title: 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.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA 92697, USA. vsubrama@uci.edu
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Sandeep B
authors.initials: SB
authors.lastname: Subramanya
authors.forename: Abhisek
authors.initials: A
authors.lastname: Ghosal
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Akihiro
authors.initials: A
authors.lastname: Harada
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D021381:Protein Transport; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D060347:Sodium-Coupled Vitamin C Transporters / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D034741:RNA, Small Interfering; C546061:Rab8a protein, mouse; C555162:SLC23A1 protein, human; C555164:Slc23a1 protein, mouse; D060347:Sodium-Coupled Vitamin C Transporters; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins; D001205:Ascorbic Acid