| Objective: Cholesterol stone is a common type of gallbladder stone,which is associated with reduced bile acid excretion by liver and increased cholesterol level in bile.Caveolae are specialized lipid rafts that form flask-shaped invaginations of the plasma membrane which play important roles in endocytosis,exocytosis,stabilization of plasma membrane,and signal transduction.Caveolae family compose of Caveolin-1(Cav-1),Caveolin-2(Cav-2),Caveolin-3(Cav-3).The number and structure of the caveolae alter under cholesterol stimulation,further affecting signal transduction via various type of receptors such as bile salt export pump(BSEP,ABCB11).Cav-1 plays a crucial role in cellular signal transduction and endocytosis and was proved to be co-localized with cholesterol-enriched membrane domains.Moreover,transportation of bile acid through bile canaliculi is an important driving force for liver to excrete bile.The primary rate limiting step of bile acid excretion by liver is the transportation through BSEP.BSEP belongs to ATP binding cassette(ABC)superfamily and is closely related to the formation of cholesterol stones.Moreover,BSEP is the most probable candidate gene for lithogenic gene Lith1.Studies have shown that BSEP in the bile canalicular side membrane of rat hepatocytes is localized in the Cav-1-rich microdomain of the cell membrane,however,the relationship between Cav-1 and BSEP is unclear.Our study aims to elucidate the roles of C av-1 and BSEP in the formation of cholesterol stones.Methods: Part I: Effects of different cholesterol concentration o n the expression,localization and transport function of BSEP.C57BL/6 mice(8 weeks old,male,SPF level),weight 20-22 g.20 mice were randomly divided into two groups: control group(n=10)fed with basal diet,experimental group(n=10)fed with high-fat and high cholesterol food(HFC)(83.5% basal food,15%fat,1%cholesterol,and 0.5%bile salt).All groups were kept in 23°C temperature room with free access to water.Two and four weeks later,5 mice from each group were randomly selected and killed,and their liver and gallbladder were removed.The mice in each group were observed for formation of gallbladder stone.Western blot and real time PCR were used to detect the expression of protein and m RNA of BSEP.Hep G2 cells were conventionally cultured.Cholesterol stock solution with different concentrations were added into culture dishes to gain different culture condition.After 48 hours,BSEP protein and m RNA expression under different cholesterol concentration were detected.BSEP on plasma membrane and in cytoplasm were extracted and protein expression was detected.Immunofluorescence single-staining was applied to observe the alteration of cellular localization of BSEP under different cholesterol concentration in Hep G2 cells.Finally,ATPase activity assay was applied to detect the transport function of BSEP under different cholesterol concentration in Hep G2 cells.Part II: Effects of different cholesterol concentration on C av-1 expression,PKCα phosphorylation and binding capacity of Hax-1 and BSEP.Mouse hepatocytes and Hep G2 cells in part I were selected for detection.Western blot was used to detect the expression of Cav-1,Hax-1,and the phosphorylation of PKCα under different concentration of cholesterol in mouse liver cell samples and in Hep G2 cells.Ele ctron microscopic examination was used to observe changes in Cav-1 under different concentration of cholesterol in Hep G2 cells.The m RNA expression in mouse liver sample and Hep G2 cells under different concentration of cholesterol(0,25,50 μg/ml)was detected.Co-immunoprecipitation was performed to assess the binding capacity of BSEP and Hax-1 under different cholesterol concentration in Hep G2 cells.Immunofluorescence double-staining was performed to observe the status of co-localization of BSEP and Hax-1 under different concentration of cholesterol in Hep G2 cells.Part III: The mechanism of the regulation of BSEP expression and function by Cav-1 under stimulation of different concentration of cholesterol in Hep G2 cells.The Cav-1 lentiviral vector was constructed,and Hep G2 was transfected to obtain a Cav-1 overexpression and knockdown stable cell line.Western blot was performed to detect the expression of BSEP,Cav-1,Hax-1 and the phosphorylation of PKCα in Cav-1 OE and wild type Hep G2 cells.Immunofluorescence staining was performed to observe the membrane localization and co-localization of Hax-1.Co-immunoprecipitation was performed to detect the binding capacity of BSEP and Hax-1.ATPase activity assay was performed to detect the transport function of Cav-1 OE cells.Rescue experiment was performed under stimulation of high concentration of cholesterol(pathological condition): Cav-1 was over-expressed followed by addition of PKCα inhibitor.Western blot was performed to detect the expression of BSEP,Cav-1,Hax-1 and phosphorylated PKCα.Results: Part I: 1.In C57BL/6 mice which were fed for two weeks,the expression of BSEP protein and m RNA in liver cells were significantly elevated in experimental group compared with control,cholesterol stones may not form in mice of experimental group.In C57BL/6 mice which were fed for four weeks,the expression of BSEP protein and m RNA in liver cells were significantly reduced in experimental group compared with control group,and cholesterol stones were seen in the gallbladders of the mice of experimental group.2.Under stimulation of low concentration(25 μg/ml)of cholesterol,the expression of BSEP protein as well as m RNA were increased in Hep G2 cells.However,under stimulation of high concentration(50 μg/ml)of cholesterol,the expression of BSEP protein and m RNA were significantly reduced in Hep G2 cells.3.Under stimulation of low concentration of cholesterol,the expression of BSEP on cell membrane as well as in cytoplasm were increased,and the membrane localization of BSEP was enhanced.However,under stimulation of high concentration of cholesterol,the expression of BSEP on cell membrane as well as in cytoplasm were significantly decreased,and the membrane localization of BSEP wa s reduced.4.When the concentration of substrate taurocholic acid(TC)was fixed,the transport function of BSEP was first strengthened then weakened along with the increase of cholesterol concentration.Under stimulation of low concentration of cholestero l,the transport function of BSEP was enhanced in both groups when the concentration of substrate TC increased,and the transport function of experimental group was significantly enhanced compared with control group.Under stimulation of high concentration of cholesterol,the transport function of BSEP was enhanced in both groups when the concentration of substrate TC increased,and the transport function of experimental group was significantly weakened compared with control group.Part II: 1.In C57BL/6 mice which were fed for two weeks,the expression of Cav-1 protein and m RNA were significantly increased and the PKCα phosphorylation was significantly decreased in liver cells in experimental group compared with control group.In C57BL/6 mice which were fed for four weeks,the Cav-1 protein and m RNA expression were significantly decreased and the PKCα phosphorylation was significantly increased in liver cells in experimental group compared with control group.High-cholesterol diet did not significantly affect the expression of Hax-1.2.Under stimulation of low concentration(25 μg/ml)of cholesterol,the expression of Cav-1 protein and m RNA were increased in Hep G2 cells.However,under stimulation of high concentration(50 μg/ml)of cholesterol,the expression of Cav-1 protein and m RNA were decreased in Hep G2 cells.The expression of Hax-1 protein did not change with the different concentration of cholesterol.3.Co-immunoprecipitation and immunofluorescence double-staining indicated that under stimulation of low concentration of cholesterol,the binding capacity of BSEP and H ax-1 was weakened in Hep G2 cells in experimental group compared with control group,and the primary co-localization of BSEP and Hax-1 was observed on cell membrane.Under stimulation of high concentration of cholesterol,the binding capacity of BSEP and Hax-1 was enhanced in experimental group compared with control group.The co-localization of BSEP and Hax-1 on cell membrane was reduced and the co-localization in cytoplasm was increased in experimental group.Part III: 1.Cav-1 over-expression and knock-down lentivirus-transfected cell lines were constructed and transfection efficiency was assessed.However,cell viability significantly decreased after Cav-1 knock down and cells died shortly.2.The expression of Cav-1 protein in Cav-1 OE group was significantly higher than that of wild-type control group,the phosphorylation of PKCα was reduced and the expression of BSEP was increased in Cav-1 OE group.No significant change of Hax-1 expression was seen in Cav-1 OE group.3.Co-immunoprecipitation examination and immunofluorescence double-staining indicated that the binding capacity of BSEP and Hax-1 in Cav-1 OE cells was weakened compared with control group and the co-localization of BSEP and Hax-1 was primarily seen on cell membrane.4.The transport function of BSEP in both Cav-1 OE and control group was enhanced with the increase of the concentration of substrate TC,and the transport function of BSEP in Cav-1 OE group was significantly higher than control.5.Rescue experiment showed that under stimulation of high concentration of cholesterol,C av-1 over-expression was able to reverse the inhibitory effect of high cholesterol concentration on BSEP expression and was capable of reverse the phosphorylation of PKCα.PKCα inhibitor can block PKCα phosphorylation and modulation of BSEP by Cav-1.Conclusion: 1.High-fat and cholesterol diet within a short period was able to stimulate the expression of Cav-1 and BSEP and decrease the PKCα phosphorylation in C57BL/6 mouse liver cell,gallbladder stone may not form.But long-term stimulation by HFC diet resulted in decrease of Cav-1 and BSEP expression and increase of PKCα phosphorylation,gallbladder stone formation was seen.2.Under stimulation of low concentration of cholesterol,protein and m RNA expression of Cav-1 and BSEP were significantly increased,PKCα phosphorylation was significantly decreased in Hep G2 cells.Under stimulation of high concentration of cholesterol,protein and m RNA expression of Cav-1 and BSEP were decreased,PKCα phosphorylation was increased in Hep G2 cells.3.Cholesterol stimulation did not significantly affect the Hax-1 expression in C57BL/6 mouse liver cell and Hep G2 cells.Under stimulation of low cholesterol concentration,the binding capacity of BSEP and Hax-1 was weakened,membrane localization as well as transport function of BSEP increased.Under stimulation of high cholesterol concentration,the binding capacity of BSEP and Hax-1 was enhanced,cytoplasmic localization of BSEP increased,the transport function of BSEP was reduced.4.In Cav-1 OE group,the protein expression of Cav-1 increased,PKCα phosphorylation decreased,expression of BSEP increased,the binding capacity of BSEP and Hax-1 weakened,membrane localization and transport function of BSEP increased compared with wild-type control.5.Cav-1 modulates expression and function of BSEP via regulation of PKCα phosphorylation in hepatocytes under cholesterol stimulation. |
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