Inhibition of intestinal ascorbic acid uptake by lipopolysaccharide is mediated via transcriptional mechanisms

Abstract

Ascorbic acid (AA) accumulation in intestinal epithelial cells is an active transport process mainly mediated by two sodium-dependent vitamin C transporters (SVCT-1 and SVCT-2). To date, little is known about the effect of gut microbiota generated lipopolysaccharide (LPS) on intestinal absorption of water-soluble vitamins. Therefore, the objective of this study was to investigate the effects of bacterially-derived LPS on AA homeostasis in enterocytes using Caco-2 cells, mouse intestine and intestinal enteroids models. Pre-treating Caco-2 cells and mice with LPS led to a significant decrease in carrier-mediated AA uptake. This inhibition was associated with a significant reduction in SVCT-1 and SVCT-2 protein, mRNA, and hnRNA expression. Furthermore, pre-treating enteroids with LPS also led to a marked decrease in SVCT-1 and SVCT-2 protein and mRNA expression. Inhibition of SVCT-1 and SVCT-2 occurred at least in part at the transcriptional level as promoter activity of SLC23A1 and SLC23A2 was attenuated following LPS treatment. Subsequently, we examined the protein and mRNA expression levels of HNF1α and Sp1 transcription factors, which are needed for basal SLC23A1 and SLC23A2 promoter activity, and found that they were significantly decreased in the LPS treated Caco-2 cells and mouse jejunum; this was reflected on level of the observed reduction in the interaction of these transcription factors with their respective promoters in Caco-2 cells treated with LPS. Our findings indicate that LPS inhibits intestinal carrier- mediated AA uptake by down regulating the expression of both vitamin C transporters and transcriptional regulation of SLC23A1 and SLC23A2 genes.

Keywords: Enteroids; SVCT-1; SVCT-2; Transport; Uptake; Vitamin C.

Figure 1. Effect of LPS on AA uptake by Caco-2 cells and native mouse jejunum tissue

A. Confluent Caco-2 cells were serum-starved overnight followed by treatment with LPS (50 μg/ml) for 48 h. AA uptake was measured as described in “Methods”. Data are means ± SE of at least three independent experiments with different passages of cells (*P < 0.01). B. Male C57BL/6 mice (12 weeks old) were injected with LPS (0.005g/kg body weight, IP) and AA uptake was performed using mouse jejunal tissue as described in “Methods”. Data are means ± SE of at least three sets of mice (**P < 0.05).

Figure 2. Effect of LPS on the level of expression of SVCT-1 and SVCT-2 protein in intestinal epithelial Caco-2 cells, native mouse jejunum tissue and enteroids

A&B. Caco-2 cells were pre-treated with LPS and after 48 h, the total protein was isolated and subjected to western blotting to determine hSVCT-1 and hSVCT-2 protein expression levels, respectively as described in “Methods”. Data are means ± SE of at least three independent experiments (*P < 0.01; **P < 0.05). C&D. Total protein was isolated from mouse jejunum tissue of LPS-treated mice and subjected to western blotting to determine mSVCT-1 and mSVCT-2 protein expression levels, respectively as described in “Methods”. Data are means ± SE of at least three sets of mice (**P < 0.05). E. Images of (1–7 days old) mouse intestinal enteroids. F&G. Enteroids were pretreated with LPS (50 μg/ml) for 48 h and the level of expression of mSVCT-1 and mSVCT-2 protein were determined as described in “Methods”. Data are means ± SE of at least three separate experiments (*P < 0.01; **P < 0.05).

Figure 3. Effect of LPS on the level of expression of SVCT-1 and SVCT-2 mRNA in Caco-2 cells, mouse jejunum tissue and enteroids

A&B. hSVCT-1 and hSVCT-2 mRNA expression levels were determined using total RNA isolated from LPS treated Caco-2 cells and real-time PCR as described in “Methods”. Data are the means ± SE of at least three independent experiments (** P < 0.05). C&D. mSVCT-1 and mSVCT-2 mRNA expression levels were determined using total RNA isolated from LPS injected mouse jejunum as described in “Methods”. Data are means ± SE of at least three sets of mice (*P < 0.01). E&F. Real-time PCR for mSVCT-1 and mSVCT-2 in LPS treated and untreated enteroids were performed using gene specific primers as described in “Methods”. Data represents the means ± SE of at least three separate experiments. (*P < 0.01; **P < 0.05).

Figure 4. Effect of LPS on heterogenous nuclear RNA (hnRNA) expression of SVCT-1 and SVCT-2

A&B. Real-time-PCR for hSVCT-1 (SLC23A1) and hSVCT-2 (SLC23A2) in the LPS treated and untreated Caco-2 cells were performed using gene-specific primers as described in “Methods”. Data represent the means ± SE of three separate experiments (*P < 0.01; **P < 0.5). C&D. Real-time-PCR for mSVCT-1 (Slc23a1) and mSVCT-2 (Slc23a2) in the LPS injected and control mouse jejunum tissues were performed using gene-specific primers as described in “Methods”. Data represents the means ± SE of three separate experiments involving three sets of mice (*P < 0.01).

Figure 5. Effect of exposure of Caco-2 cells to LPS on activity of SLC23A1 and SLC23A2 promoters

SLC23A1 (A) and SLC23A2 (B) promoter activities were determined in LPS treated and untreated Caco-2 cells. The relative firefly luciferase activity was normalized by Renilla luciferase activity, and the values are expressed as fold over basic. Data are means ± SE of at least four separate experiments (*P < 0.01).

Figure 6. Effect of LPS on the level of expression of HNF1α and Sp1 protein in Caco-2 cells and native mouse jejunum tissue

A&B. Caco-2 cells were pre-treated with LPS and western blotting was performed using hHNF1α and hSp1 specific antibodies as described in “Methods”. Data are means ± SE of at least three independent experiments (**P < 0.05). C&D. Mouse jejunum tissue total protein was isolated from LPS treated and their control animals, and western blotting was performed using mHNF1α and mSp1specific antibodies as described in “Methods”. Data are means ± SE of at least three independent experiments (*P < 0.01).

Figure 7. Effect of LPS on the nuclear factors HNF1α and Sp1 binding with SLC23A1 (−150 to +5) and SLC23A2 (−97 to +42) promoter regions in Caco-2 cells

The HNF1α (A) and Sp1 (B) binding with SLC23A1 (−150 to +5) and SLC23A2 (−97 to +42) promoter region, respectively were examined in the formaldehyde cross-linked chromatin from LPS treated and control Caco-2 cells followed by immune-precipitation with anti-HNF1α and Sp1 antibodies. The DNA was purified from the immune-precipitated complex and qPCR was performed as described in “Methods”. Data (means ± SE) were normalized relative to input DNA and presented as percentage of relative enrichment (*P < 0.01).