Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C

Liang, Weijun; Johnson, D.; Ma, Liang; Jarvis, Simon M.

doi:10.1152/ajpcell.00461.2001

Cited by 71 publications

(50 citation statements)

References 29 publications

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“…A recent report by Liang et al (11) suggests VOL. 24, 2004 hSVCT2 INHIBITS ASCORBIC ACID TRANSPORT 3155 on May 9, 2018 by guest http://mcb.asm.org/ that there is yet another mechanism of the regulation of vitamin C transport that occurs through the protein kinase C cascade.…”

Section: Discussionmentioning

confidence: 95%

A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport

Lutsenko

Cárcamo

Golde

2004

Molecular and Cellular Biology

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Vitamin C is transported as ascorbic acid (AA) through the sodium-ascorbate cotransporters (SVCT1 and -2) and as dehydroascorbic acid (DHA) through the facilitative glucose transporters. All cells have glucose transporters and take up DHA that is trapped intracellularly by reduction and accumulated as AA. SVCT2 is widely expressed in cells and tissues at the mRNA level; however, only specialized cells directly transport AA. We undertook a molecular analysis of SVCT2 expression and discovered a transcript encoding a short form of human SVCT2 (hSVCT2-short) in which 345 bp is deleted without a frame shift. The deletion involves domains 5 and 6 and part of domain 4. cDNA encoding this isoform was isolated and expressed in 293T cells, where the protein was detected on the plasma membrane. Transport studies, however, revealed that hSVCT2-short gave rise to a nonfunctional transporter protein. hSVCT2-short arises by alternative splicing and encodes a protein that strongly inhibited the function of SVCT2 and, to a lesser extent, SVCT1 in a dominant-negative manner, probably by protein-protein interaction. The expression of hSVCT2-short varies among cells. PCR analysis of cDNA isolated from melanocytes capable of transporting AA revealed a predominance of the full-length isoform, while HL-60 cells, which express SVCT2 at the mRNA level and were incapable of transporting AA, showed a predominance of the short isoform. These findings suggest a mechanism of AA uptake regulation whereby an alternative SVCT2 gene product inhibits transport through the two known AA transporters.Vitamin C is essential for human health. Most mammals produce vitamin C in the liver; however, humans and other primates are unable to synthesize ascorbic acid (AA) and must obtain it from the diet (9, 13). Vitamin C is transported into cells in the oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (GLUTs) (19,24) and as AA in specialized cells by sodium-dependent AA transporters (23). Two isoforms of the sodium-dependent vitamin C transporters (SVCTs) have been molecularly characterized in rats and humans (3,10,18,23,28,29). Kyte-Doolittle hydropathy analysis (7) of the human SVCT2 (hSVCT2) amino acid sequence predicts a topographical model of a transporter with 12 transmembrane domains with both the N and C termini intracellular. The N-terminal (102-amino-acid) and the C-terminal (81-amino-acid) tails in the cytoplasm are long and hydrophilic. The extracellular loop between transmembrane domains 3 and 4 contains two potential sites for N-glycosylation . The hSVCT1 transporter is highly homologous to hSVCT2 with the same predicted membrane topology. An obvious difference between hSVCT1 and hSVCT2 is the additional sequences of 12 and 44 amino acids present in the N terminus of hSVCT2 at positions 2 and 38, respectively (10). The two isoforms of hSVCT differ in tissue distribution, as determined by Northern blot analysis, with hSVCT2 widely expressed at the mRNA level compared to hSVCT1. For hSVCT2, a 7.5-kb transcript wa...

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Section: Discussionmentioning

confidence: 95%

A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport

Lutsenko

Cárcamo

Golde

2004

Molecular and Cellular Biology

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“…[33][34][35] For hSVCT1, decrease in AA uptake in COS-1 cells treated with PKC inhibitor is associated with a redistribution of hSVCT1 from the cell surface to intracellular membranes. 36 EP1 receptor inhibitor, AH-6809, does not affect PGE2-induced SVCT2 translocation, AA uptake, osteoblast differential markers expression, and mineralization, suggesting that PGE2-associated effects are not due to activation of EP1 receptor subtype. However, EP4 receptor inhibitor, AH-23848B abrogates PGE2-associated effects, suggesting that activation of EP4 subtype and subsequent increase of cAMP contents are involved in PGE2-associated effects.…”

Section: Resultsmentioning

confidence: 92%

“…Stained cells were examined under an Olympus confocal microscope (bars, 100 mm) Na þ -dependent vitamin C transport in a cell line derived from rabbit nonpigmented ciliary epithelium and in oocytes expressing hSVCT1 and hSVCT2. 6,39 PKC activator phorbol 12-myristate 13-acetate (PMA) causes a time-and concentration-dependent decrease in AA transport activity in COS-1 cells, for hSVCT1 but not hSVCT2, 36 the decrease in AA uptake is correlated with a redistribution of the transporter from the cell surface to intracellular membranes. In our case, the immunocomplexes precipitated by SVCT2 antibody contain cPKA under stimulation with PGE2 or without PGE2 indicating that the specifically physical interaction between SVCT2 and cPKA is PGE2-independent, however, the immunocomplexes precipitated by cPKA antibody contain SVCT2 only after exposure to PGE2 suggesting PGE2-dependent interaction between SVCT2 and cPKA.…”

Section: Resultsmentioning

confidence: 99%

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

Taniguchi

et al. 2007

Cell Death Differ

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Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.

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“…In the fi rst mechanism, vitamin C (ascorbic acid) is the specifi c substrate transported, by a high-affi nity, low-capacity system, that is sodium-and temperature-dependent (Daruwala et al, 1999;Tsukaguchi et al, 1999;Liang et al, 2002;Godoy et al, 2007). The molecular identity of this system has been delineated following cloning of two transporters (from human kidney cDNA library): the human sodium vitamin C cotransporters, SVCT1 and SVCT2 (products of the SLC23A2 and SLC23A1 genes, respectively; Daruwala et al, 1999).…”

Section: Vitamin C and Vitamin C Transportersmentioning

confidence: 99%

Vitamin C and oxidative stress in the seminiferous epithelium

et al. 2011

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In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These fi ndings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis.

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Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C

Cited by 71 publications

References 29 publications

A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport

A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

Vitamin C and oxidative stress in the seminiferous epithelium

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