Overexpression Of ESCRT System Members Enhances Cells Ability To Cope With U18666A-induced Cholesterol Accumulation

Objective:Alveolar type II?AT2?cells are tissue stem cells that maintain environmental stability in the alveolar region.One of their functions is to secrete pulmonary surfactants,prevent alveolar collapse,and participate in alveolar immune regulation.Pulmonary surfactants are a complex mixture of lipids and related proteins synthesized by AT2.Laminar bodies?Lbs?are lysosomal-associated organelles characteristic of mature AT2 cells and are a form of storage and secretion of pulmonary surfactant.The formation of lamellar bodies requires not only multivesicular bodies,but also glycogen granules and ribosomes in multivesicular vesicles.Pulmonary surfactants are composed of phospholipids(a large part of which are dipalmitoylphosphatidylcholine?DPPC?,cholesterol and surfactant-related proteins.They account for 80%,10%and 10%,respectively,as a complete functional system.The three components are important.The molecular structure of the pulmonary surfactant is closely related to the cholesterol concentration.The exact phase behavior of the pulmonary surfactant depends strongly on the exact composition of the lipid-protein mixture of the pulmonary surfactant,water content,Solution conditions and external conditions such as temperature and pressure.Minor changes in any of these parameters may result in movement of the phase boundary position.The endosomal sorting complex required for transport?ESCRT?is a highly conserved,versatile system widely found in yeast and eukaryotic plant and animal cells,including ESCRT-0,I,II,III.Complex and Vps4-Vtal cofactor family.The ESCRT system plays a key role in the formation of endosome and multivesicular bodies,membrane cleavage during cytokinesis,and a series of membrane remodeling processes,and plays an important role in the regulation of miRNA activity.The formation of multivesicular bodies is associated with a variety of cellular physiological activities such as viral budding secretion,secretion of exosomes,and formation of specific intracellular lamellar bodies.U18666A is a cell-permeable amphiphilic aminosteroid that was first marketed as a drug for the inhibition of cholesterol synthesis and then delisted due to severe side effects.The current study found that U18666A is an inhibitor of intracellular cholesterol transport,which can inhibit the transport of cholesterol from lysosomes to late endosomes and the reverse Golgi network,resulting in the accumulation of cholesterol in cell lysosomes,pathological changes of cells and NPC-1 mutations.Type N Niemann-Pick disease is similar.In type 2 alveolar epithelial cells,U18666A also promotes the accumulation of cholesterol in the plaque organelle lamellar body,which is associated with NPC-1.U18666A can interfere with the formation of multivesicular cells in the cell and inhibit the release of cells from the virus such as HIV and Hepatitis C virus?HCV?by budding.The multi-follicle-related functions that U18666A is involved in are overlapped with the ESCRT system.The relationship between the cellular physiology caused by U18666A and the ESCRT system is a question worth pondering.In this study,transgenic MLE12 cell lines overexpressing ESCRT-III complex members,Chmp1b and Vps24,were used as materials.The normal MLE12 cells and cells transfected with empty vector were used to examine the changes of cholesterol accumulation ability of U18666A-induced transgenic cells.The U18666A pair was analyzed.The influence of the viability of transgenic cells on the regulation of U18666A and ESCRT system was discussed.Methods:This study used U18666A to induce MLE12 cells.Since high doses of U18666A caused apoptosis,we induced MLE12 cells with different concentrations of U18666A.The expression of Vps4,Vps24 and Chmp1b in ESCRT system was observed by Western Blot.The drug concentration was 0.5?g/mL,1?g/mL,2?g/mL,4?g/mL and 6?g/mL.To detect the distribution of members of the ESCRT system after drug U18666A interference,we used immunofluorescence detection.Here we examined the distribution of Chmp1b protein in MLE12 cells compared with the control group after drug U18666A interference.We used flow cytometry to detect MLE12 cells interfered with 1?g/mL U18666A for 24h and DMSO.Compared with the control group,the G₂/M phase cells in the interference group increased,and G₂/M phase cell block appeared.In order to further explore the regulatory relationship between U18666A and ESCRT system,we established a transgenic cell line overexpressing Vps24 and Chmp1b,and used western blot to detect the expression of Chps1b with myc-his tag and obtain the correct overexpression transgene.Cell line.MLE12 cells,MLE12-VPS24 cells,MLE12-CHMP1B cells,and MLE12-VECTOR cells were taken.Concentration effect:U18666A was added to the experimental group of each cell line at a final concentration of 0.1,0.2,0.4,0.8?g/mL,and the same volume of DMSO was added to the control group.The cholesterol accumulation of each cell was observed after induction.Time effect:U18666A was added to each cell line at a final concentration of 0.5?g/mL.After 4 h,8 h,and 16 h of drug induction,the accumulation of cholesterol in each cell was observed.MLE12 cells,MLE12-VECTOR cells,MLE12-VPS24 cells,MLE12-CHMP1B cells,0.4%Trypan Blue,and cytometer TC20 were counted,and the cell viability of each cell at each time point was compared.Results:Western blot was performed after treatment of MLE12 cells with different concentrations of U18666A for 48 h.With the increase of U18666A concentration,the overall trend of Vps4,Vps24 and Chmp1b relative to?-tubulin in ESCRT system members was decreased.We underwent immunofluorescence staining of MLE12 cells after interference with 2?g/mL U18666A for 48h.Compared with the control group,the cells of U18666A-induced cells were distributed in the nucleus and plasma.Flow cytometry was performed on MLE12 cells interfered with by U18666A.Compared with the control group,G₂/M phase arrest was observed in the experimental group.We detected two stable cell lines of MLE12 overexpressing Chmp1b and Vps24 by western blot.MLE12,MLE12-VECTOR,MLE12-VPS24,MLE12-CHMP1B cell lines were treated with U18666A at a final concentration of 0.1,0.2,0.4,and 0.8?g/mL,respectively.The results showed that MLE12 cells and MLE12-VECTOR had significant cholesterol accumulation when the concentration of U18666A was0.2?g/mL,while MLE12-VPS24 and MLE12-CHMP1B had a concentration of0.4?g/mL at U18666A.MLE12,MLE12-VECTOR,MLE12-VPS24,MLE12-CHMP1B cell lines were treated with U18666A at a final concentration of0.5?g/mL,respectively.The results showed that MLE12 cells and MLE12-VECTOR showed cholesterol accumulation at 8h of U18666A interference,while MLE12-VPS24 and MLE12-CHMP1B had little accumulation of cholesterol when interfered with U18666A for 8h.Cholesterol accumulation occurred after 16 hours.The cells in each group were collected and counted at 24h,48h,and 72h,respectively.The analysis showed that the survival rate of each cell line decreased after U18666A treatment.However,at 72 h,MLE12-VPS24,MLE12-CHMP1B cell line survival rate was higher than MLE12,MLE12-VECTOR cell line.Conclusion:U18666A interferes with a part of the function of the ESCRT system.The ESCRT system is closely related to the biological function of U18666A.The expression of the protein associated with the endosomal sorting complex?ESCRT?system is reduced after the U18666A is used to treat MLE12 cells.The ESCRT system has an effect on intracellular cholesterol transport.Starting with the ESCRT system,it can provide new ideas for studying the pathogenesis of type C Niemanneck disease.