D4-F Contributes To ABCA1 Expression And Cholesterol Efflux In THP-1 Macrophage-derived Foam Cells

BACKGROUND: ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in Apolipoprotein A-I (apoA-I) binding activity and promotes cellular cholesterol efflux. Mounting animal evidence suggests that D-4F may serve to protect against the development of atherosclerosis. In apoE KO Mice, oral D-4F causes decease of LDL and triacylglycerol. apoA-I mimetic peptide D4-F has been proved to up-regulate the ABCA1 expression. However, the detailed mechanisms of ABCA1 regulation by D4-F are not fully understood.Cdc42 is member of RhoGTPase super-family which contributes to cellular cholesterol efflux and the molecular weight is 25KD. The Cdc42 erpression was decrease in desmocyte and macrophage of dangier patient. Early studies defined that apoA-I binding ABCA1 activates Cdc42. In addition, Our studies have confirmed that apoA-1 inceases the ABCA1 expression and enhances cholesterol efflux through cAMP / PKA pathway. Based on these studies, we investigate to evaluate the effects of D4-F on ABCA1 expression and ABCA1-dependent cholesterol efflux and examine the role of Cdc42/cAMP/ PKA pathway on the regulation of ABCA1 by D4-F in THP-1 macrophage-derived foam cells. Prat I The effect of D4-F on ABCA1 expression and cholesterol effluxOBJECTIVE: D-4F, an Apolipoprotein A-I Mimetic Peptide, whether it also affects the ABCA1 eepression and cholesterol efflux in THP-1 macrophage-derived foam cells is still unclear. Thus, in this part we are to observe the effect of D4-F on ABCA1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells. METHODS: Human THP-1 cells were differentiated into macrophages by the addition of 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 72h. Macrophages were transformed into foam cells by incubation with the presence of 50 mg/ml ox-LDL in serum-free RPMI1640 medium containing 0.3% bovine serum albumin for 48h. 1. Different concentrations of D4-F (0μg/ml﹑2μg/ml﹑4μg/ml﹑6μg/ml) incubated with cells for 24h; 2. 6μg/ml D4-F treated for different time(0﹑6﹑12﹑24h) and 5μg/ml BSA treated for 24h as the control group. 3. 6μg/ml D4-F and 20μg/ml cycloheximide incubated for 0, 1, 2h, respectively. Subsequently, real-time PCR and (or) Western blot detection of ABCA1mRNA and protein exprssion, cellular cholesterol efflux measured by liquid scintillation counting, High performance liquid chromatography assays the cellular total cholesterol, free cholesterol and cholesteryl ester.RESULTS: Results showed that D4-F had no effect on ABCA1 mRNA expression, but the ABCA1 protein expression increased signicantly and peaked at a 6μg/ml concentration or after 24h treatment. After added cycloheximide to arrest protein synthesis, we found D4-F treatment progressively scale-upped ABCA1 protein levels at the 1h and 2h time points. Using high performance liquid chromatography assays we found D4-F obviously decreased the cellular total cholesterol, free cholesterol and cholesteryl ester level, meanwhile, the scintillation counting assays showed D4-F increased the cellular cholesterol efflux in a concentration-dependent and time-dependent manner.. Prat II The possible mechanism of D4-F up-regulated ABCA1 expression and cholesterol effluxOBJECTIVE: Our previous findings showed that D4-F inceased the ABCA1 protein expression and cholesterol efflux in THP-1 Macrophage-Derived Foam Cells. Then, we are to investigate the possible mechanism of D4-F inceased the ABCA1 protein expression and cholesterol efflux in THP-1 Macrophage-Derived Foam Cells.METHODS: Human THP-1 cells were differentiated into macrophages by the addition of 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 72h. Macrophages were transformed into foam cells by incubation with the presence of 50 mg/ml ox-LDL in serum-free RPMI1640 medium containing 0.3% bovine serum albumin for 48h. 1. 6μg/ml D4-F treated the cells 0, 15, 20 and 25 minutes respectively to test the activity of PKA; 2. Transfestesd with PKA si-RNA 48 later, 6μg/ml D4-F treated for 24 hours; 3. 1.0mM PKA specific excitomotor dBcAMP and 6μg/ml D4-F incubated for 24 hours; 4. 20μg/ml cycloheximide and PKA-siRNA or dBcAMP and 6μg/ml D4-F incubated for 0, 1, 2h, respectively; 5. 6μg/ml D4-F and adenylate cyclase specific inhibitor SQ22536 or 25mM Cdc42 inhibitor Secramine B incubated for 30 minutes at 37℃. 6. 6μg/ml D4-F treated the cells for 0, 10, 15 and 20 minutes respectively to test the activity of Cdc42. Subsequently, optical density value to represent the PKA activity and Cdc42 activation assay Biochem (BK034)Kit? to test Cdc42 activity; Western blot detection of ABCA1 protein phosphorylation exprssion; immunosorbent assay to test cAMP level.RESULTS: Results showed that D4-F activated the PKA; treatment with siRNA for PKA down-regulated PKA protein expression by 85%; both PKA-siRNA and dBcAMP could not change the ability of D4-F to stabilize ABCA1 protein. PKA-siRNA, dBcAMP, SQ22536 could reverse D4-F to increase the ABCA1-induced cellular cholesterol efflux. Furtherly, SQ22536 also could reverse D4-F to raise cAMP level and PKA activity and to increase the ABCA1 phosphorylation; D4-F could effect the Cdc42 activity; Secramine B reversed D4-F to raise cAMP level and PKA activity.CONCLUSIONS:1 D4-F increase ABCA1 protein expression in a concentration-dependent and time-dependent manner;2 D4-F enhance ABCA1 serine phosphorylation and ABCA1-dependent cholesterol efflux through a Cdc42/cAMP/PKA pathway in THP-1 macrophage-derived foam cells.