The Study On Cholesterol Induced Unfolded Protein Response And ACAT2 Expression

Cholesterol is an important factor resulting in atherosclerosis. In recent years, it has been suggested that high cholesterol level resulting in the AS plaque relate with cholesterol-induced Unfolded Protein Response (UPR) and apoptosis closely. UPR has played a significant role in stringent mechanism of cells. When endoplasmic reticulum(ER) suffered from various stress, such as high cholesterol level, glucose starvation, calcium ion concentration disorder and so on, the normal protein folding function of ER is interrupted, a great many non-folding or faulty-folding proteins assembling in ER, contributing to the activation of signal transduction by way of protein receptor, producing active transcription factor to regulate many kinds of gene expression, in order to adapt to cellular environment change or resulting to cell apoptosis, this response is UPR. In mammalian cells, there are three transmembrane proteins locating on ER, that are Activating transcription factor-6(ATF6), Inositol requiring-1(IRE1)和PER-like ER kinase(PERK), they induce UPR by different signal transduction. When cells suffer from ER stress, the active part of ATF6 transferred from cytoplasm to nucleus, up-regulate the downstream gene XBP1 to transcript; a strong reactive transcription factor XBP1 is produced by alternative splicing by activated IRE1 endoribonuclease and then regulate various downstream gene expression, so XBP1 is regarded as a labeling gene of UPR; When PERK is activated, it will phosphorylate eIF2α, and then protein synthesis will be inhibited. It has been known that in acat2 promoter there be several cis-acting elements which can be recognized and combined by XBP1, and may be regulated by XBP1 in some cells. ACAT2 mainly distributes in hepatocyte and enterocyte, and it plays a significant role in cholesterol absorption and in hepatocyte metabolism, so it will be important to investigate cholesterol-induced UPR and acat2 expression to cholesterol metabolism and the research of AS mechanism.On the basis of the research on the role of cholesterol in xbp1 and acat2 expression in murine macrophage, we investigate the regulation of xbp1 and acat2 expression in hepatocyte and enterocyte incubated by free cholesterol and oxidative cholesterol, in order to investigate the effect of cholesterol on UPR and acat2 expression. RAW264.7 cell, HepG2 cell and Caco2 cell were incubated with different concentration of free cholesterol and oxidative cholesterol, 7-ketocholesterol(7-KC) for 8h, 16h, 24h respectively, and SD mice were fed with two different diet respectively ,containing 2% cholesterol and non-cholesterol diet, and the enterocytes were scrapped. The mRNA level of xbp1 and acat2 in RAW264.7 cell, HepG2 cell, Caco2 cell and the enterocytes from SD mice were detected by semi-quantitative RT-PCR. In cells incubated with FC and 7-KC, xbp1 and acat2 mRNA level in RAW264.7, HepG2 cell and Caco2 cell were up-regulated, and were dependent on the incubation temporal prolongation and concentration increase. In enterocytes from SD rats fed with diet containing 2% cholesterol, compared with those enterocytes from SD rats fed with non-cholesterol diet, xbp1 and acat2 mRNA level increased.The results suggest that cholesterol can induce UPR, and both of xbp1 and acat2 mRNA level can be up-regulated, and the results imply the possibility that acat2 is the downstream target gene of UPR, and UPR and acat2 may play an important role in the cholesterol esterification and absorption, and maybe important regulatory targets of cholesterol absorption.