Ad I Po Ph I L In Pr O M O T E S L I Pi D Ac C Um U L A T Io N T H Ro U G H E RK 1 / 2- P P A Rγ Pa T Hw A Y I N R A W 26 4. 7 C E L L S

Objective We observed the expression of peroxisome proliferator-activated receptor gamma (PPARy) and adipophilin, the activity of Extracellular signal-regulated kinasel/2(ERKl/2) and the accumulation of intracellular lipids after the RAW264.7 cells treated with oxidized low density lipoprotein (Ox-LDL) for different time, or with ERK1/2 inhibitors and Ox-LDL, or with PPARy inhibitor and Ox-LDL, or with PPARy agonist and Ox-LDL. In order to further measure the expression of PPARy and adipophilin, the activity of ERK1/2 and the accumulation of intracellular lipids, we established recombinant retroviral vectors, PQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA. And we also obtained both overexpression and knockdown of adipophilin RAW264.7 cell lines. We want to explore that whether adipophilin promoted intracellular lipids accumulation through ERKl/2-PPARy signaling pathway, which can clarify the mechanism of adipophilin in foam cell formation.Methods RAW264.7 cells were incubated with 50μg/ml Ox-LDL for different time, or were preincubated with ERK1/2 inhibitors or PPARy inhibitors/agonists for 2 h following with 50μg/ml Ox-LDL incubated for 24 h. The lipids accumulation of cells were stained by Oil red 0 staining, the expression of mRNA and proteins of adipophilin and PPARy were detected by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and western blot respectively. ERK1/2 and its phosphorylation involved in the pathogenesis of atherosclerosis were measured by western blotting. The recombinant retroviral vetors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA were tested and verified by the methods of enzyme-shearing. Transfected the recombinant retroviral vetors into packaging cell PA317 was mediated by SofastTM, which can induce retrovirus release. The recombinant retroviral viruses were collected. Then we used the retroviruses to infect RAW264.7 cells and achieved adipophilin gene overexpression and knockdown RAW264.7 cell lines applying puromycin screening. After the infected RAW264.7 cells of overexpression and knockdown adipophilin incubated with 50μg/ml Ox-LDL for 24 h, the lipids accumulation of cells was stained by Oil red O staining, the expression of mRNA and proteins of adipophilin and PPARγwere detected by semi-quantitative RT-PCR and western blot respectively, and phosphorylation of ERK1/2 was analyzed by western blot too.Results The amount of cytoplasmic lipid droplets, the expression of mRNA and proteins of adipophilin and PPARγ, and the phosphorylation of ERK1/2 were significantly increased, as the incubation time of Ox-LDL was extended in RAW264.7 cells, which were reversed by the treatment of ERK1/2 inhibitor and PPARγinhibitor, and the phosphorylation of ERK1/2 reduced by inhibitors too. Whereas PPARγagonists increased the number of cytoplasmic lipid droplets, the expression of mRNA and proteins of adipophilin and PPARγand the phosphorylation of ERK1/2. The results of enzyme-shearing confirmed the recombinant retroviral vectors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA as expected. The viruses were released from packaging cells PA317 after transfection. In the situation of Dutch fat, pQCXIP and pSuper-scramble siRNA transfection had no effect on PPARγexpression and intracellular lipids accumulation. Transfected cells with pQCXIP-HA-Adipophilin significantly increased the accumulation of lipids, but reduced the expression of PPARγand the phosphorylation of ERK1/2, which were reversed in cells by pSuper-retro-adipophilin siRNA transfection.Conclusion Ox-LDL increased the activity of ERK1/2. PD98059, inhibitor of ERK1/2, inhibited the expression of mRNA and proteins of adipophilin and PPARγand intracellular lipids accumulation. PPARγagonist increased the number of cytoplasmic lipid droplets, the expression of mRNA and proteins of adipophilin and PPARγand the phosphorylation of ERK1/2, which were reversed by the treatment of PPARγinhibitor. Overexpressed adipophilin significantly increased intracellular lipids and reduced the phosphorylation of ERK1/2, while low expression of adipophilin obviously decreased intracellular lipids and increased ERK1/2 activation. These results of findings indicated that adipophilin involves in the formation of foam cells. It suggests that Ox-LDL induce the increasement of adipophilin level via ERKl/2 activation, which is one of the mechanisms of accumulation of intracellular lipid droplets in RAW264.7 cells.