Regulation Of Cholesterol Transport-related Proteins Expressions By Oleic Acid-induced Endoplasmic Reticulum Stress In CaCo-2 Cells

Oleic acid is a main component of dietary oil.It has been shown that Oleic acid is arecognized potent inducer to promote the synthesis and secretion oftriacylglycerol-rich lipoprotein,in which unesterified and esterified cholesterolderived from the plasma membrane were increased in intestinal mucosa epithelialcells incubated with oleic acid.Furthermore,it causes a modest increase of thesynthesis of cholesterol in the absence of micellar cholesterol.It has been reportedthat high concentration of Oleic acid decreases the percentage of cholesterolabsorption in animals fed by high amount of cholesterol.Epidemiological studies hasshown that a certain concentration of oleic acid can reduce the concentration ofplasma LDL cholesterol.So,oleic acid together with polyunsaturated fatty acid isknown as the"healthy fatty acid".However,the mechanism for how oleic acidinflences the absorption and transport of cholesterol is not clear.Hypercholesterolemia is a significant risk factor for atherosclerosis.In addition tode novo cholesterol synthesis,cholesterol derived from the diet also contributes to theamount of cholesterol circulating in plasma.Moreover,reducing the intestinalabsorption of dietary and biliary cholesterol will decrease plasma cholesterol level.It is clear,therefore,that the intestine plays an important role in maintainingwhole-body cholesterol homeostasis.In the past few years,Niemann-Pick C1-Like1 (NPC1L1),which resides in the brush-border membrane of intestine cells,has beenidentified as a cholesterol transporter that plays an important role in the absorption ofcholesterol by intestine.The NPC1L1 null mice are completely resistant todiet-induced hypercholesterolemia and do not get hypercholesterolemia in response toa high-cholesterol diet.ABCG5 and ABCG8 also expressed on the brush-border membrane of intestine cells,which function as a heterodimer,regulate the absoptionof cholesterol by facilitating the efflux of cholesterol from absorptive cells back intothe lumen.HMGCR is the rate-limiting enzyme of cholesterol synthesis.Part ofabsorbed and synthesized cholesterol is converted to cholesterol ester by ACAT2 inendoplasmic reticulum.Microsomal triglyceride transfer protein (MTP) is absolutelyessential for the assembly and secretion of triglyceride-rich lipoproteins in theintestine and liver.Sterol regulatory element binding protein-2 (SREBP-2) is amembrane-bound transcription factor that upon proteolytic processing can activate theexpression of genes involved in cholesterol biosynthesis and uptake.It has beenshown to up-regulate the expression of NPC 1 L1 and HMGCR,and negatively regulatethe expression of MTP when SREBP-2 is actived.Endoplasmic reticulum(ER) is the principal site for protein synthesis and folding,biosynthesis of cholesterol and fatty acids,as well as Ca²⁺ storage and signaling.Anyperturbation that interferes with these activities leads to ER stress and activates theunfolded protein response (UPR),resulting in transcriptional activation of geneswhose products promote the capacity of ER to alleviate the stress.It has beendocumented that OA- induced hepatic ER stress inhibits the secretion ofapolipoprotein B 100.However,whether OA would induce ER stress and further affectthe expression of cholesterol transport-related proteins in the intestinal mucosaepithelial cells is not clear.To explore the effect of OA on the expression of cholesterol transport-relatedprotein in intestinal mucosa epithelial cell and the associated mechanism,CaCo-2cells serve as a model and were incubated with various lipid micelles in this paper.CaCo-2 cells were incubated with various types of micelles containing differentconcentration of oleic acid with or without 0.2mM cholesterol.Western blottinganalysis was applied to determine the protein masses of NPC1L1,ABCG8,BRP78,mature SREBP-2 and ATF6.RT-PCR was used to detect the spliced XBP1,CHOP,NPC1L1,ABCG8,HMGCR,ACAT-2,MTP mRNA.4 - phenyl-butyric acid (PBA) wasemployed to confirm whether the expression variation of cholesterol transport-relatedproteins was induced by UPR or not. The research indicated that both mRNA and protein expression of ACAT-2,SREBP-2 was not altered in CaCo-2 cells incubated with different lipid micelles.Inthe presence or absence of micellar cholesterol,high concentration of oleic acid(0.5mM,1mM ) increased the amount of spliced XBP lmRNA,mature ATF-6 protein,BIP protein and CHOP mRNA expression,resulting in ER stress.In the presence of micellar cholesterol,OA increased the expression of ABCG8protein and MTP mRNA and decreased the expression of NPC1L1 and HMGCR in aconcentration-dependent manner in CaCo-2 cells incubated with OA/CHOL.Incontrast,in the absence of micellar cholesterol,OA increased the expression ofNPC1 L1 and HMGCR in a dose-dependent manner in cells incubated with OA alone.Treatment of CaCo-2 cells with PBA supressed OA-induced incrases of XBP 1 andthe protein mass ofmATF6,as well as BIP and CHOP expression.UPR inhibitor PBA diminished the effect of OA-induced UPR on the expression ofNPC1L1,HMGCR and MTP mentioned above in cells incubated OA/CHOL or OAalone.In a word,in the presence or absence of micellar cholesterol,the expression ofNPC1L1,HMGCR,MTP was diffenently regulaged by oleic acid-inducedendoplasmic reticulum stress in CaCo-2 cells.In the presence of micellar cholesterol,OA-induced UPR increased MTP expression and decreased the expression ofNPC1L1 and HMGCR in a concentration-dependent manner in CaCo-2 cellsincubated with OA/CHOL.In contrast,in the absence of micellar cholesterol,OA-induced UPR increased the expression of NPC1L1 and HMGCR in adose-dependent manner in cells incubated with OA alone.