水通道蛋白9磷酸化对哺乳动物细胞中砷摄入的影响

AQP9 Protein Phosphorylation Decreases Arsenic Influx in Mammal Cells

目的研究水通道蛋白9(AQP9)mRNA表达水平、水通道蛋白9及p38蛋白表达及其磷酸化水平对肝癌细胞HepG2和肝正常细胞L-02砷摄入的影响,探讨水通道蛋白9磷酸化的调控机制。方法采用电感藕合等离子体质谱法(ICP-MS)测定细胞内砷含量。采用实时定量PCR、免疫印迹法和免疫共沉淀技术分别检测不同处理后两细胞株中水通道蛋白9 mRNA、水通道蛋白9和p38蛋白表达水平及其磷酸化水平。采用SPSS统计软件分析实验数据。结果 HepG2细胞内砷含量及摄入速度高于L-02细胞。HepG2细胞的水通道蛋白9 mRNA表达水平在6 h内随NaAsO2处理时间延长而显著增加(P<0.05),而在L-02细胞无明显变化。在2 h时,HepG2细胞水通道蛋白9基因表达水平在各NaAsO2处理浓度均显著增加(P<0.05)。免疫沉淀实验结果显示,HepG2细胞中水通道蛋白9蛋白磷酸化水平随NaAsO2处理时间和浓度的增加而增加,而L-02细胞在各时间和浓度处理点水通道蛋白9蛋白磷酸化水平与对照相比明显增加,但处理间无明显差异;p38的磷酸化水平在两种细胞中均随砷处理时间延长而增加;SB203580抑制p38活性后能完全取消L-02细胞水通道蛋白9蛋白的磷酸化,而对HepG2细胞水通道蛋白9蛋白磷酸化无明显影响。结论水通道蛋白9的表达及磷酸化水平可能在调节细胞砷摄入中发挥重要作用,在不同细胞中水通道蛋白9蛋白磷酸化的调控机制有所差异。

OBJECTIVE To detect the levels of aquaglyceroporin 9 （ AQP9 ） mRNA expression, AQP9 and p38 proteins and their phosphorylation in HepG2 and L-02 cells treated with NaAs02, and to investigate the association of these expression levels with arsenic intake and the AQP9 phosphorylation mechanism. METHODS The intracellular arsenic content was determined by inductively cou- pled plasma mass spectrometry （ ICP-MS ）. Real-time quantitative PCR, Western blotting and immunopreeipitation techniques were used to detect AQP9 mRNA, AQP9 and p38 protein levels and their phosphorylation levels in HepG2 and L-02 cells. SPSS statistical software was used to analyze experimental data. RESULTS Intracellular arsenic content and intake rate in HepG2 ceils were faster than those in L-02 cells. AQP9 mRNA levels in L-02 cells was increased with time within 6 h after NaAs02 treatment （ P 〈 0.05 ） , while no significant change was observed in L-02 cells. Two hours after treatment, AQP9 gene levels in HepG2 cells were all signifi- cantly increased at different coocentrations of NaAs02. The phosphorylation levels of AQP9 in HepG2 cells were increased with treating time and concentration of NaAsO2. While the phosphorylation levels of AQP9 in L-02 cells significantly increased compared to control at each time point and concentration, but no significant difference was shown between the treatments, p38 phosphorylation levels in both cells were increased with time. Inhibition of p38 activity by SB203580 completely abolished AQP9 protein phosphorylation in L-02 cells, while it had no significant effect on HepG2 cells. CONCLUSION AQP9 expression and phosphorylation levels may play an important role in regulating arsenic influx ; regulation mechanism of AQP9 phosphorylation may be different in different cells.