Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: studies utilizing Caco-2 cells and Rab8a knockout mice. Dig Dis Sci 2013 Mar;58(3):641-9

Date

09/28/2012

Pubmed ID

23014846

Pubmed Central ID

PMC3547156

DOI

10.1007/s10620-012-2388-9

Scopus ID

2-s2.0-84876673386   7 Citations

Abstract

BACKGROUND: Ascorbic acid (AA) is required for normal human health and development. Human intestine expresses two sodium-dependent vitamin C transporters (hSVCT-1 and -2) that mediate cellular AA transport, with hSVCT1 targeting to the apical membrane of polarized epithelia. Studies have shown a role for the Rab8a in the apical membrane targeting of transporters in intestinal cells.

AIMS: The purpose of this study was to determine whether Rab8a impacts the function and/or targeting of hSVCT1, and intestinal AA uptake.

METHODS: We used human intestinal cells and cells from a Rab8a knockout mouse. (14)C-AA uptake was performed to determine functionality. PCR and western blotting were performed to determine RNA and protein expression, respectively. Confocal imaging was performed to determine co-localization.

RESULTS: We show that hSVCT1 co-localized with Rab8a in intestinal cells. Knockdown of Rab8a lead to a significant inhibition in AA uptake and cell surface biotinylation studies revealed a lower cell surface expression of hSVCT1 in Rab8a siRNA-treated cells. Similarly, in the small intestine of a Rab8a knockout mouse, AA uptake was significantly inhibited. This effect again resulted from a decreased expression level of mSVCT1 protein, even though mRNA expression of SVCT1 was similar in intestinal cells from Rab8a knockout and wild-type litter-mates. The latter data are suggestive of enhanced lysosomal degradation of hSVCT1 protein in Rab8a-deficient cells; indeed, confocal imaging of Rab8a siRNA-treated intestinal cells revealed a strong overlap between hSVCT1-YFP and LAMP1-RFP.

CONCLUSIONS: These findings show a role for Rab8a in the physiological function of hSVCT1 in intestinal epithelia.

Author List

Subramanian VS, Subramanya SB, Ghosal A, Marchant JS, Harada A, Said HM

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acids
Animals
Ascorbic Acid
Blotting, Western
Caco-2 Cells
Gene Knockdown Techniques
Gene Silencing
Humans
Intestinal Mucosa
Mice
Mice, Knockout
Protein Transport
RNA, Small Interfering
Real-Time Polymerase Chain Reaction
Sodium-Coupled Vitamin C Transporters
rab GTP-Binding Proteins
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a