Abstract: BACKGROUND: Human proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of cholesterol metabolism, modulates cholesterol uptake and plasma lipid levels by binding to and promoting the degradation of the low-density lipoprotein receptor. Cholesterol efflux is mediated by transporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI), with trafficking kinesin protein 2 (TRAK2) potentially acting as an adaptor protein in this process. Investigating whether PCSK9 regulates cholesterol efflux through the TRAK2/ABCA1 pathway provides novel insights into its role in atherosclerosis.
OBJECTIVE: To explore the effect of PCSK9 on cholesterol efflux in human monocyte-derived macrophages and its underlying mechanisms.
METHODS: THP-1 cells were centrifuged and added with phorbol ester for 48 hours to induce differentiation into THP-1-derived macrophages, and then incubated with serum-free medium containing oxidized low-density lipoprotein for 24 hours to induce foam cell formation. (1) The foam cells were divided into four groups: control group, PCSK9 protein group, negative control group (non-targeted siRNA transfected foam cells), and PCSK9 siRNA transfection group. RT-qPCR and western blot assay were used to detect the mRNA and protein expressions of TRAK2, ABCA1, ABCG1, and SR-BI. (2) The foam cells were divided into three groups: negative control group, TRAK2 siRNA transfection group, and (TRAK2+PCSK9) siRNA co-transfection group. RT-qPCR and western blot assay were used to detect the mRNA and protein expressions of ABCA1. (3) The foam cells were divided into eight groups: control group, PCSK9 protein group, PCSK9 siRNA transfection group, TRAK2 siRNA transfection group, ABCA1 siRNA transfection group, (TRAK2+PCSK9) siRNA co-transfection group, (PCSK9+ABCA1) siRNA co-transfection group, and (TRAK2+ABCA1) siRNA co-transfection group. 25-NBD-cholesterol was used to analyze the cholesterol efflux rate of foam cells. 
RESULTS AND CONCLUSION: (1) The TRAK2 mRNA and protein expression levels in the PCSK9 group were higher than those in the control group (P < 0.05), whereas the expression levels of ABCA1, ABCG1, and SR-BI showed no significant difference compared to the control group. In the PCSK9 siRNA transfection group, TRAK2 mRNA and protein expression levels were lower than in the negative control group (P < 0.001 and P < 0.05, respectively), while ABCA1 mRNA and protein expression levels were higher than in the negative control group (P < 0.001); no significant difference was observed in the mRNA and protein expression levels of ABCG1 and SR-BI compared to the negative control group. (2) In the TRAK2 siRNA transfection group, ABCA1 mRNA and protein expression levels were higher than those in the negative control group (P < 0.001 and P < 0.05, respectively), and in the (TRAK2+PCSK9) siRNA group, ABCA1 mRNA and protein expression levels were higher than those in the negative control group (P < 0.001 and P < 0.05, respectively). There was no significant difference in ABCA1 mRNA and protein expression levels between the (TRAK2+PCSK9) siRNA group and the TRAK2 siRNA group. (3) There was no significant difference in cholesterol efflux between the PCSK9 group and the control group. Cholesterol efflux in the PCSK9 siRNA, TRAK2 siRNA, and PCSK9+TRAK2 siRNA groups was higher than in the control group (P < 0.001), with no significant difference in cholesterol efflux between these groups. Cholesterol efflux in the ABCA1 siRNA group was lower than in the control group (P < 0.001); cholesterol efflux in the (PCSK9+ABCA1) siRNA group was lower than in the PCSK9 siRNA group (P < 0.001), and cholesterol efflux in the (TRAK2+ABCA1) siRNA group was lower than in the TRAK2 siRNA group (P < 0.001). These findings indicate that PCSK9 may regulate cholesterol efflux via the TRAK2/ABCA1 pathway.

Key words: PCSK9, TRAK2, macrophage, cholesterol metabolism, ABCA1, cholesterol efflux, foam cell, THP-1