蛋白磷酸酶2AB5613全酶调控氯化镉诱导肝细胞毒性的作用研究

The role of protein phosphatase 2A B56β holoenzyme in the regulation of heavy metal CdCl2 induced cytotoxicity

目的探讨蛋白磷酸酶2A（proteinphosphatase2A，PP2A）B56β全酶调控CdCl2诱导肝细胞毒性的作用及其机制。方法采用改良四甲基偶氮唑盐（M啊比色法检测CdCl2对人正常肝细胞（L-02）、黄曲霉素诱导转化细胞（L-02RT—AFB1）及肝肿瘤细胞（Bel7402）的毒性，利用病毒感染法在L．02或Bel7402上构建丝氨酸／苏氨酸蛋白激酶B（AKT）低表达（L-02SHAKT）、B56p低表达（L．02SHB5613）和B56p高表达（L-02RT-AFB1-B5613、Bel7402．B56p）的细胞株。在浓度为0、20、40、80、160μmol/L的CdCl2处理下，检测人正常肝细胞、转化的肝细胞和肝肿瘤细胞的相对细胞活力。在2．5、5．0μmol/L的渥青霉素和40μmol／LCdCl3共同作用下检测L-02细胞中各蛋白相对表达量。采用Westemblot法检测这些细胞株中B56B、金属硫蛋白（metallothionein，MT）、AKT、AKT磷酸化（P．AKT）的表达水平。结果在40μmol／LCdCl：作用下，L．02RT。AFB，和Bel7402细胞中MT表达水平分别为0．12±0．02、0．06±0．06，低于对照组（0．92±0．14）（F=I148．16，P〈O．001）。在40μmol／LCdCl2作用下，AKT干扰细胞株L．02SHAKT-1、L-02SHAKT．2中p-AKT水平分别为0．08＋0．02、0．08±0．05，低于对照组（0．18±0．15）（F=724．70，P〈O．001）；两种细胞MT的表达水平均为0．62±0．16，高于对照组（0．22±0．14）（F=94．73，P〈O．001）。在2．5、5．0μmol／L渥青霉素和40μmol／LCdCl：共同作用下，L-02细胞中D．AKT的表达水平分别为0．28±0．07、0．15±0．11，低于未用渥青霉素处理组（0．52±0．11）（F=578．57，P〈0．001）；MT的表达水平分别为1．62±0．80、1．08±0．15，高于未用渥青霉素处理组（0．69±0．18）（F=12．34，P〈O．001）。在40μmol／LCdCl2作用下，B56p低表达细胞株L．02SHB5613．1、L-02SHB5613—2中β—AKT的表达水平分别为0．57±0．13、0．59±0．02，高于对照细胞L-02SHGFP（0．32±0．02）（F=87．16，P〈0．001）；MT的表达水平分别为0．35±0．07、0．20＋0．03，低于对照细胞L．02SHGFP（1．51±0．13）（仁2457．10，P〈0．001）。在40μmol／LCdCl：作用下，B56B高表达细胞株L．02RT—AFB1-B56B、Bel7402．B56B中p-AKT的表达水平分别为0．10±0．11、0．09±0．01，低于对照细胞L．02RT—AFB。（0．36±0．01）、Bel7402（0．43±0．11）（F=877．62，P〈0．001）；MT的表达水平分别为0．92±0．13、0．95±0．08，高于对照细胞L．02RT—AFB，（0．44±0．12）、Bel7402（0．77±0．06）（F=51．97，P〈O．001）。结论特异的PP2AB56β全酶介导AKT的去磷酸化调控MT的表达，最终影响重金属CdCl2诱导的肝细胞毒性。本研究揭示了一条PP2A参与CdCl2诱导肝细胞毒性的关键信号通路。

Objective To investigate the role of holoenzyme containing protein phosphatase 2A B56β in regulating CdC12 induced cytotoxicity. Method CdC12-induced cytotoxicity in normal human cell line L-02,AFBrtransformed hepatic cell line L-02 RT-AFB, and tumor cell line Be17402 was measured by modified MTT assay. Stable cell lines L-02 SHAKT, L-02 SHB5613, L-02 RT-AFB1-B56β and Be17402-B5613 were generated by infecting L-02 cells or Be17402 cells with retroviral vectors encoding lentiviral AKT shRNA, lentiviral B5615 shRNA and B5613. The relative cell viability was measured in normal human cell line AFB,-transformed hepatic cell line and tumor cell line when treated by CDC12（0,20,40,80, 160 txmol/L）. After treated by wortmannin （2.5,5.0 μmol/L） combined with 40 μmol/L CdC12,Western blot was applied to measure the expression of associated protein in L-02.Western blot was applied to measure the expression of B56J3, MT （metallothionein）, AKT, and p-AKT in these cell lines treated by CdC12. Results The levels of MT were 0.12±0.02, 0.06±0.06 in L-02 RT-AFBI and Be17402,which were lower than L02 （0.92±0.14） （F=I 148.16,P〈0.001）when treated by 40 μmol/L CdCL.When treated by 40 μmol/L CdC12,the expression of p-AKT in L-02 SHAKT-1 and L-02 SHAKT-2 were 0.08±0.02, 0.08±0.05,which levels were lower than L-02 SHGFP（0.18±0.15）（F=724.70,P〈O.OO1）;and the expression of MT were both 0.62±0.16 in L-02 SHAKT-1 and L-02 SHAKT-2,which levels were higher than L-02 SHGFP （0.22±0.14） （F=94.73,P〈 0.001）.After treated by wortmannin （2.5,5.0 μmol/L） combined with 40 μmol/L CdCl2, the expression of p-AKT in L-02 were 0.28±0.07, 0.15±0.11,which levels were lower than wortmannin untreated cells （0.52± 0.11） （F=578.57,P〈O.OO1）;and the expression of MT were 1.62± 0.80, 1.08± 0.15,which levels were higher than wortmannin untreated cells （0.69±0.18） （F=12.34,P〈0.001）.When treated by 40 μmol/L CdC12,the levels of p-AKT in L-02 SHB5613-1 and L-02 SHB5613-2 were 0.57±0.13, 0.59±0.02,which were higher than L-02 SHGFP（0.32±0.02）（F=87.16,P〈0.001）; and the levels of MT were 0.35±0.07, 0.20±0.03 in L-02 SHB5613-1 and L-02 SHB5613-2,whieh were lower than L-02 SHGFP （1.51 ±0.13） （F=2 457.10,P〈 0.001）. After treated by 40 μmol/L CdC12, the expression of p-AKT in L-02 RT-AFBI-B5613 and Be17402-B5613 were 0.10±0.11, 0.09±0.01,which were lower than L-02 RT-AFB1 （0.36±0.01） and Be17402 （0.43±0.11） （F=877.62,P〈0.001）; and the levels of MT were 0.92±0.13, 0.95±0.08 in L-02 RT-AFB1-B5613 and Be17402-B5613,which were higher than L-02 RT-AFB1 （0.44±0.12） and Be17402 （0.77±0.06） （F=51.97, P〈0.001）. Conclusion Protein phosphatase 2A complexes containing B5613 participated in the regulation of MT expression through direct dephosphorylation of AKT, finally affected the cytotoxicity responding to CdC12. Our study revealed a key signaling pathways of PP2A involved in heavy metals induced cytotoxieity.