Counter-regulatory effect of sodium butyrate on tumour necrosis factor-alpha (TNF-alpha)-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells - PubMed (original) (raw)

Counter-regulatory effect of sodium butyrate on tumour necrosis factor-alpha (TNF-alpha)-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells

A Andoh et al. Clin Exp Immunol. 1999 Oct.

Abstract

The various biological activities of butyrate have been well documented. In this study, we tested the effects of butyrate on TNF-alpha-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells. The biosynthesis of C3, factor B and IL-8 was evaluated at the protein and mRNA levels. To evaluate transcriptional activation, the nuclear run-on assay was performed. The transcription factor-DNA binding activity was assessed by an electrophoretic gel mobility shift assay (EMSA). In the intestinal epithelial cell lines HT-29, T84 and Caco-2, sodium butyrate enhanced TNF-alpha-induced C3 secretion, but suppressed TNF-alpha-induced factor B and IL-8 secretion. Nuclear run-on assay revealed that transcriptional regulatory mechanisms are involved in the effects of sodium butyrate. The EMSAs indicated that sodium butyrate suppressed TNF-alpha-induced nuclear factor (NF)-kappaB- and activation protein (AP)-1-DNA binding activity, but enhanced TNF-alpha-induced activation of CCAAT/enhancer-binding protein (C/EBP)beta (NF-IL-6)-DNA binding activity. Sodium butyrate induced a counter-regulatory effect on TNF-alpha-induced C3 and factor B biosynthesis in human intestinal epithelial cells. Butyrate action has been discussed with its activity to induce histone hyperacetylation, but its counter-regulatory effect on complement biosynthesis may be closely associated with the modulation of transcription factor activation.

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Figures

Fig. 1

Effects of sodium butyrate on TNF-α-induced C3 (a), factor B (b) and IL-8 (c) secretion in HT-29 cells. Cells were incubated with medium alone, TNF-α (25 ng/ml), or TNF-α (25 ng/ml) plus increasing concentrations of sodium butyrate for 24 h. The amounts of C3, factor B and IL-8 were determined by ELISA. Values were expressed as mean ± s.d. of quadruplicate cultures.

Fig. 2

Effects of sodium butyrate on TNF-α-induced C3 (a) and factor B (b) secretion in T84 and Caco-2 cells. Cells were incubated with medium alone, sodium butyrate 5 m

, TNF-α 25 ng/ml, or sodium butyrate 5 m

plus TNF-α 25 ng/ml for 24 h. The amounts of C3 and factor B were determined by ELISA. Values were expressed as mean ± s.d. of quadruplicate cultures. □, T84; ▪, Caco-2.

Fig. 3

Northern blot analysis of C3, factor B and IL-8 mRNA expression in HT-29 cells. Cells were incubated with medium alone, TNF-α 25 ng/ml, sodium butyrate 5 m

, or TNF-α 25 ng/ml plus sodium butyrate 5 m

for 6 h, and then total cellular RNA was extracted. Lane 1, medium alone; lane 2, TNF-α; lane 3, sodium butyrate 5 m

; lane 4, TNF-α 25 ng/ml plus sodium butyrate 5 m

Fig. 4

Nuclear run-on assays of C3, IL-8 and factor B gene transcription in HT-29 cells. Confluent HT-29 monolayers were stimulated with TNF-α 25 ng/ml or TNF-α 25 ng/ml plus sodium butyrate 5 m

for 3 h, and then nuclei were prepared. Run-on transcription was performed in 5.0 × 106 nuclei in the presence of α-32P-uridine triphosphate for 60 min, and nuclear RNA was isolated and hybridized to filter-bound DNA probe. Plasmid, a 2.9-kb empty plasmid (pAT153/Pvu) isolated from human C3 cDNA clone pHLC3.11.

Fig. 5

Electrophoretic gel mobility shift assays (EMSAs) for NF-κB (a) and AP-1 (b) DNA binding activities. HT-29 cells were incubate with medium alone, sodium butyrate 5 m

, TNF-α 25 ng/ml, or sodium butyrate 5 m

plus TNF-α 25 ng/ml for 1.5 h, and then nuclear extracts were prepared. (a) Lane 1, medium alone; lane 2, sodium butyrate; lane 3, TNF-α; lane 4, sodium butyrate plus TNF-α; lane 5, TNF-α plus cold probe; lane 6, TNF-α plus anti-p50 antibody; lane 7, TNF-α plus anti-p65 antibody. (b) Lane 1, medium alone; lane 2, sodium butyrate; lane 3, TNF-α; lane 4, sodium butyrate plus TNF-α; lane 5, TNF-α plus cold probe; lane 6, TNF-α plus anti-c-jun antibody. Thin arrow on the left indicates specific band, and thick arrow on the right indicates supershift band.

Fig. 6

Electrophoretic gel mobility shift assays (EMSAs) for C/EBP (a) and Sp1 (b) DNA binding activities. HT-29 cells were incubate with medium alone, sodium butyrate 5 m

, TNF-α 25 ng/ml, or sodium butyrate 5 m

plus TNF-α 25 ng/ml for 1.5 h, and then nuclear extracts were prepared. (a) Lane 1, medium alone; lane 2, sodium butyrate; lane 3, TNF-α; lane 4, sodium butyrate plus TNF-α; lane 5, TNF-α plus cold probe; lane 6, TNF-α plus anti-C/EBP α antibody; lane 7, TNF-α plus anti-C/EBPβ (NF-IL-6) antibody; lane 8, TNF-α plus anti-C/EBP δ antibody. (b) Lane 1, medium alone; lane 2, sodium butyrate; lane 3, TNF-α; lane 4, sodium butyrate plus TNF-α; lane 5, TNF-α plus cold probe; lane 6, TNF-α sample plus anti-Sp1 antibody. Thin arrow on the left indicates specific band, and thick arrow on the right indicates supershift band.

References

1. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol. 1992:411–52. - PubMed
1. Murch SH, Lamkin VA, Savagr MO, et al. Serum concentrations of tumor necrosis factor-α in childhood chronic inflammatory bowel disease. Gut. 1991;32:913–6. - PMC - PubMed
1. Braegger CP, Nicholls SW, Murch SH, et al. Tumor necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet. 1992;339:89–91. - PubMed
1. Breese EJ, Michte CA, Nicholls SW, et al. Tumor necrosis factor α-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology. 1994;106:1455–66. - PubMed
1. Morgan BP. The complement system. In: Morgan BP, editor. Complement: clinical aspects and relevance to disease. London: Academic Press; 1990. pp. 1–35.

Counter-regulatory effect of sodium butyrate on tumour necrosis factor-alpha (TNF-alpha)-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells - PubMed (original) (raw)