Adipophilin Facilitate ACAT1 Expression In RAW264.7 Cells

Background:The accumulation of neutral lipid, such as cholesterol, plays a key role in the foam-cell formation and in the development of atherosclerotic lesion. In eukaryocytes, the deposited lipid is in the form of lipid droplets. The content of adipophilin is the highest among some 40 lipid droplet-related proteins. Its main function is to promote lipid accumulation and inhibit cholesterol efflux.ACAT(acyl-coenzyme A:cholesterol acyltransferase, ACAT) is the main intracellular cholesterol ester synthetase .It catalyzes free cholesterol and long-chain fatty acid to synthesize cholesterol ester so that it can influences the lipid accumulation. The concordance of adipophilin and ACAT in function and subcellar location suggests that there is close correlation between them.But the mechanism of action between them is still unknown.Objective: To explore whether adipophilin affects the expression of ACAT1 mediated by PKC signal pathway and illuminate the lipid-accumulation mechanism mediated by adipophilin,our study,firstly,obtained a RAW264.7 cell line high expressing adipophilin gene by infection and then overviewed the expreesion of ACAT1 in RAW cells which overexpressed adipophilin.Method: The recombinant retroviral vetor pQCXIP-HA-Adi were tested with the methods of enzyme-shearing and RT-PCR.Transfecting the recombinant retroviral vetor into packaging cell PA317 mediated by SofastTM,which can induce retrovirus release.The recombinant retroviral virus was collected.Then we used this retrovirus to infect RAW264.7 cell and achieved adipophilin gene high expression RAW264.7 cell line afer Puromycin screening. The mRNA level of adipohilin and ACAT1 was determined by reverse transcription-polymerase chain reaction(RT-PCR).The adipophilin and ACAT1 proteins were analyzed by Western Blot.The cells were incubated with Atorvastatin and PKC inhibitor Calphostin C,respectively.Investigated the expression of ACAT1.Result: The results of enzyme-shearing and RT-PCR confirmed the recombinant retroviral vector pQCXIP-HA-Adi were as expected.The virus released from packaging cell PA317 after transfection.Using RT-PCR and Western Blot detected infected RAW264.7 cells, which indentified adipophilin mRNA and protein both up-regulated.According to RT-PCR and Western Blot results,pQCXIP transfection had no effect on ACAT1 expression,but pQCXIP-HA-Adi transfection significantly up-regulated ACAT1 expression.The expression of ACAT1 still increased in pQCXIP-HA-Adi transfection after used Atorvastatin to inhibit the composition of the substrate for removing the effect of the substrate on ACAT1.Adding PKC inhibitor Calphostin C the expression of ACAT1 was down-regulated in RAW264.7 cells loaded with lipid or not by RT-PCR and Western Blot. Conclusion: The retroviral vector pQCXIP-HA-Adi was successfully constructed and infected RAW264.7 cell, obtaining a new RAW264.7 cell lines which highly express adipophilin protein.Overexpressed adipophilin upregulated ACAT1 expression in RAW264.7 cells.It suggests that adipophilin accumulated cellular cholesteryl ester by ACAT1 and the PKC signal patheway maybe invovled in it.