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Vitamin E is a dietary lipid that is essential for vertebrate health and fertility. The biological activity of vitamin E is thought to reflect its ability to quench oxygen- and carbon- based free radicals, and thus to protect the organism from oxidative damage. However, recent reports suggest that vitamin E may also display other biological activities. Here, to examine possible mechanisms that may underlie such non-classical activities of vitamin E, we investigated the possibility that it functions as a specific modulator of gene expression. We show that treatment of cultured hepatocytes with RRR-α-tocopherol alters the expression of multiple genes and that these effects are distinct from those elicited by another antioxidant. Genes modulated by vitamin E include those that encode key enzymes in the cholesterol biosynthetic pathway. Correspondingly, vitamin E caused a pronounced inhibition of de novo cholesterol biosynthesis. The transcriptional activities of vitamin E were mediated by attenuating the post-translational processing of the transcription factor SREBP-2 that, in turn, led to a decreased transcriptional activity of sterol responsive elements in the promoters of target genes. These observations indicate that vitamin E possesses novel transcriptional activities that affect fundamental biological processes. Cross talk between tocopherol levels and cholesterol status may be an important facet of the biological activities of vitamin E.
The term vitamin E refers to a family of structurally related neutral plant lipids that are critical for vertebrate health and fertility. Numerous studies established that members of the vitamin E family are efficient chain-breaking radical scavengers both in vitro and in vivo, and led to the definition of vitamin E as the most important lipid-soluble antioxidant (1). While all isoforms of vitamin E possess comparable radical trapping activity in vitro (2), RRR-α-tocopherol (denoted herein as αTOH) exhibits the highest potency in biological assays (3). This vitamer preference stems from the combined activities of the hepatic α-tocopherol transfer protein (TTP) that selectively retains αTOH (3–5), and the catabolic actions of hepatic enzymes that degrade other forms of vitamin E to water-soluble products (6).
The selectivity for αTOH raises the possibility that this vitamer has unique biological roles, in addition to its anti-oxidant function. Indeed, novel regulatory properties have been ascribed in recent years to αTOH, including the modulation of apoptosis, cell adhesion, and specific enzymatic activities (cf. 7, 8). Moreover, αTOH was shown to regulate the expression levels of several mRNAs and proteins, such as collagen α1 (9), the scavenger receptors SR-BI (10) and CD36 (11, 12), α-tropomyosin (13), the nuclear receptor PPARγ (14), and the adhesion molecule VCAM-I (15). These anecdotal observations were further supported by several studies that documented genomic responses to vitamin E using expression-profiling approaches (16–23). However, interpretation of such analyses is complicated by the known genomic responses to changes in cellular redox status (24). Indeed, treatment with vitamin E elicits pronounced changes in the expression of known antioxidant-responsive genes, such as glutathione-S-transferase, cytochrome P450-1A2, catalase, superoxide dismutase, and metallothioprotein (18, 19, 22). Thus, at present, only very limited information is available regarding the molecular mechanisms that mediate genomic responses to vitamin E. Here, we report the identification of specific genomic activities of αTOH that are distinct from those induced by another anti-oxidant, and provide insights into the molecular mechanisms that mediate these activities.
HepG2 C3A cells were cultured at 37 °C and 5% CO2 with Dulbecco’s modified Eagle’s medium (CellGro) supplemented with 10% FBS (Atlanta Biologicals) or LPDS (Sigma Chemical Co.). Antioxidants were added as either pre-formed serum complexes (25–27) or from ethanol stocks directly to the media. Both methods resulted in comparable uptake efficiencies as determined by accumulation of radioactively labeled tocopherol. Final ethanol concentrations did not exceed 0.2% (v/v). Unless otherwise indicated, treatments lasted 48 hours, with fresh media change after 24 hours. Cell viability was evaluated using an MTT reduction conversion assay (28, Sigma Chemical Co.).
Total RNA was isolated using the TRIzol reagent (Invitrogen) and RNA quality was evaluated using the 2100 Bioanalyzer (Agilent Technologies). Double-stranded cDNA was synthesized using a SuperScript II Reverse Transcriptase Kit (Invitrogen). Phase Lock Gel, phenol/chloroform extraction, and ethanol precipitation were employed to purify the resultant cDNA. cRNA was synthesized using the GeneChip IVT Labeling Kit (Affymetrix). In vitro transcription was carried out for four hours at 37°C, using biotinylated ribonucleotides (Enzo Diagnostics) for labeling. Labeled cRNA was purified with an RNeasy kit (Qiagen), and fragmented in fragmentation buffer for 30 minutes at 94 °C. Fragmented cRNA was hybridized to human U133A GeneChip micro-arrays (Affymetrix) in a rotating hybridization oven for 16 hours at 45°C. Staining was performed on Affymetrix fluidics station utilizing streptavidin/phycoerythrin conjugate (Molecular Probes), followed by biotinylated antibody to streptavidin (Vector Laboratories), and finally via a second streptavidin/phycoerythrin conjugate. Stained micro-arrays were scanned on a Hewlett Packard’s GeneArray Scanner, and data was compiled with Affymetrix Microarray Suite 5.0 software. Each treatment condition was repeated in three independent cultures, each of which was subjected to three independent hybridization reactions, for a total of nine micro-arrays per experimental condition.
Intensity data from each of the nine micro-arrays was uploaded to GeneTraffic version 2.8 (Iobion). Raw data were normalized using Robust Multichip Analysis, and p-values were calculated using an F-class ratio with variance stabilization, which served as a measure of the difference between the normalized intensity-value means of various treatment groups relative to the variability of intensity values of each replicate within a given treatment group. Significantly regulated gene lists (p < 0.05, absolute fold change greater than 1.2) were then manually clustered based upon known cellular function.
Triplicate wells of HepG2 C3A cells in 96-well plates were serum-starved for 72 hours, washed twice with minimum essential medium lacking phenol red (CellGro), and supplemented with various concentrations of NAC or αTOH (delivered from ethanolic stocks) for 24 hours. The cell-permeable dye DCFDA (Molecular Probes) was then added to 5 μg/mL in Hank’s buffered saline solution lacking phenol red (Sigma Chemical Co.) for two hours at 37°C. DCFDA rapidly diffuses across cell membranes, where the diacetate moiety is cleaved by intracellular esterases to yield 2′,7′-dichlorodihydrofluorescein that oxidizes upon interaction with ROS to the highly fluorescent DCF that serves as a sensitive quantitative read-out for ROS levels in live cells (29, 30). DCF fluorescence was measured with a Tecan SpectraFluor Plus microplate reader (excitation = 485 nm; emission = 535 nm). Assay range and linearity were ensured using known concentrations of hydrogen peroxide.
Total RNA was isolated from treated cells, and cDNA was prepared as described above. The expression profiles of selected genes were quantified, in triplicate, on a 7500 Real Time RT-PCR System instrument (Applied Biosystems). FAM dye-labeled Taqman MGB probes (Applied Biosystems) were utilized to assess mRNA levels of the genes encoding HMG-CoA reductase, the low-density lipoprotein (LDL)-receptor, and A-kinase anchor protein 12 (AKAP12). Threshold cycle (Ct) values were determined for each replicate and normalized to β-actin or 18S mRNA levels detected by the same procedure. These measurements were then converted to fold change values. Where indicated, actinomycin D (Sigma) was added from DMSO stock to 1 μM.
HepG2 C3A cells grown in MEM/10% LPDS were transiently transfected with a pGL3-basic vector encoding firefly luciferase under the control of the SREBP2 promoter (generous gift of Dr. Timothy Osborne, University of California Irvine; see (31) for details) using the Fugene6 reagent (Roche). Two hours after transfection, triplicate wells were treated as indicated for 36 hours, lysed and luciferase expression was assayed using a commercial kit (Promega) on a Berthold luminometer. Where indicated, the single sterol response element (SRE) sequence in this promoter was mutated to change the wild-type SRE (5′ATCACCCCAT3′) to a non-functional response element (5′ATAAAAAAAT3′) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All constructs were verified by sequencing.
Chinese hamster ovary (CHO) cells were propagated in Ham’s F-12 media supplemented with 10% FBS, then adapted to LPDS for two days. Mevalonic acid (50 μM; Sigma Chemical Co.) and other treatments were added as indicated, after which the cells were treated with 10 μM MG132 for 5 additional hours. The cells were then harvested and nuclear extracts prepared as described previously (32). Briefly, scraped cells were washed with cold PBS and resuspended in 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 M EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After swelling for 15 minutes on ice, NP-40 was added to 0.58%, the sampled were mixed vigorously and microfuged at 3000 × g for 30 seconds. The pellet was resuspended in 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF, rocked at 4°C for 15 minutes, and the nuclear extract were obtained after microcentrifugation at 14,000 rpm for 5 minutes at 4° C. The presence of processed SREBP-2 in the nuclear fraction was measured by and anti-SREBP-2 immunoblotting (Caymen Chemicals, antibody 10007663). Equal loading was ensured by immunoblotting with antibodies for proliferating cell nuclear antigen (PCNA, Cell Signaling clone PC10). Fractionation of HepG2 cells into nuclear and membranous fractions (Figure 4A) was done according to the method of Goldstein and Brown (33). Immunoblots were quantified by densitometry using Scion Image software (Scion Corp.).
De novo biosynthesis of cholesterol was measured by following the incorporation of radio-labeled acetate into cholesterol following the protocol of Goldstein and Brown (34). HepG2 C3A cells were cultured in 6-well plates for 18 hours, washed with PBS and media was changed to DMEM + 10% lipoprotein-deficient serum (LPDS, Sigma Chemical Co.). Twenty-four hours later, triplicate wells were treated with either of the following: 1–100 μM αTOH, 10 μg/μl cholesterol + 0.1 μg/μl 25-hydroxycholesterol, 1 mM NAC, or vehicle control (ethanol). After 24 hours, cells were pulsed with 2 μCi of [1-14C]acetic acid (2.11 GBq/mmol, 57.0 mCi/mmol, GE Amersham) for 6 hours. The cells were washed with PBS and lipids were extracted by incubation with 2 mL of 3:2 hexane:isopropanol at 25°C for 30 minutes. Solvent was then evaporated under N2 stream, and the lipids were resuspended in 60 μL hexane and resolved by thin layer chromatography (TLC) developed with 80:20:1 petroleum ether:diethyl ether:acetic acid. Resolved lipids were visualized with iodine vapor, and radioactive incorporation into cholesterol was quantified by scintillation counting.
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, 36–38). Data in Figure 1 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.
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 (Table 1). Treatment with αTOH caused specific and significant reduction in the expression levels of 17 genes that play central roles in regulating cellular lipid status (Table 2). 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 (Table 2). 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.
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 (Figure 2). 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, Figure 2A). 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 (Figure 2B). 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.
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 (Figure 3A), 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 (Figure 3A). This feedback inhibition of SRE-containing promoters by sterols is an established feature of cholesterol homeostatic regulation (31, 44–46). 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 (Figure 3A). 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 (Figure 3B). Importantly, mutating the SRE sequence also completely abolished transcriptional responses to αTOH (Figure 3B). 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.
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 Figure 4. 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 (Figure 4).
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.
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 (Figure 5). 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 (Figure 5). 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.
It is commonly believed that the biological requirement for vitamin E stems from the ability of the vitamin to scavenge intracellular free radicals. By counteracting the harmful actions of reactive oxygen species and lipid radicals, the vitamin is thought to prevent cellular damage and the onset of related pathologies. In support of this notion, vitamin E deficient humans and animals display pathologies that are commonly attributed to elevated oxidative stress, such as atherosclerosis and neuronal degeneration. However, recent observations suggest that members of the vitamin E family possess biological activities in addition to those elicited by other antioxidants. Thus, tocopherol was shown to influence apoptosis (48, 49, 50) and proliferation (51), and to modulate the activity of some enzymes (52–55). An additional striking ‘non-classical’ effect of vitamin E is its documented ability to modulate the expression of certain mRNAs and proteins. Recently, a number of groups utilized cDNA micro-array approaches to determine the effect of vitamin E on mRNA expression profiles. Gohil et al (16, 21–23) compared the gene expression profiles in brains and livers from TTP−/− (vitamin E- deficient) mice with those from wild-type mice and reported pronounced differences (2- to 20-fold) in the levels of multiple transcripts that play important roles in myriad biological functions. Similarly, Barella and colleagues compared the expression profile of vitamin E- depleted rats with those obtained from normal animals (17–19, 56). A major confounding factor in interpreting the data from these studies stems from the established genomic responses to changes in redox status. Vertebrate cells have evolved sensitive mechanisms to detect changes in the levels of oxygen, oxygen radicals and reducing agents, and to relay this information to specific transcription factors, such as the hypoxia responsive HIF-1 (24), the redox responsive NF-kB (57) and the anti-oxidant responsive Nrf2 (58, 59). Thus, treatment of cells with vitamin E can (and is likely to) lead to transcriptional responses that originate from changes in cellular redox status, and are not vitamin E–specific. Indeed, the aforementioned expression profiling experiments documented pronounced changes in the expression of multiple known redox-sensitive genes.
To isolate genomic responses that are specific to vitamin E, we focused our attention to αTOH-responsive mRNAs that were not affected by treatment with another antioxidant, NAC. The data revealed a limited number of transcripts that are attenuated specifically by vitamin E. Of special interest are the observations that genes involved in maintaining lipid homeostasis are down-regulated by vitamin E. Of these, ten encode enzymes that catalyze important steps in the de-novo biosynthesis of cholesterol, including the rate-limiting enzyme of this pathway, HMG-CoA reductase. The data further demonstrate that these specific genomic responses to vitamin E are mediated by the sterol-responsive transcription factor SREBP-2. Moreover, αTOH appears to elicit its regulatory action on SREBP-2 through a mechanism similar to that employed by sterols, namely by attenuating the proteolytic activation of SREBP-2 and its translocation into the nucleus. The transcriptional activities of vitamin E have a marked physiological impact in that treatment with αTOH caused a pronounced inhibition of de novo cholesterol biosynthesis.
The precise molecular mechanisms by which vitamin E influences SREBP-2 activity are not completely understood. It is possible that αTOH shares the sensing mechanism utilized for sterols, i.e. binding to sterol-sensing domains such as that found in the sterol sensor SCAP (60, 61). Alternatively, novel and specific mechanisms may exist that convey information about tocopherol levels to the transcriptional machinery.
Our observations suggest that a ‘cross-talk’ exists between the physiological levels of vitamin E and the mechanisms that regulate cholesterol homeostasis. Thus, one would expect that, in whole animals, vitamin E levels will impact overall cholesterol status. Indeed, it was reported that increased dietary intake of vitamin E is accompanied by a dose-dependent reduction in total serum cholesterol in the rat, quail and rabbit. The impact of vitamin E on the health risk in human populations still awaits complete resolution (62–73). The observations described in this study reinforce the notion of physiologically relevant ‘non-classical’ activities of vitamin E, and reveal that key roles of the vitamin in supporting human health may be mediated by its ability to regulate the rates of transcription of specific target genes.
This work was supported by award DK067494 from the National Institutes of Health.
We thank Timothy Osborne (University of California Irvine) for reagents, the University of Rochester’s Micro-array Core Facility for their assistance with the profiling experiments, and Noa Noy, Samantha Morley and Julie Beckenstein for critical readings of the manuscript.