Verapamil Increases the Apolipoprotein-Mediated Release of Cellular Cholesterol by Induction of ABCA1 Expression Via Liver X Receptor-Independent Mechanism

Abstract

Objective-Release of cellular cholesterol and phospholipid mediated by helical apolipoprotein and ATP-binding cassette transporter (ABC) A1 is a major source of plasma HDL. We investigated the effect of calcium channel blockers on this reaction. Methods and Results-Expression of ABCA1, apoA-I-mediated cellular lipid release, and HDL production were enhanced in cAMP analogue-treated RAW264 cells by verapamil, and similar effects were also observed with other calcium channel blockers. The verapamil treatment resulted in rapid increase in ABCA1 protein and its mRNA, but not the ABCG1 mRNA, another target gene product of the nuclear receptor liver X receptor (LXR). By using the cells transfected with a mouse ABCA1 promoter-luciferase construct (Ϫ1238 to ϩ57bp), verapamil was shown to enhance the transcriptional activity. However, it did not increase transcription of LXR response element-driven luciferase vector. Conclusions-The data demonstrated that verapamil increases ABCA1 expression through LXR-independent mechanismand thereby increases apoA-I-mediated cellular lipid release and production of HDL. Key Words: calcium channel blocker Ⅲ verapamil Ⅲ ABCA1 Ⅲ HDL Ⅲ cholesterol Ⅲ apolipoprotein Ⅲ macrophage I t is a well-known fact that the risk of cardiovascular disease inversely correlates with the plasma level of high-density lipoprotein (HDL). 1,2 The background hypothesis for this finding is that HDL functions to transport cholesterol from somatic cells to the liver for its conversion to bile acids; therefore, it is believed that HDL also removes cholesterol pathologically accumulated in the cells of arterial walls as an initial stage of atherosclerosis. 3 HDL removes cellular cholesterol by two independent mechanisms: bidirectional exchange of cholesterol molecules between cell surface and HDL, in which cholesterol acyl-esterification on HDL creates its net efflux from the cells, and the interaction of helical apolipoproteins, perhaps dissociated from HDL with cellular surface to generate new HDL particles with the cellular lipid. 4 The latter reaction, initially described for macrophages 5 and then for other types of cells, 6 was found defective in the cells from the patients with genetic HDL deficiency, Tangier disease, 7,8 and in those treated with HDL-lowering drug probucol. 9,10 Therefore, the reaction is assumed as a main source of plasma HDL. Mutations in ATP-binding cassette transporter (ABC) A1, one of the ABC superfamily members, were identified in Tangier disease and other genetic HDL deficiencies to indicate that this membrane protein is a key for generation of plasma HDL. [11][12][13] Forced expression of ABCA1 led to the increase of apolipoprotein-mediated lipid release from cells, 14,15 and its overexpression in mice resulted in a mild elevation of HDL cholesterol, 16,17 implicating that expression level of this protein is a rate-limiting factor for production of plasma HDL. Accordingly, the increase of ABCA1 expression has been shown to protect the animals against atherosclerosis in certain limite...

Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis

“…Verapamil and cyclosporine A, two molecules known to inhibit P-gp activity, were recently shown to modulate ABCA1 transporter activity (20,21), a key protein in cholesterol homeostasis. The reevaluation of the role of P-gp in cholesterol homeostasis using P-gpinducible cells has led us to conclude that P-gp does not play a major role in cholesterol homeostasis.…”

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

“…However, the regulation of expression of ABCA proteins is multifactorial, including the liver X receptor/retinoid X receptor system (21-24), cAMP (18)(19)(20), the calcium-signaling pathway (34), and the peroxisome proliferator-activated receptor ␣ -related system (35,36). The content of cholesterol in the product HDL is regulated somewhat independently of the HDL assembly reaction itself, potentially with the involvement of such factors as caveolin-1 (11), protein kinase C-related signals (8)(9)(10), and intracellular cholesterol level and its esterification (10,12).…”

FGF-1 induces expression of LXRα and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes

“…To obtain a reporter construct with mutation of apoE DR4 element (DR4mu), site-directed mutagenesis was performed to introduce the DR4 at 2448 to 2433 bp of the 2690 to 19 reporter gene using a Quick Change II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and the respective primers, DR4 of forward: 5′-CCGGGGATGGGGAGttaaCACCGTGGCAGAGGAATCACTA-3′, reverse: 5′-TAGTGATTCCTCTGCCACGGTGttaaCTCCC-CATCCCCGG-3′ (lowercase letters indicate mutated nucleotides). The plasmid for expression of LXRa and the reporter plasmid containing four tandem repeats of LXREs upstream of the thymidine kinase (TK) promoter and its mutant, LXRE-TK and LXREmut-TK, the promoter plasmid containing sterol regulatory element (SRE), were prepared as previously described (19,20). 3T3 cells were grown to 60-70% confluence in 24-well plates, transfected with 1 mg of plasmid DNA including 0.2 mg LXRa expression vector, 0.77 mg reporter gene construct, and 30 ng hRluc-TK (Promega; for normalization) using Lipofectamine 2000 (Invitrogen).…”