Effects of dimethyl sulfoxide on apolipoprotein A-l in the human hepatoma cell line, HepG2

Exposure of HepG2 cells to 1 % (v/v) dimethyl sulfoxide (DMSO), an effective free radical scavenger, for 24 h resulted in a 2-fold increase in the levels of apolipoprotein (apo) A-I mRNA and secreted protein, with no significant change in apoA-11, apoB, and apoE mRNA and protein levels. The induction of apoA-I was accompanied by a 50% increase in secreted HDL. Nuclear run-off assays indicated that the transcription rate of the apoA-I gene was also increased 2-fold in DMSO-treated cells. Consistent with nuclear run-off assays, transient transfection experiments, using a series of pGL2-derived luciferase reporter constructs containing the human apoA-I proximal promoter, demonstrated that DMSO treatment increased apoA-I promoter activity 2-fold. We have identified a potential ‘antioxidant response element’ (ARE) in the apoA-I promoter that may be responsible for the increase in apoA-I transcriptional activity by DMSO. Gel mobility shift assays with an apoA-I-ARE revealed increased levels of a specific protein-DNA complex that formed with nuclear extracts from DMSO-treated cells. The formation of this complex is sequence specific as determined by DNA competition studies. When a copy of the ARE was inserted upstream of the SV40 promoter in a luciferase reporter plasmid, a significant 2-fold induction in luciferase activity was observed in HepG2 cells in the presence of DMSO. In contrast, a plasmid containing a mutated apoA-I-ARE did not confer responsiveness to DMSO treatment. Furthermore, pGL2 (apoA-1-250 mutant ARE), in which point mutations eliminated the ARE in the apoA-I promoter, showed no increase in luciferase activity in response to D M S 0 . I These results implicate protein-DNA interactions at the antioxidant response element region in the transcriptional induction of human apoA-I gene expression by DMSO.--Tam, S-P., X. Zhang, C. Cuthbert, Z. Wang, and T. Ellis. Effects of dimethylsulfoxide on apolipoprotein A-I in the human hepatoma cell line, HepG2. ./. Lipid Res. 1997. 38: 2090-2102. Supplemenkey words erosclerosis apolipoproteins gene expression athDimethyl sulfoxide (DMSO) is a dipolar aprotic solvent and an effective scavenger of hydroxyl radicals (1). It has great permeability which allows it to penetrate all membranes impenetrable to other antioxidants, particularly enzymatic antioxidants. DMSO has biologically diverse effects on mammalian cells. Addition of 2% (v/v) DMSO to the culture medium of primary cultures of rat hepatocytes maintained biochemical and differentiated functions for extended periods (2). Results of Isom and co-workers (3) suggested that DMSO promoted the persistence of some liver-specific mRNAs in cultured hepatocytes. DMSO also partially protected the loss of total cytochrome P450 content in cultured rat hepatocytes (4, 5 ) , and induced the alcohol-inducible P450IIE1 in vivo (6). In addition, increase in haem synthesis in HepG2 cells in response to DMSO has been shown to be a transcriptionally activated event (7) . Several reports have suggested that DMSO may also be effective in inhibiting cholesterol-induced atherosclerosis in experimental animals (8-10). However, the mechanisms by which DMSO inhibits choIestero1-induced atherosclerosis are still not clear. Studies have indicated that DMSO slows the development of dietary cholesterol-induced atherosclerosis in rabbits and reduces the accumulation of cholesterol in tissues despite severe hypercholesterolemia. By far the most prominent effect of DMSO in rabbits on an atherogenic diet was the marked decrease in both the number of foam cells and their content of lipid droplets (10). The inhibition of atherosclerosis in the presence of high plasma cholesterol levels would suggest that DMSO may act via mechanisms that prevent the uptake of cholesterol by macrophages or endothelial cells of the aorta. The “oxidation hypothesis of atherosclerosis” proposes that oxidative modification of low density lipoprotein (OxAbbreviations: apoA-I, apolipoprotein A-I; ARE, antioxidant response element; DMSO, dimethyl sulfoxide; DTI’, dithiothreitol; FBS, fetal bovine serum; GST, glutathione-S-transferase; HDL, high density lipoprotein; MEM, minimal essential medium; Na2 EDTA, disodium ethylene-diamine tetraacetate; Ox-LDL, oxidized low density lipoprotein; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; XRE, xenobiotic response element. ‘To whom correspondence should be addressed. 2090 Journal of Lipid Research Volume 38, 1997 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom LDL) is crucial in the pathogenesis of the atherosclerotic lesion (11). Ox-LDL is a prerequisite for macrophage uptake and cellular accumulation of cholesterol (12, 13). However, oxidized high density lipoprotein is not avidly taken up by macrophages and does not lead to foam cell formation (14). Furthermore, HDL has been reported to inhibit endothelial cell-mediated LDL modification (14,15) and to reduce cellular uptake and degradation of native and Ox-LDL (14,16). HDL is also capable of protecting against LDL peroxidation in vitro (14, 17) and in vivo (18). We hypothesize that DMSO would prevent the formation of Ox-LDL either directly or indirectly through the modulation of HDL production. These two possibilities, though not mutually exclusive, are supported by the studies of Layman, Alam, and Newcomb (9), and De La Vega and Mendoza-Figueroa (19). The first study indicated that the addition of 2% DMSO to human skin fibroblasts resulted in a greater than 35% reduction in uptake and binding of lZ5I-labeled LDL and a 26% decrease in its degradation (9). Two simple explanations for this observation are i ) DMSO treatment causes a reduction in the number of LDL receptors on the fibroblasts, or ii) DMSO slows the oxidation process of LDL. The second study, which supports the latter hypothesis, has shown that adult rat hepatocytes cultured on a feeder layer of lethally treated 3T3 cells increases the de novo synthesis and secretion of total lipids by 2to %fold after 14 days of 2% DMSO treatment (19). Although the secretion of various lipoprotein classes by 3T3 hepatocytes was not determined, the investigators suggested that an increase in VLDL and HDL would be expected (19). To test this hypothesis, we have examined the effects of DMSO on the synthesis of apoA-I by using the human hepatoma cell line, HepG2, as an in vitro model. This cell line is highly differentiated and retains many of the specialized functions normally lost by hepatocytes in culture (20, 21). The current study explores some of the molecular mechanisms by which DMSO modulates human apoAI production. We have demonstrated that exposure of HepG2 cells to 1% DMSO resulted in a 2-fold increase in apoA-I transcription rate and a corresponding increase in both apoA-I mRNA and secreted apoA-I. Evidence summarized below suggest that the expression of the human apoA-I gene may be modulated by DMSOinduced nuclear factor. We have identified a putative antioxidant response element (ARE) located in the apoA-I promoter between nucleotides -142 and -132 relative to the transcription start site of the gene. This element was first identified by Rushmore, Morton, and Pickett (22) during short-term transfection studies on the regulation of the rat glutathione-S-transferase (GST) Ya subunit gene in response to metabolites of planar aromatic compounds and phenolic antioxidants. Here, we describe experiments showing that the putative ARE is necessary and sufficient for DMSO-mediated induction of human apoA-I gene expression.