Regulatory Role Of Oxidative Stress On The Methylation Of Protein Phosphatase 2A Catalytic Subunit

Protein phosphatase 2A(PP2A)is an intracellular important and universal expression serine/threonine phosphatase,which consists of structural subunit(A subunit),regulatory subunit(B subunit)and a catalytic subunit(C subunit,PP2Ac).Modification of the catalytic subunit plays an important role to PP2A holoenzyme regulation.Studies have shown that the methylation of PP2A catalytic subunit was associated with a variety of cellular processes.There are studies suggest that PP2A is an oxidation-sensitive protein,but whether or not oxidative stress can lead to the methylation of catalytic subunit changes is unclear.H2O2 is the major intracellular reactive oxygen species,either as a direct result of biological macromolecules oxidative stress,or as a second messenger involved in oxidative stress-related signaling pathways regulated.This study will provide understanding on oxidative stress induced PP2A dependent biological effect and provide new clue on prevention and treatment of oxidative stress related disease and chemical damage.Objective1.examine the effect of oxidative stress on the methylation of PP2A catalytic subunit.2.explore the mechanism of the methylation of PP2A catalytic subunit changes under oxidative stress.MethodsWe selected mouse embryonic fibroblasts(NIH/3T3 cells)and human epithelial fibroblasts(HPF cells)as the object of study.Resazurin reducing rate was observed to determine the cell proliferation.Expression of demethylated PP2Ac and total PP2A c was observed by Western blot.PME-1 inhibitor AMZ30 and antioxidant NAC were used to block the effect of H2O2,resazurin reducing rate was observed to determine the cell proliferation,expression of un-methylated PP2Ac and PP2Ac was observed by Western blot.Confocal laser scanning was used to observe the changes of demethylation PP2Ac and total PP2Ac in normal control group,1mmol·L-1H2O2 group,1mmol·L-1H2O2+1mmol·L-1NAC group,lmmol·L-1H2O2+10?mol-L-1 AMZ30 group in NIH3T3 cells.Confocal laser scanning was used to observe HPF cells the changes of demethylation PP2Ac,total PP2Ac,PME-1,LCMT in normal control group,1mmol·L-1 H2O2 group,lmmol·L-1H2O2+1mmol·L-1 NAC group,lmmol·L-1H2O2+10?mol·L-1AMZ30 group.ResultsNIH/3T3 cell viability were significant restrained at the present of 0.1-25mmol·L-1 H2O2(P<0.01).After H2O2 treatment for h,demethylated PP2Ac expression were significant increased at 1 and 5mmol·L-1 H2O2,compared with the control group.Both NAC(0.1-5mmol·L-1)and AMZ30(5-10?mol·L-1)significantly inhibited the demethylation of PP2Ac induced by lmmol·L-1 of H2O2,but only NAC show cell survival benefit against H2O2.Confocal laser scanning found that H2O2 increased unmethylated PP2Ac expression both in the cytoplasm and in the nucleus,while the nuclear/cytoplasmic ratio was significantly lower than that of the control group.after using NAC to antagonize H2O2,nuclear/cytoplasmic ratio of PME-1 and unmethylated PP2Ac were significantly increased;while AMZ30+H2O2 group remained similar to that of H2O2 treatment.nuclear/cytoplasmic ratio of total PP2Ac and LCMT did not show significant differences among the groups.Conclusion1.H2O2 could cause intracellular PP2Ac demethylation,while antioxidants NAC and PME-1 inhibitors AMZ30 could antagonize the demethylation of PP2Ac induced by H2O2.2.H2O2 decreased nuclear/cytoplasmic ratio of un-methylated PP2Ac in cells.Antioxidant NAC could reverse the nuclear to cytosolic ratio of PP2Ac back to control level,but AMZ30 did not show significant effect.