The Effects of Highexpression and Knockdown Adipophilin in The Activity of ERK1/2 and Expression of PPARγ and Lipid Accumulation in Cells*

Abstract

Our previous studies have showed that adipophilin promoted intracellular lipids accumulation through ERK1/2-PPARγ signaling pathway.In the study we explored that whether highexpression and knockdown adipophilin affects the activity of ERK1/2 and the expression of PPARγ and lipid accumulation in RAW264.7 cells,and further certified that adipophilin promoted intracellular lipids accumulation through this pathway.The recombinant retroviral vetors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA were verified by the methods of enzyme-digesting.The recombinant retroviral vetors were transfected into PA317 cell by mediating SofastTM,which can induce retroviruses release.Then we used the collected retroviruses to infect RAW264.7 cells and achieved adipophilin gene highexpression and knockdown RAW264.7 cell lines applying puromycin screening.After the infected RAW264.7 cells incubated with 50 mg/L Ox-LDL for 24 h,the lipids accumulation were measured by Oil red O staining and HPLC,the expression of mRNA and proteins of adipophilin and PPARγ were detected by semi-quantitative RT-PCR and Western blot respectively,and phosphorylation of ERK1/2 was analyzed by Western blot too.The results of enzyme-digesting confirmed the recombinant retroviral vectors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA as expected.In the situation of Dutch fat,transfected cells with pQCXIP-HA-Adipophilin significantly increased the accumulation of lipids,but reduced the expression of PPARγ and the phosphorylation of ERK1/2,which were reversed in cells with pSuper-retro-adipophilin siRNA transfection.Our results showed that ERK1/2 and PPARγ may be related with lipids accumulation caused by adipophilin expression in macrophages incubated with modified LDL.Therefore,adipophilin might contribute,in vivo,to lipid accumulation in the intima of the arterial wall through the ERK1/2-PPARγ signaling pathway.