Differentiation of Promonocytic U937 Cells to Monocytes Is Associated with Reduced Mitochondrial Transport of Ascorbic Acid.

Growth of promonocytic U937 cells in the presence of DMSO promotes their differentiation to monocytes. After 4 days of culture in differentiating medium, these cells ceased to proliferate, displayed downregulated ryanodine receptor expression, and responded to specific stimuli with enhanced NADPH-oxidase-derived superoxide formation or cytosolic phospholipase A2-dependent arachidonic acid release. We found that the 4-day differentiation process is also associated with downregulated SVCT2 mRNA expression, in the absence of apparent changes in SVCT2 protein expression and transport rate of ascorbic acid (AA). Interestingly, under the same conditions, these cells accumulated lower amounts of the vitamin in their mitochondria, with an ensuing reduced response to external stimuli sensitive to the mitochondrial fraction of AA. Further analyses demonstrated an unexpected increase in mitochondrial SVCT2 protein expression, however, associated with reduced SVCT2-dependent AA uptake in isolated mitochondria. A decrease in the transporter Vmax, with no change in affinity, was found to account for this response. Differentiation of promonocytic cells to monocytes is therefore characterized by decreased SVCT2 mRNA expression that, even prior to the onset of SVCT2 protein downregulation or apparent changes in plasma membrane transport activity, impacts on the mitochondrial accumulation of the vitamin through a decreased Vmax of the transporter.

SVCT2 transporter expression is post-natally induced in cortical neurons and its function is regulated by its short isoform.

Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium-vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post-natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1-15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed. The sodium-vitamin C cotransporter, SVCT2, is induced in neurons within the inner layer of the brain cortex during post-natal development, mainly in pyramidal cortex neurons. Two different isoforms, SVCT2wt and SVCT2sh, were detected. Using in vitro studies, we suggest a molecular interaction between SVCT2wt and SVCT2sh, which may regulate the affinity of vitamin C uptake.

The molecular mechanism underlying the proliferating and preconditioning effect of vitamin C on adipose-derived stem cells.

Although adipose-derived stem cells (ASCs) show promise for cell therapy, there is a tremendous need for developing ASC activators. In the present study, we investigated whether or not vitamin C increases the survival, proliferation, and hair-regenerative potential of ASCs. In addition, we tried to find the molecular mechanisms underlying the vitamin C-mediated stimulation of ASCs. Sodium-dependent vitamin C transporter 2 (SVCT2) is expressed in ASCs, and mediates uptake of vitamin C into ASCs. Vitamin C increased the survival and proliferation of ASCs in a dose-dependent manner. Vitamin C increased ERK1/2 phosphorylation, and inhibition of the mitogen-activated protein kinase (MAPK) pathway attenuated the proliferation of ASCs. Microarray and quantitative polymerase chain reaction showed that vitamin C primarily upregulated expression of proliferation-related genes, including Fos, E2F2, Ier2, Mybl1, Cdc45, JunB, FosB, and Cdca5, whereas Fos knock-down using siRNA significantly decreased vitamin C-mediated ASC proliferation. In addition, vitamin C-treated ASCs accelerated the telogen-to-anagen transition in C3H/HeN mice, and conditioned medium from vitamin C-treated ASCs increased the hair length and the Ki67-positive matrix keratinocytes in hair organ culture. Vitamin C increased the mRNA expression of HGF, IGFBP6, VEGF, bFGF, and KGF, which may mediate hair growth promotion. In summary, vitamin C is transported via SVCT2, and increased ASC proliferation is mediated by the MAPK pathway. In addition, vitamin C preconditioning enhanced the hair growth promoting effect of ASCs. Because vitamin C is safe and effective, it could be used to increase the yield and regenerative potential of ASCs.

Downregulation of vitamin C transporter SVCT-2 in doxorubicin-induced cardiomyocyte injury.

Vitamin C (Vit C) has been shown to be protective against doxorubicin (Dox)-induced cardiotoxicity. However, Vit C uptake into cardiomyocytes is poorly understood. Furthermore, whether the antioxidant enzyme reserve is enhanced by Vit C is also not known. The present study investigated an influence of Dox on Vit C transporters, expression of endogenous antioxidant reserve as well as enzymes, oxidative stress, and apoptosis in isolated cardiomyocytes. Cardiomyocytes isolated from adult Sprague-Dawley rats were exposed to control (culture medium 199 alone), Dox (10 µM), Vit C (25 µM), and Vit C + Dox for 24 h. Vit C transporter expression and localization, oxidative stress, antioxidant enzymes, and apoptosis were studied. Expression and localization of sodium-dependent vitamin C transporter-2 (SVCT-2) in the sarcolemma was reduced by Dox, but Vit C supplementation was able to blunt this change. There was a decrease in the expression of antioxidant enzymes glutathione peroxidase (GPx), catalase, and Cu/Zn superoxide dismutase (SOD) due to Dox, but only GPx expression was completely prevented and Cu/Zn SOD was partially rescued by Vit C. Dox-induced decrease in antioxidant reserve and increase in oxidative stress were partially mitigated by Vit C. Dox-induced apoptosis was ameliorated by Vit C. It is suggested that cardioprotection offered by Vit C in Dox-induced cardiomyopathy may involve an upregulation of SVCT-2 transporter followed by a reduction in oxidative stress as well as blunting of cardiomyocyte injury.

CpG methylation at the USF-binding site mediates cell-specific transcription of human ascorbate transporter SVCT2 exon 1a.

SVCT2 (sodium-vitamin C co-transporter 2) is the major transporter mediating vitamin C uptake in most organs. Its expression is driven by two promoters (CpG-poor exon 1a promoter and CpG-rich exon 1b promoter). In the present study, we mapped discrete elements within the proximal CpG-poor promoter responsible for exon 1a transcription. We identified two E boxes for USF (upstream stimulating factor) binding and one Y box for NF-Y (nuclear factor Y) binding. We show further that NF-Y and USF bind to the exon 1a promoter in a co-operative manner, amplifying the binding of each to the promoter, and is absolutely required for the full activity of the exon 1a promoter. The analysis of the CpG site located at the upstream USF-binding site in the promoter showed a strong correlation between expression and demethylation. It was also shown that exon 1a transcription was induced in cell culture treated with the demethylating agent decitabine. The specific methylation of this CpG site impaired both the binding of USF and the formation of the functional NF-Y-USF complex as well as promoter activity, suggesting its importance for cell-specific transcription. Thus CpG methylation at the upstream USF-binding site functions in establishing and maintaining cell-specific transcription from the CpG-poor SVCT2 exon 1a promoter.