Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC 2010 May 18.
Published in final edited form as:
PMCID: PMC2872164

The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1


The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-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-κB 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-SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. 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.

Virtually unique among mammalian cell types, the intestinal epithelium coexists in intimate contact with a normal flora of ~1012 prokaryotic organisms. The greatest numbers and diversity are in the cecum and ascending colon where bacterial density can reach 1011 cells/g of contents, yielding a biomass of >1 kg. This bacterial community of ~500–1000 species has diverse beneficial roles including vitamin synthesis, bile salt metabolism, and degradation of complex carbohydrates (1). Furthermore, through studies with germfree or gnotobiotic mice, it is also now established that the enteric flora can fundamentally affect epithelial gene transcription, ultimately affecting enterocyte proliferation and homeostasis (2, 3).

The normal flora thrives in a largely anaerobic environment, generating energy by the fermentation of luminal complex carbohydrates. The end products of fermentation are a spectrum of organic acids, including short chain fatty acids (SCFAs)3 such as butyrate, succinate, and propionate, as well as other terminal products such as lactate (4, 5). These organic compounds are an important energy source for the colonic epithelium and may influence various aspects of gut physiology. For example, butyrate and other SCFAs have well-known differentiating and growth-promoting activities in vitro and in vivo, a biological effect ascribed to histone deacetylase activity (6). Additionally, SCFAs have been noted to have immunomodulatory effects on colonic inflammation, suppressing inflammatory cytokine secretion in cultured epithelial cells, and ameliorating model colitis in mice, suggesting that these molecules contribute to the ability of the mucosa to tolerate the presence of vast quantities of living microorganisms and associated microbial-associated molecular patterns (MAMPs) (6, 7). Furthermore, luminal instillation of butyrate has been shown to be a promising experimental therapy in ulcerative colitis and related inflammatory disorders (8, 9). How these naturally occurring bacterial metabolic products influence epithelial homeostasis are unknown and are a topic of considerable research interest.

A key pathway that modulates inflammatory and/or growth survival in the intestine is the NF-κB or Rel signaling pathway. The NF-κB pathway is controlled by regulated degradation of a physically associated inhibitor molecule, IκB-α, which when phosphorylated is ubiquitinated by a specific ubiquitin ligase complex designated E3-SCFβ-TrCP. Ubiquitinated IκB-α is targeted for degradation by the proteasome (10, 11). E3-SCFβ-TrCP and other E3-SCF complexes are themselves regulated by transient covalent modifications. The ubiquitin paralog NEDD8 must be conjugated to the Cullin-1 (Cul-1) subunit of the E3-SCF complex for ubiquitin ligase activity (1215). NEDD8 modification of Cul-1 has been demonstrated to be necessary for ubiquitination of IκB-α and p100/p105, and for the subsequent activation of NF-κB in mammalian cells (1620).

We have demonstrated that viable commensal bacteria can block IκB-α ubiquitination by blockade of neddylation of the Cul-1 subunit of E3-SCFβ-TrCP, accounting for the attenuation of NF-κB activation (21). We further reported that loss of Cul-1 neddylation was mediated by rapid and reversible oxidative inactivation of Ubc12, the ubiquitin-like conjugating enzyme responsible for the neddylation of Cullin subunits (22). Herein, we demonstrate that butyrate and other SCFAs supplemented to model epithelia in vitro and human tissue ex vivo also cause loss of neddylated Cul-1 resulting in consequent inhibition of the NF-κB pathway. We also show that de novo and transient reactive oxygen species (ROS) generation mediate these regulatory effects of butyrate. These results suggest that metabolic products from the normal intestinal flora (and potentially other complex microbial communities) can influence mammalian signaling pathways by modulating the activation of a rate-limiting enzymatic step.

Materials and Methods


Butyric, lactic, propionic, and succinic acid were obtained from Sigma-Aldrich. MG-262 was obtained from Affinity. TNF-α was from R&D Systems. Flagellin was purified as described previously (23). N-acetyl cysteine (NAC) and diphenyleneiodonium (DPI) were purchased from Sigma-Aldrich.

Cell culture

Confluent monolayers of T84 cells were grown in a polarized fashion on permeable supports as previously described (24). HeLa, Caco-2, and IEC-6 epithelial cells were grown in standard tissue culture vessels and maintained as recommended by the American Type Culture Collection. Human promyelocytic leukemia cells (HL-60) were maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS and 100 U/ml penicillin and 100 U/ml streptomycin. The cultures were maintained in humidified 95% air, 5% CO2, at 37°C by passage of 2 × 105 cells/ml every other day. Differentiation was induced by treatment with PMA (20 ng/ml) for 48 h and the extent of differentiation was measured by attachment of cells to the substratum.

Human biopsy material

Healthy human colonic mucosa was obtained from the outer borders of fresh distal colonic specimens resected for neoplastic disease. Mucosa was dissected free from the underlying gut wall and 3-mm punches were incubated for 1 h in HBSS+ supplemented with butyrate at the indicated concentration. Tissue was immediately lysed in denaturing SDS-PAGE buffer.

Transient transfections and luciferase assays

HeLa cells were transiently transfected using Lipofectamine 2000 (Invitrogen) or FuGENE 6 (Roche) according to the manufacturer’s instructions. Briefly, plasmid DNA was mixed with serum-free DMEM and transfection reagent. After 15 min, the mixture was added in a dropwise fashion to the growth medium of ~70–90% confluent adherent cells in 24- or 6-well plates or 100-mm dishes. The cells were allowed 16–24 h for transfection and expression of transfected genes. For luciferase reporter assays, cells were transfected with pNF-κB-Luc plasmid (Stratagene), allowed 18 h for expression, treated with TNF-α (20 ng/ml) for 6 h, and lysed according to the manufacturer’s protocol. All transfections were balanced to contain 200 ng of DNA with empty vector. Luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega).

Western blot analysis

Following experimental treatment, epithelial cells were washed in cold HBSS+ and whole cell extracts were prepared by rapid lysis in denaturing SDS-PAGE buffer. Cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose using standard protocols. Immunoreactive proteins were detected with Abs to IκB-α (Santa Cruz Biotechnology), phospho-IκB-α (Cell Signaling), Cul-1 (Zymed), NEDD8 (Zymed), Ubc12 (Rockland), β-tubulin (Sigma-Aldrich), or β-actin (Sigma-Aldrich) using the enhanced chemiluminescent protocol (ECL; Amersham) and a HRP-conjugated secondary Ab. Blots were exposed to film for 1 s to 30 min.

Immunofluorescence studies

Immunofluorescent labeling of p65 in adherent HeLa cells grown on 12-mm glass coverslips was performed as follows: cells were fixed for 20 min in 3.7% paraformaldehyde in PBS, washed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed again. Fixed samples were incubated in blocking solution (5% normal goat serum in PBS) overnight at 4°C. A 1-h incubation with each Ab diluted in blocking buffer followed: 1/500 rabbit anti-p65 (Rockland); 1/200 fluorescein (FITC)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Cells were washed three times between each Ab. The coverslips were mounted on glass slides and stained cells were observed by laser confocal epifluorescence microscopy (Zeiss).

Monitoring ROS generation

For monitoring ROS generation, IEC-6 cell cultures were plated onto 24-well plates and treated with butyrate for the duration indicated. Following treatment, the cells were further incubated with 5 μM each of the fluorescent ROS indicators 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (DCF) (Molecular Probes) or dihydroethidium (DHE) for 15 min. After loading the ROS dyes, cultures were washed three times with warm HBSS+ and the fluorescence intensity was acquired using a confocal laser-scanning microscope (Zeiss LSM 510). For DCF imaging, images were captured with a 10× objective using an excitation wavelength of 488 nm and an emission wavelength of 513 nm. For DHE, images were captured with a 40× objective using an excitation wavelength of 490 nm and an emission wavelength of 610 nm. For determination of ROS generation in mitochondria, we used the mitochondrial superoxide dye indicator MitoSOX Red (Molecular Probes). Cells were treated with butyrate for the times indicated, after which they were loaded with 5 μM MitoSOX Red in HBSS+ for 10 min at 37°C. Cells were washed with warm HBSS+ and fluorescent images were captured with a 63× objective using an excitation wavelength of 510 nm and an emission wavelength of 580 nm.

Kinetic measurements of ROS

ROS generation was measured with DCF as described by Wang et al. (25), with the following modification. Caco-2 cells grown to confluence on a 24-well plate were incubated in DMEM supplemented with 0.4% serum and containing 1 mM DCF dye. After incubation for 2 h in 5% CO2 at 37°C, the cells were washed and treated with Lactobacillus rhamnosus GG (multiplicity of infection (MOI) = 1) or with butyrate (10 mM) in Krebs-Ringer-HEPES buffer. An increase in fluorescence units was measured by using a fluorescence microplate reader (SpectraMax M2; Molecular Devices) after various time intervals with excitation at 475 nm and emission at 525 nm.

Examination of thioredoxin1 and thioredoxin 2 redox states

For Thioredoxin (Trx) 1 (BD Biosciences) analysis, Caco-2 cells treated with butyrate were washed with cold 1×PBS and whole cell lysates were prepared in G-lysis buffer (6 M guanidine-HCl, 50 mM Tris (pH 8.3), 3 mM EDTA, 50 mM iodoacetamide (IAA)) as described in (26). Briefly, lysates were incubated for 30 min at 37°C, after which excess IAA was removed using a G-25 spin column (GE Healthcare). The samples were electrophoresed on a native gel, transferred to nitrocellulose membrane, probed with mouse anti-human Trx1 (BD Biosciences), and detected with an Alexa Fluor 680 anti-mouse secondary Ab. Trx2 redox states were analyzed as described in a previous report (26). Following treatment with butyrate, total cell protein was precipitated with 10% trichloroacetic acid. Samples were centrifuged, washed with acetone, and resuspended in 20 mM Tris (pH 8.0) buffer containing 15 mM 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid Molecular Probes)]. Samples were incubated for 30 min at room temperature. Oxidized and reduced forms of Trx2 were separated by nonreducing SDS-PAGE. Trx2 was detected using rabbit anti-human Trx2 Ab and Alexa Fluor 680 anti-rabbit secondary Ab. Bands corresponding to Trx1 and Trx2 were visualized using an Odyssey scanner (LI-COR) and band densometric values were applied to the Nernst equation as described elsewhere (27).


Butyrate mediates loss of Cul-1 neddylation

We had previously shown that colonization of epithelial cells with viable commensal bacterial strains in vitro and in vivo caused a rapid and reversible loss of epithelial Cul-1 neddylation (21). During the course of these experiments, we observed a reduction of Cul-1 neddylation if bacteria were physically separated from the epithelial cells by use of Transwell inserts mounted with a permeable membrane support (data not shown). This indicated that the bacterial effects, at least in part, were mediated by a small diffusible molecule. To identify this soluble signal, we analyzed bacterially conditioned supernatants by gas chromatography/mass spectroscopy, among other approaches. These studies revealed the presence of significant amounts of lactate in culture supernatants. Consistently, the pH of supernatants after a minimum of 30 min of coculture was 4.5– 6.0, indicating the microbial production of acidic compounds. It is well known that the fermentation products of the intestinal flora include lactate, as well as SCFAs such as butyrate (C4), succinate (C4), and propionate (C3) (4, 5). Furthermore, butyrate mediates well-known effects on inflammation, including repression of NF-κB in vivo and in vitro (8, 9, 28, 29). These observations suggested the possibility that the loss of neddylated Cul-1, at least partially, resulted from the exposure of the cells to bacterial fermentation products. When fully polarized T84 epithelial cells were treated with 5 mM concentrations of butyrate, loss in Cul-1 neddylation was observed within 15 min (Fig. 1A). Typically Cul-1 appears as a doublet of 85 and 90 kDa, with the higher molecular band representing the neddylated Cul-1. When an identical blot was probed with anti-NEDD8 Ab, the upper band reacted with the Ab, confirming the identity of this band as neddylated Cul-1 (Fig. 1A, bottom panel). Similar kinetics of butyrate-mediated Cul-1 deneddylation was also observed in HeLa cells (Fig. 1B, top panel) and in human promyelocytic leukemia cells (HL-60) differentiated to form macrophage-like cells (Fig. 1B, bottom panel). Interestingly, deneddylation of Cul-1 observed upon treatment with butyrate could be reversed if the butyrate was washed off and the cells allowed to recover in HBSS+ for another 30–60 min (Fig. 1C). The kinetics of reversibility of Cul-1 neddylation was similar as we observed with viable bacteria (21). Furthermore, loss in Cul-1 neddylation within 1 h was also observed when T84 cells were cultured in HBSS+ supplemented with at least 3 mM concentrations of other organic acids, such as lactate, propionate, and succinate (Fig. 1D). In all of these in vitro conditions, the molarity of the organic acids was within the range reported for the physiological gut (8, 30).

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 ...

In T84 cells, butyrate at a concentration of ~3–5 mM (resulting in a pH of ~4.5–5.0) was effective in causing loss of Cul-1 neddylation (Fig. 1E, top panel). Interestingly, when effective concentrations of butyrate were buffered to neutral pH, the ability of this agent to affect Cul-1 neddylation was lost (Fig. 1E, middle panel). To rule out artifacts caused by acidic pH conditions, T84 cells were similarly treated with HBSS+ supplemented with hydrochloric acid to pHs at which butyrate could induce loss of Cul-1 neddylation (Fig. 1E, lower panel). These conditions had no effect on Cul-1 neddylation, indicating that although the inhibitory effect of butyrate was pH dependent, it was not a function of pH alone, likely because butyrate at pHs approaching its pKa (4.5) becomes protonated and is freely diffusible across cellular membranes. Thus, these data suggest that the butyrate effects observed likely do not require a receptor or active transport.

To determine whether the presence of SCFAs could influence Cul-1 neddylation in intact mucosa, we similarly treated human colonic mucosal segments ex vivo in medium supplemented with butyrate and assayed Cul-1 neddylation by immunoblot (Fig. 1F). Reduction of Cul-1 neddylation was observed at concentrations of 1.5–3 mM butyrate. The lower concentrations required in this experiment presumably reflect the differences between intact, fully differentiated mucosa and cultured cells. Thus, by supplementing medium with SCFAs, we were able to recapitulate the effects of viable bacteria on the neddylation of Cul-1.

Butyrate represses NF-κB activation by blockade of IκB-α ubiquitination

Butyrate is known to be a potent inflammatory modulator in vivo; it has been shown to block up-regulation of proinflammatory mediators (i.e., IL-8) and NF-κB activation in stimulated cultured intestinal epithelial cells (31). We thus undertook a mechanistic analysis of NF-κB signaling to determine whether SCFA effects on Cul-1 neddylation affected the NF-κB pathway. As shown in Fig. 2A, in cells pretreated with 3 mM butyrate at pH 5.6 or with lactate at pH 5.0, we observed inhibition of p65 translocation, as evaluated by immunofluorescence staining of the normal TNF-α induced cytoplasmic to nuclear translocation, consistent with effects observed with intestinal epithelial cell colonization with live bacteria (32). Furthermore, as seen in Fig. 2A, this butyrate-mediated block in p65 translocation was abrogated if butyrate was buffered to pH 7.4 or with lactate at pH 6.0, similar to the observed effects of pH on neddylation of Cul-1 in Fig. 1D. This blockade was functionally relevant because the same or lower concentration of butyrate inhibited activation of a NF-κB- dependent reporter gene in a transient transfection assay (Fig. 2B). Thus, butyrate pretreatment of cultured cells inhibited activation on NF-κB, consistent with previous reports of butyrate-mediated effects on inflammatory cytokine induction.

Loss of Cul-1 neddylation results in inhibition of TNF-α-induced p65 nuclear translocation and IκB-αubiquitination and degradation. The effective concentration of TNF-α and purified flagellin used for these experiments ...

Activation of the NF-κB pathway is tightly regulated by the phosphorylation, ubiquitination, and proteolysis of its physically associated inhibitor molecule, IκB-α. Yin et al. (29) reported that prolonged exposure (24 h) of HT29 cells to butyrate suppressed the NF-κB activation via attenuation of the cellular proteasome activity. To determine whether short-term exposure (as used in our study) of intestinal epithelial cells to butyrate inhibited cellular proteasome activity, we treated intestinal epithelial Caco-2 cells with butyrate and monitored cellular proteasome activity by observing the accumulation of polyubiquitin-conjugated proteins. Pretreatment of Caco-2 cells with 2.5 mM butyrate for 1 h had no gross effects on accumulation of polyubiquitinated proteins (Fig. 2C, compare lanes 1 and 5). We also monitored the accumulation of the ubiquitinated proteins in the presence of MG-262, an inhibitor of the 26S proteasome. In cells pretreated with butyrate and further incubated with MG-262 for several hours, we saw no gross differences in accumulation of polyubiquitinated proteins (Fig. 2C, compare lanes 2– 4 to 6–8, respectively). These data indicate that within the time frame used in this study butyrate does not suppress accumulation of cellular polyubiquitinated proteins.

Next, to determine whether butyrate-mediated inhibition of NF-κB was related to ubiquitination of IκB-α, a process controlled by Cul-1 neddylation, we undertook an analysis of IκB-α ubiquitination. Pretreatment of cells with butyrate before stimulation with proinflammatory inducers such as TNF-α and flagellin resulted in stabilization of IκB-α in contrast to near to complete degradation observed with TNF-α and flagellin, respectively (Fig. 2D, top panel). To allow further study of phosphorylated and ubiquitinated IκB-α adducts, we pretreated our model epithelia with MG-262. Under these conditions, degradation of IκB-α in response to TNF-α and flagellin was attenuated (Fig. 2D, bottom panel). Furthermore, very high molecular bands consistent with polyubiquitinated IκB-α was observed in cells stimulated with TNF-α and flagellin, as previously reported (33) (Fig. 2D, bottom panel). However, these higher molecular mass species representing polyubiquitinated IκB-α were absent in cell lysates derived from butyrate-treated cells (Fig. 2D, bottom panel). Since ubiquitination of IκB-α is dependent upon phosphorylation of IκB-α on two serine residues (Ser32 and 36), we also examined the phosphorylation of these residues with a phospho-specific Ab. Again, when we pretreated the cells with MG-262 before induction with TNF-α or flagellin, we observed an increase in high-molecular mass (HMM) ubiquitinated species that was attenuated in the presence of butyrate (Fig. 2E). Importantly, phosphorylated IκB-α (denoted with asterisks in Fig. 2E) was abundant in control and enhanced in butyrate-treated cell lysates (Fig. 2E). These results are consistent with the bacterial mediated inhibition of NF-κB previously described (21, 33). Collectively, these data indicate that butyrate prevents IκB-α degradation by blocking ubiquitination of phospho-IκB-α.

Butyrate causes generation of ROS and induces redox changes in cultured epithelial cells

Neddylation of Cul-1, required for activity of the E3-SCFβ-TrCP ubiquitin ligase, is dependent on sequential transfer of thiolester NEDD8 from the NEDD8-charging enzyme Uba3-APP/BP-1 to a catalytically active cysteine residue of the NEDD8-conjugating enzyme Ubc12. Ubc12 then catalyzes the formation of an isopeptide bond between the C-terminal glycine residue of NEDD8 and a target lysine (Lys720) residue present on Cul-1 (34). We have previously demonstrated that viable commensal bacteria cause transient oxidative inactivation of Ubc12 with resultant effects on neddylation of Cul-1 (22). Given that butyrate treatment resulted in potent Cul-1 deneddylation, we asked whether SCFAs were also stimulating ROS generation with resultant oxidation of Ubc12. To monitor ROS generation, IEC-6 intestinal epithelial cells were cultured with physiological concentrations of butyrate and monitored for changes in DCF fluorescence, a dye sensitive to ROS, especially H2O2. An increase in fluorescence of DCF was observed within 15 min of treatment with 10 mM butyrate (Fig. 3, A and D) as compared with untreated controls. An increase in ROS was also confirmed with the use of an alternative ROS sensitive dye, DHE (Fig. 3B). DHE is freely permeable and is oxidized to fluorescent ethidium bromide by superoxides. We have previously shown that commensal bacteria elicited a rapid and transient increase in ROS within 5–30 min of contact with intestinal epithelial cells (22). In comparison to treatment with L. rhamnosus at MOI = 1, butyrate stimulated ROS with a slower kinetics that occurred over a period of 30 – 60 min after contact with intestinal epithelial cells (Fig. 3D). To evaluate the sources of butyrate-mediated ROS generation, we used a mitochondria-specific ROS fluorescent dye, MitoSOX Red, that specifically detects superoxide generation in mitochondria. Using this dye, we observed mitochondrial ROS generation within 15 min of treatment with butyrate, suggesting the mitochondria as a potential source of induced ROS (Fig. 3C).

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 μM of DCF (A), 10 μM DHE (B), and 10 μM Mito-Sox Red (C). Fluorescence ...

Accumulation of intracellular levels of ROS in cells causes changes in the oxidation profiles of the antioxidants. To evaluate changes in such intracellular redox pools, we assayed the thiol/disulfide redox states of thioredoxins, a major thiol-dependent antioxidant system of cells. Steady-state Eh values for cytosolic Trx1 (Fig. 3E) and mitochondrial Trx2 (Fig. 3F) were calculated using the Nernst equation and show that treatment of cells with butyrate resulted in oxidation of both Trx1 and Trx2 over a 1-h time course. For Trx1, the Eh increased from –283 mV in untreated cells to−265 mV within 30 min in cells treated with 10 mM butyrate. For Trx2, the Eh increased from −359 mV to −336 mV within 15 min with 10 mM butyrate treatment. These cumulative data indicated that butyrate treatment induced ROS in cultured epithelial cells predominantly from mitochondrial sources and induced compensatory changes in the intracellular redox balance, albeit with slower kinetics and to a lesser degree as ROS induced by viable commensal bacteria.

Butyrate-induced Ubc12 oxidation

We next sought to examine whether butyrate-mediated ROS signals were being transduced via transient oxidation of Ubc12. To evaluate the effects of butyrate on endogenous Ubc12, HeLa cells were treated with 5 and 10 mM butyrate and cell lysates were analyzed under reducing and nonreducing conditions (absence of DTT) to allow visualization of thiol modifications as previously described (22). When cells were treated with 5 and 10 mM butyrate, we observed a loss of the 30-kDa NEDD8~Ubc12 thiolester form when cell lysates were prepared and analyzed under nonreducing conditions (Fig. 4A, top panel, denoted with an asterisk). Significantly, this loss of NEDD8~Ubc12 thiolester form corresponded with the appearance of a marked dose-dependent shift in the electrophoretic mobility of Ubc12 to HMM-oxidized species (Fig. 4A, top panel, denoted with an arrow). We have previously confirmed the identity of the NEDD8~Ubc12 thiolester form in a similar immunoblot probed with anti-NEDD8 Ab under nonreducing conditions (22). In cell lysates prepared and analyzed under standard reducing conditions (with DTT), the HMM-oxidized species were abolished (Fig. 4A, bottom panel), indicating that these forms of the enzymes are mixed disulfides between Ubc12 and HMM proteins. Furthermore, in cell lysates analyzed under reducing conditions, we did not observe the 30-kDa NEDD8~Ubc12 thiolester form, further confirming the identity of this form of Ubc12 as being neddylated.

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 ...

Finally, to link the observed induction of intracellular ROS with deneddylation of Cul-1, HeLa cells were treated with butyrate in the presence of a ROS scavenger NAC and a flavoprotein inhibitor DPI. As shown in Fig. 4B, pretreatment of cells with NAC or DPI attenuated butyrate-mediated deneddylation of Cul-1. Collectively, these data demonstrate that butyrate-mediated ROS generation causes attenuation of Ubc12 activity in the same manner as of live bacteria.


Herein, we have described an important eukaryotic regulatory node, namely, ubiquitin-mediated degradation by E3-SCF ubiquitin ligases, that is influenced by the product of a complex prokaryotic community. Active E3-SCF ubiquitin ligases are required for the regulation of the NF-κB, β-catenin, Snail, Twist, and Hedgehog pathways and presumably others (35). The activity of the E3-SCF ubiquitin ligases is modulated by neddylation of its Cul-1 subunit (17). Neddylation is a posttranslation modification specific to substrates of the Cullin family in which NEDD8, a small 8-kDa ubiquitin-like protein, is attached to the substrates and is emerging as a key regulatory event in cellular processes that are controlled by ubiquitin-mediated degradation of proteins. In a variety of experimental models, including yeast (36), Arabidopsis (37), Caenorhabditis elegans (38), Drosophila (39), mice (40), and human cells in vitro (41), mutations leading to loss-of-function of Cul-1 neddylation (or deneddylation) status can have profound functional consequences. However, only limited evidence is available implicating environmental signals in the physiological regulation of Cul-1 neddylation. For example, yeast Cullin was shown to be hyperneddylated in response to UV exposure (42). In Arabidopsis studies, wild-type plants showed marked Cul-1 deneddylation when reared in darkness, while mutant plants that fail to respond to light were shown to have an abnormal accumulation of neddylated Cul-1 (43). Furthermore, our previous results demonstrate that interactions of commensal bacteria with mammalian intestinal epithelia modulate Cul-1 neddylation (21) along with exogenous H2O2 (22), adenosine (44), and, in this report, bacterial fermentation products. We found commensal bacteria were able to mediate effects on Cul-1 neddylation via soluble bacterial fermentation products. Such SCFAs, particularly butyrate, have long been known to provide a nutritive source for the mammalian gut. Moreover, butyrate has well-known immunomodulatory and trophic effects on gut homeostasis (6). In this sense, these compounds possess the ability to act as effectors or signaling intermediates in the symbiotic cross-talk between the mammalian host and the normal flora. Unlike other intermediates in the dialog between host and microbe, such as eukaryotic pattern recognition receptors and bacterial MAMPs, butyrate-mediated signaling occurs via largely unknown processes.

Recent years have seen the establishment of the paradigm stating that inducible and “deliberate” generation of ROS have a wide variety of physiological signaling functions. Several eukaryotic signaling proteins, including growth factors, hormones, and cytokines, are capable of stimulating ROS generation in various cell types including epithelial cells (4549). Physiological generation of these molecules can derive from mitochondrial sources, dedicated enzymes homologous to the phagocytic NADPH oxidase, or by 5′-lipoxygenase (50, 51). ROS are short-lived molecules and can exhibit exquisite microspatial localization within cells, allowing specific targeting of signaling effects (51). ROS signaling can be transduced by an increasingly recognized subset of enzymes that can be transiently inactivated by reversible oxidation of catalytic cysteine residues within the active sites (52, 53). Such enzymes include a variety of tyrosine phosphatases such as PTEN (54), the DUSP family of MAPK phosphatases (45), antioxidants such as thioredoxins and peroxiredoxins (55), and, as we and others have shown, members of the Ubc family of proteins (22, 56).

In the gut, sources of ROS could be exogenous, deriving from the metabolic processes of certain bacteria themselves or from infiltrating neutrophils during an acute inflammatory event. Alternatively, we have found ROS generated within epithelial cells stimulated by exogenous agents such as commensal bacteria (22), soluble fermentation products (this report), and sterile components of bacterial cell walls (N-K. Young and A. S. Neish, unpublished data). This is likely a highly conserved process, since ROS generation as a response to environmental stressors, including bacteria, is present in plants and lower metazoans (57). ROS generation by butyrate has been reported, although with far more delayed kinetics. Stimulation of ROS generation has been observed upon exposure of human tongue cancer cells to 8 and 16 mM butyrate for a period of 4 –24 h (58, 59). Of note, although in our hands ROS generation elicited by butyrate was at least as strong as ROS induced by known ROS-dependant agonists such as insulin, epidermal growth factor, flagellin, or platelet-derived growth factor (A. Kumar, M.-K. Young, and A. S. Neish, unpublished results), butyrate-induced ROS was markedly less than the amounts detected after cells were stimulated with viable bacteria and occurred with noticeably slower kinetics. As suggested by our data, butyrate may induce ROS via the mitochondrial pathway rather than by receptor-mediated events.

The physiochemical environment of the intestinal lumen in immediate contact with the epithelium is largely anaerobic and is suffused with not just with a complex ecosystem of living bacteria, but also a miasma of MAMPs, small bacterially produced compounds, and a spectrum of metabolic by-products that varies markedly over the greater than 1 m length of the organ (4, 5, 8). The ascending colon contains a highly fermentative, “saccharolytic” bacterial milieu which produce high concentrations (up to 140 mM for butyrate) of organic acids including lactate, acetate, and SCFAs (succinate, propionate, butyrate), resulting in a physiological luminal pH of 5.5– 6.5, as measured by a series of radiotelemetry studies in healthy human volunteers (reviewed in Ref. 30). As the carbohydrate-fermentative substrates are consumed through β oxidation and bicarbonate is secreted by the epithelia, the luminal environment shows a progressive decline in SCFA concentration (to 40 mM for butyrate) and a corresponding rise in pH, which in the descending colon is near neutral. SCFAs are weak organic acids, with pKa values generally in the range of 4.5–5.0, not coincidentally overlapping the range where loss of Cul-1 neddylation was observed (60). At these pH’s, the protonated forms of the acids are lipid soluble, membrane permeable, and exhibit maximal bioactivity (61, 62). Thus, different regions of the colon are exposed to significantly different concentrations of bioavailable SCFAs and no doubt other chemical parameters as well. Possibly, regions of the colon exposed to maximal bioavailable SCFA concentrations may have attenuated inflammatory potential. Intriguingly, this anatomical distribution of the gradient of SCFA concentrations is the precise inverse to the anatomical distribution of the inflammatory lesions of acute ulcerative colitis, which is invariably seen in the distal colon and progressively attenuates proximally (63).

Excessively high concentrations of SCFAs may have deleterious clinical consequences. Necrotizing enterocolitis is a spontaneous complication of prematurity that has been suggested to be mediated by excessive levels of SCFAs (64, 65). Empirically, the pHs of luminal contents in patients with this disorder have been measured at 3.8 – 4.6. Other workers have induced intestinal injury in rodents with very high doses of butyrate (300 mM/pH 4.0 with normal being 70 –140 mM/pH 5.5 in the proximal colon) and other SCFAs (66). Interestingly, it was also shown that butyrate anion at neutral or alkaline pH had no effect nor did low pH alone, consistent with our observations in vitro (67). The transient nature of Cul-1 deneddylation indicates that enterocytes can compensate and adapt to dynamic changes; however, extreme conditions may limit the ability of enterocytes to adapt. Given the wide variety of cellular processes other than NF-κB and the Wnt signaling pathway activation that are regulated by SCF-mediated protein-degradative processes, it is not surprising that excessive exposure to very high levels of compounds that cause loss of Cul-1 neddylation could inhibit many enzymatic processes and be rapidly toxic to the cell.

Luminal physiochemical properties are dynamic and may be manipulated in a therapeutic fashion by high-starch dietary supplements (prebiotics) or oral supplementation with live bacteria (probiotics) (68, 69). Interestingly, one group of microbes with therapeutic value are the lactic acid bacteria, well-known Gram-positive enteric commensals that include the genera Lactobacillus, Enterococcus, and Bifidobacteria, among others, and are defined by a common ability to produce lactic acid under microaerophilic to anaerobic conditions (70). As part of the human enteric flora, they contribute to the physiological acidic pH of the intraluminal colonic environment (68) and lactate produced by this subset of organisms may also serve as substrate for butyrate production by others, a process termed metabolic cross-feeding (71). More recently, these organisms have been recognized as possessing marked probiotic activity both clinically (reviewed in Ref. 69) and in animal models (72). Conditioned medium from L. rhamnosis GG has been shown to repress proinflammatory gene expression in cultured epithelial cells (73). It has also been reported that conditioned medium from two of these bacteria, Bifidobacterium breve and Streptococcus thermophilus, exhibited inhibitory effects on NF-κB activation in vitro (74). Furthermore, this activity was attributed to a small (<3 kDa), soluble metabolite. These workers quantified lactate in the conditioned medium at 4.5–9 mM, but were unable to show NF-κB inhibition at these concentrations. Finally, the bacterial metabolites butyrate, propionate, and acetate have been shown to have inhibitory activity on proinflammatory signaling pathways. These compounds have been used therapeutically (by intraluminal instillation) to dampen intestinal inflammation in inflammatory bowl diseases (8, 9, 75) and have been shown to block NF-κB activation in vitro (28, 29).

Our observations are likely limited to effects of large bacterial communities with substantial metabolic outputs necessary to ferment sufficient quantities of organic acids and perhaps other products. As such, our observations probably do not reflect events in most acute enteric infections. Invasive enteric pathogens such as Salmonella, Shigella, etc., do not reach biomass densities of 1011 organisms/ml before executing their effects on host cells, although pathogens that exist in microbedense biofilms could plausibly generate local microenvironments of higher metabolite concentrations.

The relationship of the normal flora to the intestinal epithelia is a prime example of a complex microbial community associated with a higher eukaryote (76). It may be an oversimplification to view this system as a binary host-pathogen interaction, with exchanges of individual and discrete biochemical signals, such as secreted effector proteins, toxins, or even small molecules with specificity toward eukaryotic cellular processes. The intestinal luminal flora produces a massive and diverse metabolic output that varies according to anatomical location, nutrient composition, and host health. It may be more illuminating to view the normal flora as a holistic community rather than as a collection of semi-independent organisms mediating “beneficial” or “detrimental” effects. Our results suggest a novel mechanism by which this complex community can collectively influence key epithelial signaling pathways. Metabolic products/small molecules produced at the eukaryotic/prokaryotic interface may account for some of the widely known effects of the bacterial flora on normal intestinal function (3) and may influence a range of eukaryotic regulatory processes.


Human Biopsy Samples were provided by Dr. Shanti Srinivasan. We thank Kirsten Gerner-Smidt for help with the confocal microscope.


3Abbreviations used in this paper: SCFA, short chain fatty acid; Cul-1, cullin-1; MAMP, microbial-associated molecular pattern; ROS, reactive oxygen species; NAC, N-acetyl cysteine; DPI, diphenyleneiodonium; DCF, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester; DHE, dihydroethidium; MOI, multiplicity of infection; IAA, iodoacetamide; HMM, high molecular mass; Eh, redox potential; Trx, Thioredoxin.


The authors have no financial conflict of interest.

1This work was supported in part by National Institutes of Health Grants DK-71604 and AI-64462 (to A.S.N.).


1. Falk PG, Hooper LV, Midtvedt T, Gordon JI. Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology. Microbiol Mol Biol Rev. 1998;62:1157–1170. [PMC free article] [PubMed]
2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science. 2001;291:881–884. [PubMed]
3. Hooper LV, Gordon JI. Commensal host-bacterial relationships in the gut. Science. 2001;292:1115–1118. [PubMed]
4. Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr. 2002;22:283–307. [PubMed]
5. Topping DL, Clifton PM. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev. 2001;81:1031–1064. [PubMed]
6. Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ. Review article: the role of butyrate on colonic function. Aliment Pharmacol Ther. 2008;27:104–119. [PubMed]
7. Wong JM, de Souza R, Kendall CW, Emam A, Jenkins DJ. Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol. 2006;40:235–243. [PubMed]
8. Wachtershauser A, Stein J. Rationale for the luminal provision of butyrate in intestinal diseases. Eur J Nutr. 2000;39:164–171. [PubMed]
9. Vernia P, Annese V, Bresci G, d’Albasio G, D’Inca R, Giaccari S, Ingrosso M, Mansi C, Riegler G, Valpiani D, Caprilli R. Topical butyrate improves efficacy of 5-ASA in refractory distal ulcerative colitis: results of a multicentre trial. Eur J Clin Invest. 2003;33:244–248. [PubMed]
10. Winston JT, Strack P, Beer-Romero P, Chu CY, Elledge SJ, Harper JW. The SCFβ-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro. Genes Dev. 1999;13:270–283. [PubMed]
11. Spencer E, Jiang J, Chen ZJ. Signal-induced ubiquitination of IκBα by the F-box protein Slimb/β-TrCP. Genes Dev. 1999;13:284–294. [PubMed]
12. Pan ZQ, Kentsis A, Dias DC, Yamoah K, Wu K. Nedd8 on Cullin: building an expressway to protein destruction. Oncogene. 2004;23:1985–1997. [PubMed]
13. Liakopoulos D, Doenges G, Matuschewski K, Jentsch S. A novel protein modification pathway related to the ubiquitin system. EMBO J. 1998;17:2208–2214. [PubMed]
14. Kamitani T, Kito K, Nguyen HP, Yeh ET. Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J Biol Chem. 1997;272:28557–28562. [PubMed]
15. Parry G, Estelle M. Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins. Semin Cell Dev Biol. 2004;15:221–229. [PubMed]
16. Ohh M, Kim WY, Moslehi JJ, Chen Y, Chau V, Read MA, Kaelin WG., Jr An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells. EMBO Rep. 2002;3:177–182. [PubMed]
17. Read MA, Brownell JE, Gladysheva TB, Hottelet M, Parent LA, Coggins MB, Pierce JW, Podust VN, Luo RS, Chau V, Palombella VJ. Nedd8 modification of cul-1 activates SCFβ(TrCP)-dependent ubiquitination of IκBα Mol Cell Biol. 2000;20:2326–2333. [PMC free article] [PubMed]
18. Furukawa M, Zhang Y, McCarville J, Ohta T, Xiong Y. The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol Cell Biol. 2000;20:8185–8197. [PMC free article] [PubMed]
19. Amir RE, Iwai K, Ciechanover A. The NEDD8 pathway is essential for SCFβ-TrCP-mediated ubiquitination and processing of the NF-κB precursor p105. J Biol Chem. 2002;277:23253–23259. [PubMed]
20. Amir RE, Haecker H, Karin M, Ciechanover A. Mechanism of processing of the NF-κB2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCFβ-TrCP ubiquitin ligase. Oncogene. 2004;23:2540–2547. [PubMed]
21. Collier-Hyams LS, Sloane V, Batten BC, Neish AS. Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1. J Immunol. 2005;175:4194–4198. [PubMed]
22. Kumar A, Wu H, Collier-Hyams LS, Hansen JM, Li T, Yamoah K, Pan ZQ, Jones DP, Neish AS. Commensal bacteria modulate Cullin-dependent signaling via generation of reactive oxygen species. EMBO J. 2007;26:4457–4466. [PubMed]
23. McSorley SJ, Ehst BD, Yu Y, Gewirtz AT. Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo. J Immunol. 2002;169:3914–3919. [PubMed]
24. Zeng H, Carlson AQ, Guo Y, Yu Y, Collier-Hyams LS, Madara JL, Gewirtz AT, Neish AS. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J Immunol. 2003;171:3668–3674. [PubMed]
25. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biol Med. 1999;27:612–616. [PubMed]
26. Halvey PJ, Watson WH, Hansen JM, Go YM, Samali A, Jones DP. Compartmental oxidation of thiol-disulphide redox couples during epidermal growth factor signalling. Biochem J. 2005;386:215–219. [PubMed]
27. Watson WH, Jones DP. Oxidation of nuclear thioredoxin during oxidative stress. FEBS Lett. 2003;543:144–147. [PubMed]
28. Segain JP, Raingeard de la Bletiere D, Bourreille A, Leray V, Gervois N, Rosales C, Ferrier L, Bonnet C, Blottiere HM, Galmiche JP. Butyrate inhibits inflammatory responses through NFκB inhibition: implications for Crohn’s disease. Gut. 2000;47:397–403. [PMC free article] [PubMed]
29. Yin L, Laevsky G, Giardina C. Butyrate suppression of colonocyte NF-κB activation and cellular proteasome activity. J Biol Chem. 2001;276:44641–44646. [PubMed]
30. Nugent SG, Kumar D, Rampton DS, Evans DF. Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs. Gut. 2001;48:571–577. [PMC free article] [PubMed]
31. Weng M, Walker WA, Sanderson IR. Butyrate regulates the expression of pathogen-triggered IL-8 in intestinal epithelia. Pediatr Res. 2007;62:542–546. [PMC free article] [PubMed]
32. Collier-Hyams LS, Neish AS. Innate immune relationship between commensal flora and the mammalian intestinal epithelium. Cell Mol Life Sci. 2005;62:1339–1348. [PubMed]
33. Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, Madara JL. Prokaryotic regulation of epithelial responses by inhibition of IκB-α ubiquitination. Science. 2000;289:1560–1563. [PubMed]
34. Wada H, Yeh ET, Kamitani T. A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo. J Biol Chem. 2000;275:17008–17015. [PubMed]
35. Cardozo T, Pagano M. The SCF ubiquitin ligase: insights into a molecular machine. Nat Rev Mol Cell Biol. 2004;5:739–751. [PubMed]
36. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Deshaies RJ. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science. 2001;292:1382–1385. [PubMed]
37. Bostick M, Lochhead SR, Honda A, Palmer S, Callis J. Related to ubiquitin 1 and 2 are redundant and essential and regulate vegetative growth, auxin signaling, and ethylene production in Arabidopsis. Plant Cell. 2004;16:2418–2432. [PubMed]
38. Kurz T, Pintard L, Willis JH, Hamill DR, Gonczy P, Peter M, Bowerman B. Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science. 2002;295:1294–1298. [PubMed]
39. Ou CY, Lin YF, Chen YJ, Chien CT. Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development. Genes Dev. 2002;16:2403–2414. [PubMed]
40. Tateishi K, Omata M, Tanaka K, Chiba T. The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice. J Cell Biol. 2001;155:571–579. [PMC free article] [PubMed]
41. Cope GA, Deshaies RJ. COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003;114:663–671. [PubMed]
42. Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R, Kisselev AF, Tanaka K, Nakatani Y. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003;113:357–367. [PubMed]
43. Wang X, Feng S, Nakayama N, Crosby WL, Irish V, Deng XW, Wei N. The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development. Plant Cell. 2003;15:1071–1082. [PubMed]
44. Khoury J, Ibla JC, Neish AS, Colgan SP. Antiinflammatory adaptation to hypoxia through adenosine-mediated Cullin-1 deneddylation. J Clin Invest. 2007;117:703–711. [PubMed]
45. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M. Reactive oxygen species promote TNFα-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell. 2005;120:649–661. [PubMed]
46. Mahadev K, Wu X, Zilbering A, Zhu L, Lawrence JT, Goldstein BJ. Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3–L1 adipocytes. J Biol Chem. 2001;276:48662–48669. [PubMed]
47. Mahadev K, Zilbering A, Zhu L, Goldstein BJ. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem. 2001;276:21938–21942. [PubMed]
48. Chen KC, Zhou Y, Xing K, Krysan K, Lou MF. Platelet derived growth factor (PDGF)-induced reactive oxygen species in the lens epithelial cells: the redox signaling. Exp Eye Res. 2004;78:1057–1067. [PubMed]
49. Goldstein BJ, Mahadev K, Wu X, Zhu L, Motoshima H. Role of insulin-induced reactive oxygen species in the insulin signaling pathway. Antioxid Redox Signal. 2005;7:1021–1031. [PMC free article] [PubMed]
50. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004;4:181–189. [PubMed]
51. Terada LS. Specificity in reactive oxidant signaling: think globally, act locally. J Cell Biol. 2006;174:615–623. [PMC free article] [PubMed]
52. Tonks NK. Redox redux: revisiting PTPs and the control of cell signaling. Cell. 2005;121:667–670. [PubMed]
53. Chiarugi P, Buricchi F. Protein tyrosine phosphorylation and reversible oxidation: two cross-talking posttranslation modifications. Antioxid Redox Signal. 2007;9:1–24. [PubMed]
54. Seo JH, Ahn Y, Lee SR, Yeol Yeo C, Chung Hur K. The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway. Mol Biol Cell. 2005;16:348–357. [PMC free article] [PubMed]
55. Veal EA, Day AM, Morgan BA. Hydrogen peroxide sensing and signaling. Mol Cell. 2007;26:1–14. [PubMed]
56. Bossis G, Melchior F. Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Mol Cell. 2006;21:349–357. [PubMed]
57. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–399. [PubMed]
58. Jeng JH, Kuo MY, Lee PH, Wang YJ, Lee MY, Lee JJ, Lin BR, Tai TF, Chang MC. Toxic and metabolic effect of sodium butyrate on SAS tongue cancer cells: role of cell cycle deregulation and redox changes. Toxicology. 2006;223:235–247. [PubMed]
59. Hsiao CH, Li W, Lou TF, Baliga BS, Pace BS. Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species. Exp Hematol. 2006;34:264–273. [PubMed]
60. Velazquez OC, Lederer HM, Rombeau JL. Butyrate and the colonocyte: production, absorption, metabolism, and therapeutic implications. Adv Exp Med Biol. 1997;427:123–134. [PubMed]
61. Stein J, Zores M, Schroder O. Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism. Eur J Nutr. 2000;39:121–125. [PubMed]
62. Charney AN, Micic L, Egnor RW. Nonionic diffusion of short-chain fatty acids across rat colon. Am J Physiol. 1998;274:G518–G524. [PubMed]
63. Cotran R, Kumar V, Collins T. The Pathologic Basis of Disease. Saunders; Philadelphia: 1999.
64. Clark DA, Thompson JE, Weiner LB, McMillan JA, Schneider AJ, Rokahr JE. Necrotizing enterocolitis: intraluminal biochemistry in human neonates and a rabbit model. Pediatr Res. 1985;19:919–921. [PubMed]
65. Sanderson IR. The physicochemical environment of the neonatal intestine. Am J Clin Nutr. 1999;69:1028S–1034S. [PubMed]
66. Nafday SM, Chen W, Peng L, Babyatsky MW, Holzman IR, Lin J. Short-chain fatty acids induce colonic mucosal injury in rats with various postnatal ages. Pediatr Res. 2005;57:201–204. [PubMed]
67. McCafferty DM, I, Zeitlin J. Short chain fatty acid-induced colitis in mice. Int J Tissue React. 1989;11:165–168. [PubMed]
68. Teitelbaum JE, Walker WA. Nutritional impact of pre- and pro-biotics as protective gastrointestinal organisms. Annu Rev Nutr. 2002;22:107–138. [PubMed]
69. Sartor RB. Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology. 2004;126:1620–1633. [PubMed]
70. Klein G, Pack A, Bonaparte C, Reuter G. Taxonomy and physiology of probiotic lactic acid bacteria. Int J Food Microbiol. 1998;41:103–125. [PubMed]
71. Flint HJ, Duncan SH, Scott KP, Louis P. Interactions and competition within the microbial community of the human colon: links between diet and health. Environ Microbiol. 2007;9:1101–1111. [PubMed]
72. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology. 1999;116:1107–1114. [PubMed]
73. Pena JA, Versalovic J. Lactobacillus rhamnosus GG decreases TNF-α production in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism. Cell Microbiol. 2003;5:277–285. [PubMed]
74. Menard S, Candalh C, Bambou JC, Terpend K, Cerf-Bensussan N, Heyman M. Lactic acid bacteria secrete metabolites retaining anti-inflammatory properties after intestinal transport. Gut. 2004;53:821–828. [PMC free article] [PubMed]
75. Scheppach W, Weiler F. The butyrate story: old wine in new bottles? Curr Opin Clin Nutr Metab Care. 2004;7:563–567. [PubMed]
76. McFall-Ngai MJ. Unseen forces: the influence of bacteria on animal development. Dev Biol. 2002;242:1–14. [PubMed]