势（P〈0.05）。结论：PP2A B 56α在调控Cd Cl诱导的细胞毒2性中起着重要作用,其作用机制可能通过介导JNK的去磷酸化调控MT的表达而发挥作用。本研究揭示了PP2A参与重金属应激的关键靶点和信号通路的调控。

OBJECTIVE：To identify involvement ofprotein phosphatase 2A B56αin the regulation of cytotoxicity induced by cadmium chloride （CdCl2） and address the underlying molecular mechanism.METHODS：Stable cell lines were generated by infecting HEK cells with lentiviral shRNA targeting B56α subunit. Modified MTT was performed to detect the cytotoxicity induced by CdCl2. Immunoblotting analysis was applied to examine the expressions of B56α,p-JNK and MT in cells treated with differentconcentrations ofCdCl2or co-treated with SP600125.RESULTS：Immunoblotting results verified that stable cell lines HEK-SHB56α-1 and HEK-SHB56α-2 were successfully established. We found that suppression of PP2A B56α reduced the cytotoxicity induced by CdCl2. In addition,the phosphorylation status of c-Jun N-terminal kinases （JNK）and the expression level of MT weresignificantly increased in response to CdCl2. Suppression of B56α led to a 2.78 or 1.26-fold（P〈0.05） increase of p-JNK in cells treated with CdCl2 for 12 h,and a 1.36 or 1.19-fold （P〈0.05） increase of MT. Upon SP600125 treatment,the expression level of p-JNK and MT werereduced by 35%-38% （P〈0.05） and 13%-35%（P〈0.05）,respectively,whilecytotoxicity induced by CdCl2 was enhanced（P〈0.05）. More-over,the expression of B56α was lowered（P〈0.05）,but p-JNK and MT were increased（P〈0.05） inatime-dependent manner upon CdCl2 treatment. CONCLUSION：PP2A B56α regulated MT expression via dephosphorylating JNK,and affecting the cytotoxicity induced by CdCl2. Our study demonstratedthat PP2A B56α participated in regulating the targets and pathways in response to metallic stress.