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. 2013 Mar;58(3):641-9.
doi: 10.1007/s10620-012-2388-9. Epub 2012 Sep 27.

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

Veedamali S Subramanian  1 , Sandeep B Subramanya, Abhisek Ghosal, Jonathan S Marchant, Akihiro Harada, Hamid M Said
Affiliations

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

Veedamali S Subramanian et al. Dig Dis Sci. 2013 Mar.

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.

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Figures

Figure 1
Figure 1. Distribution of hSVCT1 and Rab8a in intenstinal epithelial cells
Caco-2, HT-29, NCM460 and HuTU-80 cells were transiently co-transfected with hSVCT1-YEP and DsRed-Rab8a constructs. Data are from n > 6-10 transfected cells. Scale bar is 10 μm
Figure 2
Figure 2. Effect of impairment of Rab8a expression on 14C-AA uptake in vitro and in vivo
A) 14C-AA uptake (32 μM) was performed on Rab8a siRNA treated hSVCT1 expressing Caco-2 cells. Data are mean ± SE of at least three separate experiments performed on different batches of cells on separate occasions. *p < 0.02. B) Real-time PCR was performed using gene specific primers for Rab8a and β-actin from total RNA isolated from Rab8a siRNA and control siRNA (scrambled) treated Caco-2 cells. Data are mean ± SE of at least three independent experiments. *p < 0.01. C, Top, Western blot analysis was performed on cell extract (60 μg) isolated from control (left) and Rab8a siRNAs treated Caco-2 cells (right). Blots were incubated with rabbit polyclonal anti-human Rab8a specific antibodies (top) along with β-actin antibodies (bottom). Bottom, densitometric quantification of the immunoreactive bands. Data are mean ± SE of at least three separate sample preparations. *p < 0.01. D) Carrier-mediated 14C-AA uptake (32 μM) by Rab8a KO mice jejunal portion was performed as described in “Materials and Methods”. Values are mean ± SE of at least three separate uptake determinations from multiple sets of mice. *p < 0.01. E) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type (litter-mate) mice jejunal mucosa using mouse Rab8a and β-actin gene-specific primers. Data are mean ± SE of at least three separate samples from three different mice. *p < 0.01. F, Top) Western blot analysis was performed on Rab8a KO (right) and wild-type litter-mate (left) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit polyclonal anti-mouse Rab8a antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three sets of samples from three different mice.
Figure 3
Figure 3. Effect of Rab8a knockdown and knockout on the level of expression of hSVCT1 mRNA and protein in Caco -2 cells and intestinal specific Rab8a KO mice jejunum
A) Real-time PCR was performed using primers for hSVCT1, hSVCT2 and β-actin on total RNA isolated from control (scrambled) and Rab8a siRNAs treated Caco-2 cells. Data are mean ± SE of multiple experiments performed on different batches of cells. B, Top, western blot analysis was performed on cell extract (60 μg) isolated from control and Rab8a siRNAs treated Caco-2 cells. Blots were incubated with rabbit anti-human hSVCT1 specific antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of multiple experiments performed on separately isolated samples.*p < 0.01. C) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type litter-mate mice jejunal mucosa using mouse SVCT1, SVCT2 and β-actin primers. Data are mean ± SE of at least three separate samples from three different mice. D, Top, western blot analysis was performed on Rab8a KO and wild-type (litter-mate) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit anti-mouse SVCT1 antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three separate samples from three mice. *p < 0.02.
Figure 4
Figure 4. Effect of Rab8a siRNA on the cell surface expression level of hSVCT1 in Caco-2 cells
Caco-2 cells were transiently transfected with Rab8a siRNA or control siRNA. Forty eight hours after transfection cells were processed for biotinylation. Equal amount of protein was loaded onto pre-made 4-12% mini-gel and western blot was performed using anti-hSVCT1 antibody. The level of cell surface expression was normalized relative to the total amount of cellular hSVCT1 protein. Densitometric values are from mean ± SE of four independent experiments. Inset shows representative western blot images. *p < 0.01.
Figure 5
Figure 5. Effect of silencing Rab8a gene on hSVCT1 intracellular trafficking in intestinal cells
Hu-Tu-80 cells were co-transfected with hSVCT1-YEP, LAMP1-RFP, control or Rab8a siRNAs. Live cell confocal imaging (laterl sections-xy) was performed after 48 h of transfection. Data are from n > 6-10 transfected cells and representative images were shown. Scale bar is 10 μm.
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