Adipophilin Blocks Nceh1Hydrolyzing Intracellular Lipid In RAW264.7Cells

Objective With co-immunoprecipitation and bioinformatic to explore whetheradipophilin interact with Neutral cholesterol ester hydrolase1(Nceh1). We observedthe expression of Nceh1and the accumulation of intracellular lipids after theRAW264.7cells treated with100nmol/L PMA40min, In order to further measure theexpression Nceh1and the accumulation of intracellular lipids, we establishedrecombinant retroviral vectors, PQCXIP-HA-Adipophilin andpSuper-retro-adipophilin siRNA. And we also obtained both overexpression andknockdown of adipophilin RAW264.7cell lines. We want to explore that whetheradipophilin promoted intracellular lipids accumulation through Nceh1, which canclarify the mechanism of adipophilin in foam cell formation.Methods With co-immunoprecipitation to explore whether adipophilin interactwith Nceh1; With bioinformatics to predict three-dimensional structure of adipophilinand Nceh1, and observe the regions of them combination. The lipids accumulation ofcells were stained by High performance liquid chromatography(HPLC), theexpression of mRNA and proteins of adipophilin and Nceh1were detected bysemi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) andwestern blot respectively. The recombinant retroviral vetors pQCXIP-HA-Adipophilinand pSuper-retro-adipophilin siRNA were tested and verified by the methods ofenzyme-shearing. Transfected the recombinant retroviral vetors into packaging cellPA317was mediated by SofastTM, which can induce retrovirus release. Therecombinant retroviral viruses were collected. Then we used the retroviruses to infectRAW264.7cells and achieved adipophilin gene overexpression and knockdownRAW264.7cell lines applying puromycin screening. After the infected RAW264.7cells of overexpression and knockdown adipophilin incubated with PKC agonist100nmol/L PMA for24h, adipophilin and its phosphorylation involved in the pathogenesis of atherosclerosis were measured by western blotting, The lipidsaccumulation of cells were stained by High performance liquidchromatography(HPLC), the expression of mRNA and proteins of adipophilin andNceh1were detected by semi-quantitative reverse transcription-polymerase chainreaction(RT-PCR) and western blot respectively. RAW264.7cells were incubated with50ng/ml oxidized low density lipoprotein (ox-LDL） for24h, following with100nmol/L PMA preincubated40min, intracellular distribution of adipophilin andNceh1were detected by confocal laser scanning.Results Co-immunoprecipitation showed that there were interactions betweenadipophilin and Nceh1in RAW264.7mouse macrophages,bioinformatics predictionfound adipophilin and Nceh1interactions through the hydrophobic region residues.The results of enzyme-shearing confirmed the recombinant retroviral vectorspQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA as expected. Theviruses were released from packaging cells PA317after transfection. In the situationof Dutch fat, pQCXIP and pSuper-scramble siRNA transfection had no effect onNceh1expression and intracellular lipids accumulation. Transfected cells withpQCXIP-HA-Adipophilin significantly increased the accumulation of lipids, butmRNA and protein expression of Nceh1aslo did not change significantly. After with100nmol/L PMA pretreatment transfected cells with pQCXIP-HA-Adipophilin,phosphorylated adipophilin expression was significantly increased and adipophilin isactivated, the number of lipid droplets reduce, mRNA and protein expression Nceh1did not change significantly. RAW264.7cells were incubated with50mg/L ox-LDLfor24h, confocal laser scanning found that adipophilin and Nceh1distributed in lipiddroplets surface; and Nceh1transferred from the cytoplasm to the lipid dropletsurface.Conclusion Adipophilin can be combined with Nceh1, adipophilin expressionchanges did not affect the expression of Nceh1, adipophilin can be phosphated byPKC, and the number and size of lipid droplets showed a downward trend, but theexpression of Nceh1did not change significantly. Nceh1transferred from thecytoplasm to the lipid droplet surface When the lipid droplets appear.These results of findings indicated that adipophilin involves in the formation of foam cells. It suggeststhat adipophilin promote lipid accumulation mechanism is blocking cholesterol esterhydrolysis by Nceh1.