The Effects And Mechanisms Of Rab18 On PLIN2-mediated Lipid Accumulation In Macrophages

Adipose differentiation-related protein PLIN2 can accelerate the formation of foam cells by promoting lipid accumulation and inhibiting cholesterol efflux from macrophages.Moreover,PLIN2 overexpression was closely correlated with plaque instability in atherosclerotic lesions.Thus,PLIN2 plays a crucial role on the initiation and progression of atherosclerosis.Previous researches had shown that Rab18 coexisted with PLIN2 in lipid droplets(LDs),overexpression of Rab18 caused exclusion of PLIN2 from LDs.Furthermore,Rab18 overexpression induced close apposition of LDs to endoplasmic reticulum.Suppression expression of PLIN2,i.e.treated with RNA interference or brefeldin A,induced the same morphological change.This suggests that there is a probable link between Rab18 and PLIN2,Rab18 may be an upstream regulator of PLIN2.Our previous research found that protein kinase C(PKC),together with Ox-LDL,could up-regulate PLIN2 expression and aggravate lipid accumulation in macrophages.In addition,some studiesfound that the function of some PKC subtypes was closely related to Rab proteins.Based on these theories,we therefore hold the hypothesis that Rab18 regulates macrophage lipid-accumulation mediated by PLIN2 through changing the expression of PKCα.Both cytological and zoological experimental methods were adopted in the study,we observed the expression levels of Rab18,PKCα and PLIN2 in atherosclerotic mice,how Rab18 affected the expression of PKCα and PLN2 thus influenced lipid accumulation in lipid-loaded macrophages and the effect of altered PKCα activity in lipid accumulation and PKCα,PLIN2 expression in lipid-loaded THP-1 macrophages with high Rab18 expression.With these,we attempted to explored possible mechanisms of Rab18 regulating lipid accumulation in macrophages mediated by PLIN2.In the animal experiments,24 ApoE-/-mice(8 weeks old)were randomly divided into control group and experimental group(8 in control group,16 in experimental group).For 12 weeks,control group mice were fed with general diet and experimental group with high-fat diet.8 mice were fasted for 48 hours randomly divided from experimental group before decapitation.Atherosclerotic lesions in aortas of ApoE-/-mice were observed under stereoscopic microscope.HE,oil red O and Masson staining were carried out to observe atherosclerotic lesions of aortic sinuses;Western bloting were applied to detect the protein expression levels of Rab18,PKCα and PLIN2.In the cell experiments,(1)THP-1macrophages were incubated with 50 mg/L Ox-LDL for different duration(0,6,12,or 24 h);(2)The eukaryotic expression vectors of Rab18,wild type(WT),GTPasedeficient mutant(Q67L)and constitutively GDP-bound mutant(S22N),were transfected into THP-1 macrophages by liposome,then transfected macrophages were treated with 50 mg/L Ox-LDL for 24h;(3)THP-1 macrophages,transfected with Rab18(Q67L),incubated with 50 mg/L Ox-LDL for 24 h,then were treated with PKC activator PMA(100 nmol/L)or inhibitor Calphostin C(300 nmol/L)for 16 h.In cell experiments,Western blot was taken to measure the protein levels of Rab18,PKCα and PLIN2,oil Red O staining for observing the depositing of lipid in THP-1 macrophages.In our study,the protein expression levels of Rab18,PKCα and PLIN2 in aortic atherosclerotic ApoE-/-mice were significantly higher.Moreover,fasting for 48 h further increased the expression of Rab18 while down-regulated PKCα and PLIN2 expression.The protein expression levels of Rab18,PKCα and PLIN2 elevated gradually with the increase of incubating time with Ox-LDL,coming with increasing accumulation of lipid in THP-1 macrophages.In addition,increased PKCα and PLIN2 expression emerged earlier than that of Rab18.The expression levels of PKCα and PLIN2 protein significantly decreased both in Rab18(WT)and Rab18(Q67L)transfection groups,correspondingly,lipid deposition in macrophages reduced.However,there was no clear difference between Rab18(S22N)transfection group and non-transfected group.100 nmol/L PMA offset the reducion of PKCα,PLIN2 protein resulted from Rab18(Q67L)overexpression,and the accumulation of lipid in THP-1 macrophages increased correspondingly;Conversely,300 nmol/L Calphostin C further enhanced this down-regulation effect induced of Rab18(Q67L),and reduced lipid accumulation even more.Our results verify that Rab18 downregulates PLN2 expression through PKCα,thus reduces macrophage lipid accumulation.