| Background:Eutrophication and cyanobacterial caused by discharging of wastewater without any treatment have become a global concern of environmental issues.Microcystins(MCs)produced by excessive cyanobacteria growth are toxic to aquatic organisms and human beings.Over 100 different structural analogues of MCs have been reported.Among them,microcystin-LR(MC-LR)is the most common and toxic variant,whose main target organ is the liver.It has been reported that MC-LR can induce serious hepatotoxicity,such as necrosis,apoptosis,and haemorrhage.The main toxic mechanism of MC-LR is its potent inhibition on protein phosphatase 2A(PP2A).PP2A,as one of the most important serine/threonine phosphatases in eukaiyotic cells,plays crucial roles in various cellular processes including cytoskeleton dynamic regulation.α4,an essential regulator of PP2A,has been demonstrated to have important roles in regulating PP2A,as it maintains the stability of PP2A activity and manages PP2A core enzyme assembly.It has been reported that elevated a4 expression induced an increase in cell proliferation and enhanced cell survival.Besides,a4 is important for regulating cell cytoskeletal organization.In our previous study,it is observed that MC-LR leads to the reorganization of cytoskeleton in a variety of cell lines,presumably due to inhibition of PP2A.We also reported that MC-LR can stimulate cellular PP2A activity at relatively low concentration in human embryonic kidney cell line(HEK293),which may be mediated by partial α4/PP2A/C dissociation.Moreover,our recent research demonstrated that HEK293 cells treated with MC-LR had shown more stable cytoskeletal structure when overexpressing a4,which suggested that the elevated a4 confers some resistance to MC-LR-induced cytosketetal change.All this arouses our consideration whether a4 plays a similar role in other cell lines exposed to MC-LR.In the present study,the effects of MC-LR on human liver cell line 7702(HL7702)under the condition of a4 overexpression was studied.The aim is to obtain a comprehensive understanding of the relationships among a4,PP2A,and cytoskeleton after exposure to MC-LR.Methods:The HL7702 cells were transfected with a4 construct plasmids for 24h using LipofectamineTM 2000 and then exposed to the different concentrations of MC-LR(0μM,1μM and 10μM)for 24 h.The HL7702 cells transfected with empty plasmid vector were served as vector control.PP2A activity kit,western blot,immunoprecipitation assay and immunofluorescence assay were used in the study.Results:PP2A activities were inhibited significantly in a4-overexpressing HL7702 cells treated with MC-LR,meanwhile,the protein level of PP2A/C and the phosphorylation level of p-PP2A/C(Y307)were increased and the methylation level of m-PP2A/C(L309)was decreased.Besides,the binding of PP2A/C and a4,and the binding of PP2A/C and PP2A/A were decreased.Accompanied by the changes of PP2A the ERK1/2,JNK and P38 MAPK pathway were activated,as the phosphorylation level of p-ERK(T202/Y204),p-JNK(T183/Y185)and p-P38(T180/Y182)all increased significantly.There are no obvious changes in actins,tubulins,and vimentin IFs in all the treated groups.The phosphorylation levels of VASP(S157),Ezrin(T567)were increased and the phosphorylation levels of VASP(S239)were decreased.Conclusion:Taken together,the changed protein level and post-translational modification of PP2A/C subunits,and the binding of PP2A/C to a4 as well as the AC core enzyme,may be the reasons for MC-LR inhibiting PP2A activities in a4-overexpressing HL7702 cells.The activaton of ERK1/2,JNK and P38 MAPK pathway may be related to PP2A inhibition.Elevated a4 expression may partly counteract the toxicity of MC-LR and HL7702 cells show more stable cytoskeletal structure,which might be correlated with the changed phosphorylation levels of VASP(S157),VASP(S239),and Ezrin(T567)due to the activation of ERK1/2,JNK and P38 MAPK pathway.The response of a4 overexpression HL7702 and HEK293 cells to MC-LR are similar but underline mechanism might be different. |
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