The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation - PubMed (original) (raw)

The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation

Amrita Kumar et al. J Immunol. 2009.

Abstract

The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination, a process catalyzed by the E3-SCF(beta-TrCP) ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-kappaB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3- SCF(beta-TrCP) ubiquitin ligase, the NF-kappaB inhibitor IkappaB-alpha. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.

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The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

Bacterial fermentation products at physiological pH and concentration cause loss of Cul-1 neddylation. A, Immunoblot of Cul-1 and NEDD8 from T84 model epithelia treated for up to 90 min with physiological concentrations of butyrate. B, Immunoblot of Cul-1 from HeLa cell lines (top panel) and HL-60 differentiated into macrophage-like cells treated for up to 60 min with physiological concentrations of butyrate. C, Immunoblot of Cul-1 from T84 model epithelia treated for up to 1 h with an effective dose of butyrate, washed, and incubated for 30 min and 1 h longer in HBSS+. D, Immunoblot of Cul-1 from T84 epithelial cells treated with HBSS+ supplemented with butyrate and other organic acids as indicated for 1 h. Decreasing acid concentration is marked (open wedge; 0–14 mM) and has an inverse relationship to pH (solid wedge; pH 4.0–7.5). E, Immunoblot of Cul-1 from T84 model epithelia treated with HBSS+ or HBSS+ supplemented with butyrate for 1 h at the indicated concentrations. The effective concentration of HBSS+ or HBSS+ supplemented with butyrate was buffered to the pH as indicated below each panel. F, Immunoblot of Cul-1 from 3-mm human distal colonic mucosal punches treated for 1 h in HBSS+ supplemented with butyrate.

FIGURE 2

FIGURE 2

Loss of Cul-1 neddylation results in inhibition of TNF-_α_-induced p65 nuclear translocation and I_κ_B-_α_ubiquitination and degradation. The effective concentration of TNF-α and purified flagellin used for these experiments are 10 and 100 ng/ml, respectively. A, Butyrate prevents TNF-_κ_-induced p65 nuclear translocation. Confocal image of cytoplasmic vs nuclear immunofluorescent staining of p65 (Rockland) in HeLa cells treated for 1 h with HBSS+ supplemented with butyrate or lactate at the indicated concentrations and challenged with TNF-α for 20 min. B, Butyrate inhibits activity of a NF-_κ_B-luciferase reporter. Lysates from HeLa cells transfected with a NF-_κ_B-luciferase reporter plasmid and treated in the presence of butyrate with TNF-α for 5 h were assayed for luciferase activity. Data represent the mean of three independent assays and are shown as percent TNF-_α_-induced NF-_κ_B-Luc. C, Butyrate does not attenuate polyubiquitination of proteins. The level of protein ubiquitination in cellular extracts of Caco-2 cells pretreated with 2.5 mM butyrate for 1 h before addition of MG-262 for additional time was determined by immunoblotting with anti-ubiquitin conjugate Ab (Affinity Research). D, Immunoblots of whole cell extracts with anti-total-I_κ_B-α Ab from Caco-2 cells pretreated with the proteasomal inhibitor MG-262 (500 ng/ml) or HBSS+ for 40 min before a 1-h incubation with 5 mM butyrate and subsequent TNF-α (T; 10 ng/ml) or flagellin (F; 100 ng/ml) challenge for 5 or 15 min. Ubiquitinated I_κ_B-α species are marked. Equal loading was confirmed by _β_-tubulin immunoblots. E, Immunoblots of whole cell extracts with anti-phospho-I_κ_B-α Ab from Caco-2 cells pretreated with the proteasomal inhibitor MG-262 (500 ng/ml) for 40 min before a 1-h incubation with 5 mM butyrate and subsequent TNF-α (T; 10 ng/ml) or flagellin (F; 100 ng/ml) challenge for 5 or 15 min.

FIGURE 3

FIGURE 3

Butyrate induces generation of epithelial ROS and causes redox changes. IEC-6 cells were treated with HBSS with or without 10 mM butyrate, washed, and incubated with 5 _μ_M of DCF (A), 10 _μ_M DHE (B), and 10 _μ_M Mito-Sox Red (C). Fluorescence was captured by confocal laser-scanning microscopy (Zeiss). D, Kinetics of butyrate-induced ROS generation in epithelial cells. Confluent Caco-2 cells were preloaded with 100 _μ_M DCF and then ROS production was measured upon colonization with Lactobacillus (MOI = 1) or treatment with 10 mM butyrate. Data represent the mean of three independent assays and are shown as percent induction of ROS upon untreated controls. E and F, Butyrate cause oxidation of Trx1 and Trx2 pools. Confluent Caco2 cells were treated with PBS with or without butyrate as indicated over a time course. Graphs show the redox potential (Eh) for Trx1 and Trx2, respectively. Data are represented as means ± SE.

FIGURE 4

FIGURE 4

Butyrate-mediated oxidation of Ubc12. A, Butyrate inhibits formation of the NEDD8~Ubc12 thiolester bond of endogenous Ubc12. Western blot analysis with Ubc12-specific antisera of whole cell protein extracted from HeLa cells treated with butyrate for 1 h at the indicated dose and lysed in SDS lysis buffer without (− DTT) or with (+DTT) reducing agents. The asterisk marks the 30-kDa DTT-sensitive NEDD8~Ubc12 thiolester form and the arrow indicates the oxidized forms of Ubc12. B, NAC and DPI prevents butyrate-mediated loss of Cul-1 neddylation. HeLa cells pretreated with NAC or DPI for 60 min and subsequently treated with 5 mM butyrate for 1 h were analyzed by immunoblotting with anti-Cul-1 Ab.

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