SREBP2-STARD4 Is Involved In Synthesis Of Cholesteryl Ester Stimulated By Mono-butyl Phthalate

Phthalate esters are a class of chemicals used as plasticizers or softeners in innumerable products.PAEs is not covalently bound to these products so that it can leach,migrate or evaporate into indoor air and atmosphere easily.PAEs can result in severe anti-androgenic effects and cause diverse profiles of reproductive abnormalities including deformed epididymis,hypospadias,and cryptorchidism.Dibutyl phthalate（DBP）is one of the major phthalate esters and its active metabolite is monobutyl phthalate（MBP）.Our previous studies have found that MBP exposure can increase the level of progesterone at about 10-7M in mouse Leydig tumor cells（MLTC-1 cells）.In steroidogenic cells,progesterone is synthesized from cholesterol which is from the hydrolysis of stored cholesteryl esters in the form of lipid droplets.It has’nt been investigated that MBP can affect the synthesis of cholesterol esters.In this study,MLTC-1 cells were used to explore whether SREBP2-STARD4signal pathway is involved in the process of cholesteryl ester synthesis disrupted by MBP.Objective:1.Explore the effect of monobutyl phthalate（MBP）in the synthesis of cholesterol esters.2.Investigate the role of steroid hormone synthesis of acute regulatory protein 4（STARD4）in the promotion of cholesterol ester synthesis by MBP.Methods:1.Mouse Leydig tumor cells（MLTC-1）was used as a cell model and the intracellular cholesterol ester content was measured by microplate reader.2.ORO staining was used to observe the distribution of cholesterol esters in MLTC-1 cells.3.The expression of STARD4 and SREBP2 mRNA was determined by qRT-PCR.4.Western Blot assay was used to detected the protein expression of STARD4and SREBP2.5.Immunofluorescence cytochemistry was used to observe the expression changes of STARD4 in MLTC-1 cells.6.The expression of STARD4 was down-regulated by RNAi technique,and MLTC-1 cells were treated with MBP(10^-7M)to observe the cholesterol ester content in MLTC-1 cells.7.The expression of SREBP2 was down-regulated by RNAi technique,and MLTC-1 cells were treated with MBP(10^-7M)to observe the protein expression level and intracellular free cholesterol content of STARD4 in MLTC-1 cells.8.Double luciferase labeling assay to detect the binding activity of SREBP2 to the STARD4 promoter region.Results:1.MBP promoted the synthesis of cholesterol esters in MLTC-1 cellsAfter exposure of 10^-7M MBP,the intracellular cholesterol ester content of MLTC-1 cells was determined by enzyme-labeled method.It was found that the intracellular cholesterol ester content was significantly increased compared with the control group（p<0.05）.ORO staining showed that lipid staining was compared with the control.It was significantly darker by MBP treatment at 10^-7M.2.The role of STARD4 in MBP-promoting cholesteryl ester synthesis in MLTC-1 cellsResults of qRT-PCR and Western Blot showed that the mRNA and protein levels of STARD4 increased markedly at 10^-7M MBP treatment compared with the control group（p<0.05）.Results of immunofluorescence cytochemical staining combined with laser confocal method showed that STARD4 increased on the endoplasmic reticulum after 10^-7M MBP treatment.RNAi experiments showed that STARD4 expression levels were correlated with intracellular cholesterol ester levels.After treatment with10^-7M MBP,STARD4 protein expression increased and the synthesis of intracellular cholesterol esters also increased（p<0.05）.3.Effect of MBP on the expression of SREBP2qRT-PCR and Western Blot experiments showed that MBP could promote SREBP2 mRNA and protein levels.After the expression of SREBP2 inhibited,the expression of STARD4 protein in MLTC-1 cells decreased.Treated with MBP(10^-7M),the expression level of SREBP2 protein was restored（p<0.05）.4.MBP promotes cholesterol ester synthesis through SREBP2-STARD4pathwayAfter inhibited the expression of SREBP2,the expression of STARD4 protein in MLTC-1 cells decreased,the content of free cholesterol in cells increased.When MLTC-1 cells were treated with MBP(10^-7M),the expression levels of SREBP2 and STARD4 protein were restored,and intracellular free cholesterol was recovered（p<0.05）.Using dual luciferase labeling experiments,it was found that SREBP2 can bind to the STARD4 promoter region,and treated with MBP(10^-7M)increased the binding activity of the SREBP2 and STARD4 promoter regions（p<0.05）.Conclusion:MBP promotes cholesteryl ester synthesis in MLTC-1 cells via the SREBP2-STARD4 signaling pathway.