The main goal of the studies presented here was to evaluate to impact of vitamin E (and, specifically, αTOH) on gene expression in human hepatocytes. A possible confounding factor in such experiments stems from the non-specific effects that antioxidants have on multiple redox-responsive cellular targets. To examine the unique activity of αTOH, we compared its transcriptional activities effects to those of another established antioxidant, NAC (35
). We hypothesized that specific targets of vitamin E actions would respond to vitamin E treatment, but not to NAC treatment. To compare the efficacy of αTOH and NAC in quenching reactive oxygen species in our cell system, we employed DCF, a cell permeable fluorescent probe that quantitatively reports on intracellular ROS levels (29
). Data in show that treatment of HepG2 cells with either NAC or αTOH caused a dose-dependent decrease in cellular ROS levels. Importantly, 100 μM αTOH elicited a ca. 50% reduction in cellular ROS levels, similar to the change induced by NAC. These results show that treatment of HepG2 cells with 1 mM NAC is an appropriate control for the antioxidant effects of 100 μM αTOH.
Quenching of intracellular reactive oxygen species by N-acetyl L-cysteine (NAC) and RRR-α-tocopherol (αTOH)
To evaluate the specific effects of vitamin E on hepatic gene expression, we determined the global expression profile of HepG2 cells that were treated with 100 μM αTOH, and compared it with the expression profile of untreated cells, or those treated with 1 mM NAC. RNA extracted from the different experimental groups was processed as described in Materials and Methods, and hybridized to Affymetrix U133A expression micro-arrays (Affymetrix) on which ca. 22,000 transcripts and ESTs are displayed, representing approximately 14,500 unique human genes.
In three independent array experiments, 57 genes displayed statistically significant differences in expression levels between the αTOH treatment group and untreated controls (p < 0.05). Of these, 29 were up-regulated by αTOH, while the expression of 28 genes was repressed by this treatment. Changes in the expression levels of these genes were in the 1.2- to 2.0-fold range. The affected genes can be grouped into four major categories when clustered on the basis of known cellular functions: cell proliferation, lipid status, protein stability and metabolism, and transcriptional regulation (). Treatment with αTOH caused specific and significant reduction in the expression levels of 17 genes that play central roles in regulating cellular lipid status (). Interestingly, 10 of these αTOH-responsive gene products catalyze key steps in the de novo
biosynthesis of cholesterol: dehydrocholesterol reductase, farnesyl diphosphate synthase, HMG-CoA synthase, isopentenyl-diphosphate delta isomerase, lanosterol synthase, lathosterol oxidase, squalene monooxygenase, squalene synthase and sterol-C4-methyl oxidase (). Notably, mRNA levels of the HMG-CoA reductase gene, encoding the rate-limiting enzyme in cholesterol biosynthesis (39
), and of the low-density lipoprotein (LDL) receptor, a key regulator of cholesterol transport (40
), were also significantly reduced by TOH treatment (Table II). Importantly, we observed that none of these transcripts were affected by NAC treatment.
Functional clustering of TOH-responsive genes
Effect of αTOH on the expression of lipid homeostatic genes
To verify the results obtained from micro-array analyses we employed quantitative, real-time RT-PCR. The data show that αTOH treatment caused a significant decrease in the levels of mRNAs encoding the HMG-CoA reductase and the LDL-receptor, and that the magnitude of the effect was similar to that observed in expression arrays (). Real-time RT-PCR of the signal transducer AKAP12 transcript revealed that αTOH treatment caused a similar effect to that observed in the expression arrays (i.e. 2–3-fold increase in mRNA levels, ). These results indicate that the effects of αTOH on gene expression observed in the micro-array experiment were both significant and reproducible. Additional experiments revealed that the vitamin E-induced changes in the HMG CoA Reductase transcript were specific, and did not occur following treatments with the soluble antioxidant NAC, or the lipid soluble antioxidant BHT (). These observations raise the possibility that the regulation of HMG CoA reductase transcript stems from specific, possibly antioxidant-independent, actions of vitamin E.
The vitamin E-induced decreases in transcript levels observed in cholesterol homeostatic genes could in principle result from inhibition of transcription, or, alternatively, from facilitation of mRNA degradation. To discriminate between these possibilities, we measured the effect of vitamin E on gene expression in the presence of a actinomycin D, a selective inhibitor of DNA-directed RNA synthesis (41
). Cells were treated with 1 μM actinomycin D together with 100 μM αTOH (or ethanol), and the levels of mRNAs encoding HMG-CoA reductase and the LDL-receptor were measured using real-time RT-PCR. We observed that in the presence of the transcriptional inhibitor actinomycin D, vitamin E did not affect the expression of these transcripts (data not shown), demonstrating that αTOH attenuates the mRNA levels of cholesterol homeostatic genes by inhibiting transcription, and not by affecting mRNA stability.
Molecular Mechanisms of αTOH Action
The 10 cholesterol homeostatic genes that are affected by vitamin E treatment share common regulatory features in their promoter regions. Specifically, the sequence 5′ATCACCCCAT3′ is found upstream of the transcription start sites of all of these genes. This conserved regulatory sequence, known as the sterol response element (SRE), is the binding site for the transcription factor SREBP-2, which mediates many established responses to sterols (42
). To examine whether SREBP-2 mediates the transcriptional activities of vitamin E, we employed a reporter construct in which the luciferase gene is under the regulation of an SRE-containing promoter (the SREBP-2 promoter, possessing a single SRE at position –293, (31
)). We transfected the reporter construct into HepG2 cells, and monitored the effect of αTOH treatment on luciferase expression. Inhibiting cholesterol biosynthesis by treatment of the cells with the HMG-CoA-reductase inhibitor lovastatin caused a ca. 2-fold increase in luciferase expression (), as anticipated from the established response of the SREBP-2 promoter to depletion of cellular sterol pools (43
). Conversely, increasing cholesterol levels by the addition of exogenous (cholesterol rich) serum lipoproteins caused a ca. 20% repression of promoter activity (). This feedback inhibition of SRE-containing promoters by sterols is an established feature of cholesterol homeostatic regulation (31
). Importantly, we observed that treatment of the cells with vitamin E lead to a dose-dependent attenuation of reporter gene expression, reaching approximately 50% inhibition at 100 μM αTOH. The inhibitory effect of αTOH on promoter activity does not reflect a general cell response to antioxidants or redox state, as NAC did not affect promoter activity (). To implicate specific promoter regions in the transcriptional responses to vitamin E, we ablated the single SRE sequence in the luciferase reporter construct using site-directed mutagenesis (see Materials and Methods). As expected, the SRE-defective promoter no longer responded to alterations in cholesterol status induced by lovastatin and serum (). Importantly, mutating the SRE sequence also completely abolished transcriptional responses to αTOH (). As the experimental treatments used in these experiments did not affect cell viability (data not shown), we conclude that αTOH inhibits the expression of these cholesterol homeostatic genes by attenuating SRE-mediated transcriptional responses.
Vitamin E inhibits transcription of a sterol-responsive element (SRE) -driven reporter gene
Physiological regulation of SREBP-2 by sterols occurs through post-translational proteolytic activation of the SREBP-2 transcription factor that controls its translocation from the endoplasmic reticulum to its site of action in the nucleus (47
). We examined whether the transcriptional effects of vitamin E, like those of sterols, are mediated by post-translational processing of SREBP-2. To this end, we fractionated treated HepG2 and CHO cells, and assayed the levels of proteolytically-activated SREBP-2 by immunoblotting. As expected, processing of SREBP-2 was enhanced when cholesterol levels were depleted (i.e. in the presence of lovastatin), and reduced upon the addition of exogenous sterols, as shown in . Treatment with vitamin E elicited a dose-responsive decrease in the amount of activated (nuclear) SREBP-2, reaching ca. 50% inhibition at 100 μM αTOH, similar to the magnitude of inhibition elicited by sterols ().
These results indicate that vitamin E attenuates the transcriptional responses of sterol response elements in the promoter regions of cholesterol homeostatic genes, and that these events are mediated by the actions of SREBP-2.
Attenuation of de novo Cholesterol Biosynthesis by αTOH in vivo
The observations that tocopherol specifically attenuates the expression of key regulators of cholesterol homeostasis raise the possibility that vitamin E levels impact on cellular cholesterol status. To evaluate the physiological significance of these findings, we examined whether treatment with vitamin E influences the rate of de novo cholesterol biosynthesis in cultured cells. HepG2 cells were incubated with the radio-labeled precursor [14C]-acetate for six hours, and incorporation of the label into cholesterol was measured after lipid extraction and thin layer chromatography. Treatment with vitamin E resulted in a dose-dependent inhibition of cholesterol biosynthesis, reaching ~30% inhibition at 100 μM αTOH (). The magnitude of inhibition by αTOH was comparable to that induced by established physiological attenuators of the cholesterol biosynthetic pathway, namely high sterols (10 μg/μL cholesterol + 0.1 μg/μL 25-hydroxycholesterol). Importantly, suppression of cholesterol biosynthesis was a specific attribute of vitamin E, as treatment with vehicle control (ethanol) or the general antioxidant NAC did not affect incorporation of acetate into cholesterol (). These findings indicate that the specific effects of αTOH on the cholesterol biosynthetic pathway are significant at the physiological end-point of the pathway, namely, the production of cholesterol.
Vitamin E attenuates de novo cholesterol biosynthesis