TNF-alpha decreases ABCA1 expression and attenuates HDL cholesterol efflux in the human intestinal cell line Caco-2

Abstract

HDL cholesterol levels are decreased in Crohn's disease, a tumor necrosis factor-alpha (TNF-alpha)-driven chronic inflammatory condition involving the gastrointestinal tract. ATP-binding cassette transporter A1 (ABCA1), one of several liver X receptor (LXR) target genes, is a cell surface transporter that mediates the rate-controlling step in HDL synthesis. The regulation of ABCA1 and HDL cholesterol efflux by TNF-alpha was investigated in the human intestinal cell line Caco-2. In response to cholesterol micelles or T0901317, an LXR nonsterol agonist, TNF-alpha decreased the basolateral efflux of cholesterol to apolipoprotein A1 (apoA1). TNF-alpha, by attenuating ABCA1 promoter activity, markedly decreased ABCA1 gene expression without attenuating the expression of LXR-alpha, LXR-beta, and most other LXR target genes, such as ABCG1, FAS, ABCG8, scavenger receptor-B1 (SR-B1), and apoC1. TNF-alpha also decreased ABCA1 mass by markedly enhancing the rate of ABCA1 degradation and modestly inhibiting its rate of synthesis. Inhibitors of the nuclear factor-kappaB (NF-kappaB) pathway, which is activated by TNF-alpha, partially reverse the effect of TNF-alpha on ABCA1 protein expression. The results suggest that TNF-alpha, the major cytokine implicated in the inflammation of Crohn's disease, decreases HDL cholesterol levels by attenuating the expression of intestinal ABCA1 and cholesterol efflux to apoA1.

Fig. 1.

Effect of TNF-α on cholesterol efflux. Cells were prelabeled for 24 h with [³H] cholesterol as described in “Experimental Procedures.” After thorough washing to remove unincorporated label, they were incubated with either taurocholate micelles containing 250 µM cholesterol or an LXR agonist T0901317 with or without 100 ng/ml of TNF-α added to both the apical and basolateral medium. ApoA1 was also added to the basolateral medium. Following 18-h incubation, the amount of labeled cholesterol recovered in the basolateral medium was estimated as described in “Experimental Procedures.” Values for each treatment represent a mean ± SE of 6 individual wells of a representative experiment from three separate experiments all showing similar results. *P < 0.05 versus control.

Fig. 2.

Effect of TNF-α on ABCA1 gene expression and promoter activity. (A) Cells were incubated for 18 h with micelles containing 250 µM cholesterol or the LXR agonist T0901317 with or without 100 ng/ml of TNF-α. Following incubation, ABCA1 mRNA levels were estimated by quantitative RT-PCR. (B) Cells were transfected with a luciferase-expressing plasmid containing the ABCA1 promoter. The cells were then incubated for 18 h with micelles containing 250 µM cholesterol in the presence or absence of 100 ng/ml of TNF-α, and promoter activity was estimated as described in “Experimental Procedures.” Values for each treatment in (A) represent a mean ± SE of six individual wells of a representative experiment of four separate experiments all showing similar results. Values for each treatment in (B) represent a mean ± SE of six individual wells. *P < 0.05 versus control.

Fig. 3.

Effect of TNF-α on LXR target genes. Cells were incubated for 18 h with micelles containing 250 µM cholesterol or the LXR agonist T0901317 in the presence or absence of 100 ng/ml of TNF-α. Following the incubation, mRNA levels for the respective genes were estimated by quantitative RT-PCR. Values for each treatment represent a mean ± SE of 6–8 individual wells. Similar results were observed for ABCA1, ABCG1, SREBP-1c, FAS, ABCG8, and NPC1 in five other separate experiments. *P < 0.05 versus control.

Fig. 4.

Effect of TNF-α on ABCA1, ABCG1, and NPC1 mass. Cells were incubated for 18 h with micelles containing 250 µM cholesterol or the LXR agonist T0901317 in the presence or absence of 100 ng/ml of TNF-α. Following incubation, ABCA1, ABCG1, and NPC1 mass was estimated by Western immunoblotting as described in “Experimental Procedures.” Values for each treatment represent a mean ± SE of six individual wells of a representative experiment from three separate experiments all showing similar results. *P < 0.05 versus control.

Fig. 5.

Effect of TNF-α on ABCA1 synthesis. Cells were incubated for 18 h with micelles containing 250 µM cholesterol in the presence or absence of 100 ng/ml of TNF-α. Following incubation, cells were pulsed with ³⁵S-methionine for 2, 4, or 6 h. The incorporation of labeled methionine into ABCA1 was estimated as described in “Experimental Procedures.” Values for each treatment represent a mean ± SE of three individual wells of a representative experiment from two separate experiments with a total of 7 individual wells all showing a decrease in the incorporation of label into ABCA1 in cells incubated with TNF-α. *P < 0.05 versus control.

Fig. 6.

Effect of TNF-α on ABCA1 degradation. Cells were incubated for 18 h with micelles containing 250 µM cholesterol in the presence or absence of 100 ng/ml of TNF-α. Following incubation, 100 µM of cycloheximide was added, and the cells were harvested over a 10 h period. ABCA1 mass was estimated by Western immunoblotting. Values for each treatment represent a mean ± SE of four individual wells of a representative experiment from two separate experiments showing similar results. *P < 0.05 versus control.

Fig. 7.

NF- kappa B pathway inhibitors attenuate the inhibitory effect of TNF-α on ABCA1 expression. Cells were incubated for 18 h with micelles containing 250 µM cholesterol in the presence or absence of 100 ng/ml of TNF-α and inhibitors of the NF- kappa B pathway AICAR (0.5 mM) or metformin (1 mM). Following the incubation, ABCA1 mRNA levels and mass were estimated. Values for each treatment represent a mean ± SE of three individual wells. The data were analyzed using one-way ANOVA. *P < 0.05 versus control.