Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 12, 15 December 2012, Pages 2318-2326
Free Radical Biology and Medicine

Expression of the α-tocopherol transfer protein gene is regulated by oxidative stress and common single-nucleotide polymorphisms

https://doi.org/10.1016/j.freeradbiomed.2012.10.528Get rights and content

Abstract

Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in most animal species. By controlling the secretion of vitamin E from the liver, the α-tocopherol transfer protein regulates whole-body distribution and levels of this vital nutrient. However, the mechanism(s) that regulates the expression of this protein is poorly understood. Here we report that transcription of the TTPA gene in immortalized human hepatocytes is induced by oxidative stress and by hypoxia, by agonists of the nuclear receptors PPARα and RXR, and by increased cAMP levels. The data show further that induction of TTPA transcription by oxidative stress is mediated by an already-present transcription factor and does not require de novo protein synthesis. Silencing of the cAMP response element-binding (CREB) transcription factor attenuated transcriptional responses of the TTPA gene to added peroxide, suggesting that CREB mediates responses of this gene to oxidative stress. Using a 1.9-kb proximal segment of the human TTPA promoter together with a site-directed mutagenesis approach, we found that single-nucleotide polymorphisms that are commonly found in healthy humans dramatically affect promoter activity. These observations suggest that oxidative stress and individual genetic makeup contribute to vitamin E homeostasis in humans. These findings may explain the variable responses to vitamin E supplementation observed in human clinical trials.

Highlights

Vitamin E is the major lipid-soluble antioxidant. ► α-Tocopherol transfer protein is the only known regulator of vitamin E status. ► Oxidative stress and promoter SNPs regulate expression of the TTPA gene. ► Our findings may explain perplexing results regarding responses to vitamin E supplementation observed in clinical trials.

Section snippets

Cell lines

Because expression of αTTP in primary hepatocytes dramatically decreases after isolation, we employed immortalized human hepatocytes (IHH) that endogenously express the protein [31] as a model system. IHH (generous gift from R. Ray, St. Louis University, St. Louis, MO, USA) were cultured in Dulbecco's modified Eagle's medium supplemented with 5% calf serum (Hyclone Laboratories, Logan, UT, USA) as described in [40].

cAMP response element-binding (CREB) transcription factor knockdown

Lentiviral short hairpin RNA (shRNA) constructs targeted against human CREB

Transcriptional regulation of the tocopherol transfer protein

Examination of murine Ttpa gene expression in 8-day-old C57BL/6 J mice revealed, in agreement with previous reports [21], [46], that αTTP mRNA and protein levels are highest in the liver; significant in the cerebellum, cortex, and kidney; and absent from the intestine and heart (Fig. 1). In all tissues, we observed close correspondence between the levels of the mRNA detected by RT-PCR and those of the protein, revealed by immunoblotting. These data indicate that the Ttpa gene is differentially

Discussion

αTTP is the only known direct regulator of whole-body vitamin E status, yet the mechanisms that regulate its expression are by and large unknown. Our results reveal that expression of the TTPA gene is induced by WY14643 and 9-cis-retinoic acid, ligands that activate the nuclear receptors PPARα and RXR, respectively. Together with the identification of a putative PPAR response element in the TTPA promoter, these data suggest that TTPA transcription is regulated by PPARα–RXR heterodimers [72].

Acknowledgments

This work was supported by NIH Award DK067494 to D.M. and by a pilot award from the Case Comprehensive Cancer Center Support Grant 5P30-CA043703 from the NIH.

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