The cells were treated with MTT, and absorbance was read at 570 nm in a microplate reader (Awareness Technology Inc, USA). SCAP between the ER and the Golgi, escorting more SREBP2 from the ER ENMD-2076 Tartrate to the Golgi for activation by proteolytic cleavages as evidenced by an increased N-terminal of SREBP2 (active form). As a consequence, the LDL receptor and HMGCoAR expression were up-regulated. Interestingly, these effects could be blocked by inhibitors of Golgi mannosidases. Our results indicated that inflammation increased native LDL uptake and endogenous cholesterol de novo synthesis, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in macrophages. These data imply that inhibitors of Golgi processing enzymes might have a potential vascular-protective role in prevention of atherosclerotic foam cell formation. Introduction Atherosclerosis, a maladaptive chronic inflammatory response in the vessel wall, is the primary cause of coronary artery disease, stroke and peripheral vascular disease and it thus represents the most common cause of morbidity and mortality worldwide [1]. Macrophage foam cell formation with cholesterol overloading is the defining pathological characteristic of atherosclerotic plaques [2]. LDL, the major carrier of plasma cholesterol, enters the vessel wall and macrophages by receptor and non-receptor-mediated mechanisms. Increased serum levels of LDL have been most closely correlated with the incidence of cardiovascular disease [3]. Traditionally, scavenger receptors mediated modified LDL (oxidized or glycosylated) uptake is recognized as the major resource for cholesterol accumulation in monocyte-derived macrophages within atherosclerotic plaques [4]. However, recent evidence has challenged this paradigm by showing that loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice [5]. Our previous studies also showed that the accelerating effects of inflammatory cytokines on lipid droplets accumulation in various peripheral cells such as human mesangial cells (HMCs), vascular smooth muscle cells (VSMCs) and macrophages [6], [7], ENMD-2076 Tartrate [8], were not be inhibited by scavenger receptors blocker, but were blocked by LDL receptor (LDLr) specific antibody (MB47) and heparin, which removes LDL bound to the cell surface [7], [8]. This suggests LDLr pathway involvement in lipid accumulation under inflammatory stress. LDLr, the primary receptor for binding and internalization of plasma-derived native LDL cholesterol and regulation of plasma LDL concentration, was initially considered unimportant in macrophage cholesterol accumulation and foam cell formation because LDLr gene expression in mammalian cells is normally under tight negative-feedback control via Sterol Regulatory Element Binding Protein (SREBP) [9]. In mammalian cells, two SREBP genes encode three different isoforms of SREBPs, known as SREBP-1a, -1c and -2. While ENMD-2076 Tartrate SREBP-1a is a potent activator of all SREBP-responsive genes, SREBP-1c preferentially enhances the transcription of genes involved in fatty acid synthesis. Conversely, SREBP-2 preferentially activates genes of LDLr involved in cholesterol uptake and 3-hydroxy-3-methyl-glutaryl- CoA reductase (HMGCoAR) involved in cholesterol biosynthesis [10]. SREBP Cleavage- Activating Protein (SCAP) is a transmembrane protein that serves as a chaperone protein of SREBP2 and sterol sensor, which plays a central role in the SREBP2 activation. When cells are depleted of cholesterol, SCAP delivers the SREBP2 from the endoplasmic reticulum (ER) to the Golgi where it is cleaved by two membrane-bound proteases (site 1 protease and site 2 protease) [11]. Meanwhile SCAP is glycosylated by the sequential action of Golgi enzymes -mannosidase I, -mannosidase II and GlcNAc transferase I [12], [13], [14], before recycling to the ER. The sequential cleavages release the active N-terminal fragment of SREBP2 (N-SREBP2) from the Golgi to the nucleus, binding to the sterol regulatory elements in the HMGCoAR and ENMD-2076 Tartrate LDLr promoters and activating these genes transcription. When intracellular cholesterol is overloaded, SCAP-SREBP2 complex is retained in the ER and SREBP2 cannot be processed by the proteases in the Golgi. Thereafter the expression of LDLr and HMGCoAR Rabbit Polyclonal to DRP1 is down-regulated and both cholesterol uptake and de novo synthesis decline. Yuan et al reported that SCAP glycosylation can be decreased by Golgi mannosidase inhibitors, which led to reduced LDLr and HMGCoAR expression and therefore intracellular cholesterol accumulation in HMCs [15]. It seems that SCAP cycling between the ER and the Golgi regulated by.

The cells were treated with MTT, and absorbance was read at 570 nm in a microplate reader (Awareness Technology Inc, USA)