Nedd4-1 regulates human sodium-dependent vitamin C transporter-2 functional expression in neuronal and epithelial cells.

The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is currently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we determined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways.

Blue light promotes vitamin C-mediated ferroptosis of melanoma through specifically upregulating transporter SVCT2 and generating Fe2.

Vitamin C (VC)-based cancer therapy is a promising therapeutic approach for a variety of cancers due to its profound effects on redox reactions and metabolic pathways. However, high administration dosage of VC for necessary therapeutic efficacy for cancers increases the risk of overt side effects and limits its clinical use. Here, we show cutaneous blue light irradiation can specifically upregulate the sodium-dependent vitamin C transporter 2 (SVCT2) of the tumor and increase effectively the VC concentration at the tumor sites by an overall low dosage administration. In the mouse melanoma model, blue light stimulates the SVCT2 expression through the nuclear factor-kappa B (NF-κB) signaling pathway both in vitro and in vivo. The increased cellular VC together with Fe2+ generated by blue light simultaneously elevate cellular oxidative stress and trigger the ferroptosis of melanoma. With this revealed mechanism, the synergistic actions of blue light on the VC transporter and Fe2+ generation lead to a ca. 20-fold reduction in the administration dosage of VC with an effective melanoma elimination and prolonged survival. The work defines the killing mechanism of blue light on VC-based cancer therapy and provides a practical approach for promoting VC uptake. This light-assisted VC therapy is not only highly efficient for melanoma but also considerable for a broad clinical utility.

Research Note: Heat stress affects immune and oxidative stress indices of the immune organs of broilers by changing the expressions of adenosine triphosphate-binding cassette subfamily G member 2, sodium-dependent vitamin C transporter-2, and mitochondrial calcium uniporter.

This study aimed to determine the mechanisms of heat-induced oxidative stress in the thymus and spleen of broilers. After 28 d, 30 broilers were randomly divided into the control (25°C ± 2°C; 24 h/d) and heat-stressed (36°C ± 2°C; 8 h/d) groups; the experiment lasted for 1 wk. The broilers in each group were euthanized, and some samples were collected and analyzed at 35 d. The results showed that the birds subjected to heat stress reduced the weight (P < 0.01) and the indices of thymus (P < 0.01), the activities of T-AOC (P < 0.01) and SOD (P < 0.05) of spleen, and levels of IL-10 (P < 0.05) and the GSH-PX (P < 0.05) in thymus and spleen, and increased the IL-6 content of thymus (P < 0.05), the MDA content (P < 0.01), and the reactive oxygen species (ROS) levels (P < 0.01) in thymus and spleen. Moreover, the expression of the IgG gene in the thymus and spleen of heat-stressed broilers was increased (P < 0.05); however, the expression of the IgM gene in the spleen was increased (P < 0.05), with no difference (P > 0.05) in the thymus of heat-stressed broilers compared with the control. Furthermore, the relative expression of adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) in the thymus and spleen both increased (P < 0.05). The sodium-dependent vitamin C transporter-2 (SVCT-2) (P < 0.01) and mitochondrial calcium uniporter (MCU) (P < 0.01) mRNA levels in the thymus of heat-stressed broilers increased, and the expression of ABCG2 (P < 0.05), SVCT-2 (P < 0.01), and MCU (P < 0.01) proteins in the thymus and spleen of heat-stressed broilers increased compared with the control group. This study confirmed that heat stress-induced oxidative stress in the immune organs of broilers, further reducing immune function.

Structural basis of vitamin C recognition and transport by mammalian SVCT1 transporter.

Vitamin C (L-ascorbic acid) is an essential nutrient for human health, and its deficiency has long been known to cause scurvy. Sodium-dependent vitamin C transporters (SVCTs) are responsible for vitamin C uptake and tissue distribution in mammals. Here, we present cryogenic electron microscopy structures of mouse SVCT1 in both the apo and substrate-bound states. Mouse SVCT1 forms a homodimer with each protomer containing a core domain and a gate domain. The tightly packed extracellular interfaces between the core domain and gate domain stabilize the protein in an inward-open conformation for both the apo and substrate-bound structures. Vitamin C binds at the core domain of each subunit, and two potential sodium ions are identified near the binding site. The coordination of sodium ions by vitamin C explains their coupling transport. SVCTs probably deliver substrate through an elevator mechanism in combination with local structural arrangements. Altogether, our results reveal the molecular mechanism by which SVCTs recognize vitamin C and lay a foundation for further mechanistic studies on SVCT substrate transport.

Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations.

Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.

Vitamin C transport in neurons and epithelia is regulated by secretory carrier-associated membrane protein-2 (SCAMP2).

The human sodium-dependent vitamin C transporter-1 (hSVCT1) is localized at the apical membrane domain of polarized intestinal and renal epithelial cells to mediate ascorbic acid (AA) uptake. Currently, little is known about the array of interacting proteins that aid hSVCT1 trafficking and functional expression at the cell surface. Here we used an affinity tagging ('One-STrEP') and proteomic approach to identify hSVCT1 interacting proteins, which resolved secretory carrier-associated membrane protein-2 (SCAMP2) as a novel accessary protein partner. SCAMP2 was validated as an accessory protein by co-immunoprecipitation with hSVCT1. Co-expression of hSVCT1 and SCAMP2 in HEK-293 cells revealed both proteins co-localized in intracellular structures and at the plasma membrane. Functionally, over-expression of SCAMP2 potentiated 14C-AA uptake, and reciprocally silencing endogenous SCAMP2 decreased 14C-AA uptake. Finally, knockdown of endogenous hSVCT1 or SCAMP2 impaired differentiation of human-induced pluripotent stem cells (hiPSCs) toward a neuronal fate. These results establish SCAMP2 as a novel hSVCT1 accessary protein partner that regulates AA uptake in absorptive epithelia and during neurogenesis.

SVCT2-mediated ascorbic acid uptake buffers stress responses via DNA hydroxymethylation reprogramming of S100 calcium-binding protein A4 gene.

Vitamin C, a key antioxidant in the central nervous system, cycles between ascorbic acid and dehydroascorbic acid under pathophysiological conditions. Clinical evidence supports that the absence of vitamin C may be linked to depressive symptoms, but much less is known about the mechanism. Herein, we show that chronic stress disrupts the expression of ascorbic acid transporter, sodium-dependent vitamin C transport 2, and induces a deficiency in endogenous ascorbic acid in the medial prefrontal cortex, leading to depressive-like behaviors by disturbing redox-dependent DNA methylation reprogramming. Attractively, ascorbic acid (100 mg/kg-1000 mg/kg, intraperitoneal injection, as bioequivalent of an intravenous drip dose of 0.48 g-4.8 g ascorbic acid per day in humans) produces rapid-acting antidepressant effects via triggering DNA demethylation catalyzed by ten-eleven translocation dioxygenases. In particular, the mechanistic studies by both transcriptome sequencing and methylation sequencing have shown that S100 calcium binding protein A4, a potentially protective factor against oxidative stress and brain injury, mediates the antidepressant activity of ascorbic acid via activating erb-b2 receptor tyrosine kinase 4 (ErbB4)-brain derived neurotrophic factor (BDNF) signaling pathway. Overall, our findings reveal a novel nutritional mechanism that couples stress to aberrant DNA methylation underlying depressive-like behaviors. Therefore, application of vitamin C may be a potential strategy for the treatment of depression.

Valproic acid upregulates sodium-dependent vitamin C transporter-2 functional expression in neuronal cells.

Neuronal uptake of ascorbic acid (AA) in humans occurs via the human sodium-dependent vitamin C transporter-2 (hSVCT2). Recent studies show that a significantly lower level of vitamin C is present in the blood of epileptic patients. Consequently, focused studies investigating the involved molecular mechanisms for hSVCT2 regulation are vital to enhance vitamin C body homeostasis. Currently, little is known about the role of valproic acid (VPA), a drug utilized to treat epilepsy and a class I histone deacetylase inhibitor (HDACi), on AA uptake in neuronal systems. Thus, this study aims to examine the effect of VPA on hSVCT2 functional expression in neuronal cells. VPA treatment upregulated the AA uptake and this increased AA uptake was associated with a significant increase in hSVCT2 expression and SLC23A2 promoter activity in SH-SY5Y cells. Knockdown of HDAC2, a predominant isoform in neuronal systems, significantly increased hSVCT2 functional expression. VPA treatment in mice displayed increased mouse (m)SVCT2 protein, mRNA and heterogenous nuclear RNA (hnRNA) expression in the brain. In addition, Yin Yang-1 (YY1), a transcription factor that drives the SLC23A2 promoter activity, protein and mRNA expression levels were markedly upregulated in VPA-treated SH-SY5Y cells and mice brain. Together, our findings suggest that VPA upregulates the functional expression of SVCT2 via HDAC2 and transcriptional mechanism(s).

Glioblastoma Invasiveness and Collagen Secretion Are Enhanced by Vitamin C.

Aims: Glioblastoma (GB) is one of the most aggressive brain tumors. These tumors modify their metabolism, increasing the expression of glucose transporters, GLUTs, which incorporate glucose and the oxidized form of vitamin C, dehydroascorbic acid (DHA). We hypothesized that GB cells preferentially take up DHA, which is intracellularly reduced and compartmentalized into the endoplasmic reticulum (ER), promoting collagen biosynthesis and an aggressive phenotype. Results: Our results showed that GB cells take up DHA using GLUT1, while GLUT3 and sodium-dependent vitamin C transporter 2 (SVCT2) are preferably intracellular. Using a baculoviral system and reticulum-enriched extracts, we determined that SVCT2 is mainly located in the ER and corresponds to a short isoform. Ascorbic acid (AA) was compartmentalized, stimulating collagen IV secretion and increasing in vitro and in situ cell migration. Finally, orthotopic xenografts induced in immunocompetent guinea pigs showed that vitamin C deficiency retained collagen, reduced blood vessel invasion, and affected glomeruloid vasculature formation, all pathological conditions associated with malignancy. Innovation and Conclusion: We propose a functional role for vitamin C in GB development and progression. Vitamin C is incorporated into the ER of GB cells, where it favors the synthesis of collagen, thus impacting tumor development. Collagen secreted by tumor cells favors the formation of the glomeruloid vasculature and enhances perivascular invasion. Antioxid. Redox Signal. 37, 538-559.

The Membrane Electrical Potential and Intracellular pH as Factors Influencing Intracellular Ascorbate Concentration and Their Role in Cancer Treatment.

Ascorbate is an important element of a variety of cellular processes including the control of reactive oxygen species levels. Since reactive oxygen species are implicated as a key factor in tumorigenesis and antitumor therapy, the injection of a large amount of ascorbate is considered beneficial in cancer therapy. Recent studies have shown that ascorbate can cross the plasma membrane through passive diffusion. In contrast to absorption by active transport, which is facilitated by transport proteins (SVCT1 and SVCT2). The passive diffusion of a weak acid across membranes depends on the electrostatic potential and the pH gradients. This has been used to construct a new theoretical model capable of providing steady-state ascorbate concentration in the intracellular space and evaluating the time needed to reach it. The main conclusion of the analysis is that the steady-state intracellular ascorbate concentration weakly depends on its serum concentration but requires days of exposure to saturate. Based on these findings, it can be hypothesized that extended oral ascorbate delivery is possibly more effective than a short intravenous infusion of high ascorbate quantities.

Calsyntenin-3 interacts with the sodium-dependent vitamin C transporter-2 to regulate vitamin C uptake.

Ascorbic acid (AA) uptake in neurons occurs via a Na+-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library. This interaction was confirmed by co-immunoprecipitation, mammalian two-hybrid (M2H), and co-localization in human cell lines. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells led to a marked increase in AA uptake. Reciprocally, siRNA targeting hCLSTN3 inhibited AA uptake. In the J20 mouse model of Alzheimer's disease (AD), mouse (m)SVCT2 and mCLSTN3 expression levels in hippocampus were decreased. Similarly, expression levels of hSVCT2 and hCLSTN3 were markedly decreased in hippocampal samples from AD patients. These findings establish CLSTN3 as a novel hSVCT2 interactor in neuronal cells with potential pathophysiological significance.

Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

BACKGROUND/AIMS: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse. METHODS: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels. RESULTS: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1. CONCLUSION: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC.

Vitamin C Enhances Antiviral Functions of Lung Epithelial Cells.

Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells-the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.

Histone deacetylase inhibitors regulate vitamin C transporter functional expression in intestinal epithelial cells.

Intestinal absorption of vitamin C in humans is mediated via the sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2). hSVCT1 and hSVCT2 are localized at the apical and basolateral membranes, respectively, of polarized intestinal epithelia. Studies have identified low plasma levels of vitamin C and decreased expression of hSVCT1 in patients with several inflammatory conditions including inflammatory bowel disease (IBD). Investigating the underlying mechanisms responsible for regulating hSVCT1 expression are critical for understanding vitamin C homeostasis, particularly in conditions where suboptimal vitamin C levels detrimentally affect human health. Previous research has shown that hSVCT1 expression is regulated at the transcriptional level, however, little is known about epigenetic regulatory pathways that modulate hSVCT1 expression in the intestine. In this study, we found that hSVCT1 expression and function were significantly decreased in intestinal epithelial cells by the histone deacetylase inhibitors (HDACi), valproic acid (VPA), and sodium butyrate (NaB). Further, expression of transcription factor HNF1α, which is critical for SLC23A1 promoter activity, was significantly down regulated in VPA-treated cells. Chromatin immunoprecipitation (ChIP) assays showed significantly increased enrichment of tetra-acetylated histone H3 and H4 within the SLC23A1 promoter following VPA treatment. In addition, knockdown of HDAC isoforms two, and three significantly decreased hSVCT1 functional expression. Following VPA administration to mice, functional expression of SVCT1 in the jejunum was significantly decreased. Collectively, these in vitro and in vivo studies demonstrate epigenetic regulation of SVCT1 expression in intestinal epithelia partly mediated through HDAC isoforms two and three.

Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake.

Vitamin C (ascorbic acid: AA) uptake in neurons occurs via the sodium-dependent vitamin C transporter-2 (SVCT2), which is highly expressed in the central nervous system (CNS). During chronic neuroinflammation or infection, CNS levels of lipopolysaccharide (LPS) and LPS-induced tumor necrosis factor-α (TNFα) are increased. Elevated levels of LPS and TNFα have been associated with neurodegenerative diseases together with reduced levels of AA. However, little is known about the impacts of LPS and TNFα on neuronal AA uptake. The objective of this study was to examine the effect of LPS and TNFα on SVCT2 expression and function using in vitro and in vivo approaches. Treatment of SH-SY5Y cells with either LPS or TNFα inhibited AA uptake. This reduced uptake was associated with a significant decrease in SVCT2 protein and mRNA levels. In vivo exposure to LPS or TNFα also decreased SVCT2 protein and mRNA levels in mouse brains. Both LPS and TNFα decreased SLC23A2 promoter activity. Further, the inhibitory effect of LPS on a minimal SLC23A2 promoter was attenuated when either the binding site for the transcription factor Sp1 was mutated or cells were treated with the NF-κB inhibitor, celastrol. We conclude that inflammatory signals suppress AA uptake by impairing SLC23A2 transcription through opposing regulation of Sp1 and NF-κB factors.

Novel Multifunctional Ascorbic Triazole Derivatives for Amyloidogenic Pathway Inhibition, Anti-Inflammation, and Neuroprotection.

Alzheimer's disease (AD) is a common neurodegenerative disorder. The number of patients with AD is projected to reach 152 million by 2050. Donepezil, rivastigmine, galantamine, and memantine are the only four drugs currently approved by the United States Food and Drug Administration for AD treatment. However, these drugs can only alleviate AD symptoms. Thus, this research focuses on the discovery of novel lead compounds that possess multitarget regulation of AD etiopathology relating to amyloid cascade. The ascorbic acid structure has been designated as a core functional domain due to several characteristics, including antioxidant activities, amyloid aggregation inhibition, and the ability to be transported to the brain and neurons. Multifunctional ascorbic derivatives were synthesized by copper (I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The in vitro and cell-based assays showed that compounds 2c and 5c exhibited prominent multifunctional activities as beta-secretase 1 inhibitors, amyloid aggregation inhibitors, and antioxidant, neuroprotectant, and anti-inflammatory agents. Significant changes in activities promoting neuroprotection and anti-inflammation were observed at a considerably low concentration at a nanomolar level. Moreover, an in silico study showed that compounds 2c and 5c were capable of being permeated across the blood-brain barrier by sodium-dependent vitamin C transporter-2.

Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer's disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

Transport of Vitamin C in Cancer.

Significance: Vitamin C is a powerful antioxidant that has an intricate relationship with cancer and has been studied for more than 60 years. However, the specific mechanisms that allow malignant cells to uptake, metabolize, and compartmentalize vitamin C remain unclear. In normal human cells, two different transporter systems are responsible for its acquisition: glucose transporters (GLUTs) transport the oxidized form of vitamin C (dehydroascorbic acid) and sodium-coupled ascorbic acid transporters (SVCTs) transport the reduced form (ascorbic acid [AA]). In this study, we review the mechanisms described for vitamin C uptake and metabolization in cancer. Recent Advances: Several studies performed recently in vivo and in vitro have provided the scientific community a better understanding of the differential capacities of cancer cells to acquire vitamin C: tumors from different origins do not express SVCTs in the plasma membrane and are only able to acquire vitamin C in its oxidized form. Interestingly, cancer cells differentially express a mitochondrial form of SVCT2. Critical Issues: Why tumors have reduced AA uptake capacity at the plasma membrane, but develop the capacity of AA transport within mitochondria, remains a mystery. However, it shows that understanding vitamin C physiology in tumor survival might be key to decipher the controversies in its relationship with cancer. Future Directions: A comprehensive analysis of the mechanisms by which cancer cells acquire, compartmentalize, and use vitamin C will allow the design of new therapeutic approaches in human cancer. Antioxid. Redox Signal. 35, 61-74.

New insights into Vitamin C function: Vitamin C induces JAK2 activation through its receptor-like transporter SVCT2.

Vitamin C (VitC) is a requisite nutrient for humans and other primates. Extensive research continuously illustrates the applications of VitC in promoting cell reprogramming, fine-tuning embryonic stem cell function, and fighting diseases. Given its chemical reduction property, VitC predominantly acts as an antioxidant to reduce reactive oxygen species (ROS) and as a cofactor for certain dioxygenases involved in epigenetic regulation. Here, we propose that VitC is also a bio-signaling molecule based on the finding that sodium-dependent VitC transporter (SVCT) 2 is a novel receptor-like transporter of VitC that possesses dual activities in mediating VitC uptake and Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 2 signaling pathway. Through interaction, SVCT2 induces JAK2 phosphorylation while transporting VitC into cells. Activated JAK2 phosphorylates the C-terminus of SVCT2, resulting in the recruitment and activation of STAT2. As a highlight, our results suggest that the activation of JAK2 synergistically promotes regulation of VitC in ROS scavenging and epigenetic modifications through phosphorylating pyruvate dehydrogenase kinase 1, ten-eleven translocation enzyme 3, and histone H3 Tyr41. Furthermore, VitC-activated JAK2 exhibits bidirectional effects in regulating cell pluripotency and differentiation. Our results thus reveal that the SVCT2-mediated JAK2 activation facilitates VitC functions in a previously unknown manner.

Sodium vitamin C transporter 2 orchestrates lactate metabolism in mouse Sertoli cells.

Transportation of vitamin C (also called ascorbic acid (AA)), an important water-soluble antioxidant and cofactor in testis, requires glucose transporter family (GLUTs) and sodium/vitamin C cotransporter family (SVCT1 and SVCT2). There is so far scant information vis-à-vis the functional roles of SVCTs in testis, although they possess higher affinity for transportation of AA compared to GLUTs. To analyze the biological effects of SVCT2 in testis, we assessed testicular expression of SVCT2 in different experimental settings and the effect of SVCT2 ablation on spermatogenesis. Persistent expression of SVCT2 was shown in the mouse testis at different stages of postnatal development, demonstrated on day 14 of testicular development in mice consistent with the appearance of pachytene spermatocytes during the first wave of spermatogenesis. Testicular expression of SVCT2 was enriched in the cytoplasm of murine Sertoli cells (SCs). We then showed that in vivo inhibition of SVCT2 in mouse testis significantly impaired male fertility by causing oligozoospermia and asthenospermia, which mainly stemmed from a deficiency in lactate production. By generating the TM4SVCT2-/- cells and by profiling TM4SVCT2-/- cells with a constitutively activated HIF-1α mutant, we demonstrated that SVCT2 deficiency led to impaired lactate synthesis and reduced expression of Ldha mRNA in SCs. Mechanistically, ablation of SVCT2 resulted in ubiquitination and subsequent degradation of HIF-1α protein in the FSH-stimulated SCs. Collectively, our data document a novel testicular site of action of SVCT2 in the control of lactate synthesis by SCs, probably via ubiquitination-dependent regulation of HIF-1α stability.