Enteric commensal bacteria induce extracellular signal-regulated kinase pathway signaling via formyl peptide receptor-dependent redox modulation of dual specific phosphatase 3

Abstract

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

FIGURE 1.

LGG and fMLF induce the generation of ROS in cultured epithelial cells in an FPR-dependent manner. A, CM-H₂DCF-DA (5 μm)-mediated detection of ROS in SK-CO15 cells treated with fMLF (500 nm) or LGG (5 × 10⁷ cfu/ml) over 30 min. B, quantitative representation of ROS production in A for fMLF at 30 min. C, quantitative representation of ROS production in A for LGG at 30 min. D, hydrocyanine3 (7.5 μm)-mediated detection of ROS in murine colonic enterocytes treated with fMLF (500 nm) or LGG (5 × 10⁷ cfu/ml) for 7 min. Fluorescence was measured at ×40 magnification by confocal laser scanning microscopy (Zeiss). DNA was stained with Syto 24 and H&E sections for tissue orientation. E, quantitative representation of ROS production in D. For B, C, and E, data are representative of three independent assays quantified with ImageJ software and are expressed in units of fluorescence. Error bars, S.E.

FIGURE 2.

Dampening of cellular ROS levels attenuates LGG- or fMLF-induced ERK pathway activation and cellular proliferation. A, immunoblot analysis for phospho-ERK in cultured SK-CO15 cells treated with NAC (20 μm) or DPI (40 μm) 30 min prior to stimulation with LGG (5 × 10⁷ cfu/ml) or fMLF (500 nm) up to 1 h. B, ERK pathway-specific luciferase reporter gene assay from transfected SK-CO15 cells treated with NAC (20 μm) 30 min prior to LGG (5 × 10⁷ cfu/ml), fMLF (500 nm), or H₂O₂ (1 mm) stimulation.*, p < 0.05; **, p < 0.001. C, immunoblot analysis for phospho-JNK in cultured SK-CO15 cells treated with NAC (20 μm) or DPI (40 μm) 30 min prior to stimulation with TNF-α (1 ng/ml) up to 30 min. D, EdU incorporation into cultured SK-CO15 cells treated with NAC (20 μm) prior to incubation for 12 h with LGG (5 × 10⁷ cfu/ml) or fMLF (500 nm). Blue, To-Pro-3 for DNA; red, EdU for proliferation. Confocal microscope images were recorded at ×63 magnification. E, quantitative representation of EdU-positive cells in C. Shown is the number of EdU-positive cells/10 fields of view at ×20 for three replicates/treatment. *, p < 0.05. Error bars, S.E.

FIGURE 3.

LGG and fMLF up-regulate DUSP3 mRNA and protein levels. A, quantitative RT-PCR analysis of DUSP3 mRNA levels in cultured SK-CO15 cells stimulated with LGG (5 × 10⁷ cfu/ml) or fMLF (500 nm) for 30 min. PCRs were performed in triplicate using two separate RNA preparations for each data point. Error bars represent S.E. B, immunoblot analysis for total DUSP3 in cultured SK-CO15 cells stimulated with LGG (5 × 10⁷ cfu/ml) or fMLF (500 nm) up to 1 h. C, quantitative RT-PCR analysis of DUSP3 mRNA levels in mouse colonic epithelial scrapings treated in vivo with 100 μl LGG (10⁷ cfu/ml) or fMLF (500 nm) for 30 min. PCRs were performed in triplicate using two separate RNA preparations for each data point. Error bars represent S.E. D, immunoblot analysis for total DUSP3 in mouse colonic epithelial cell scrapings treated in vivo with 100 μl of LGG (10⁷ cfu/ml) or fMLF (500 nm) for 30 min.

FIGURE 4.

LGG- or fMLF-induced generation of ROS oxidizes DUSP3. A, SK-CO15 cultured cells transfected with vector control or plasmids expressing DUSP3 or mDUSP3 assayed by immunoblotting for basal levels of phospho-ERK. B, ERK-responsive luciferase reporter gene assay for basal levels of ERK stimulation from SK-CO15 cells transfected with vector control or plasmids expressing DUSP3 or mDUSP3.*, p < 0.05; **, p < 0.001. Error bars, S.E. C, SK-CO15 cultured cells transfected with vector control or plasmids expressing DUSP3 or mDUSP3 or DUSP3 treated with NAC (20 μm) 30 min prior to stimulation with LGG (5 × 10⁷ cfu/ml) or fMLF (500 nm) up to 30 min. Lysates were then assayed for DUSP3 oxidation status by immunoblotting for myc in nonreducing conditions or phospho-ERK by immunoblotting in reducing conditions. All cells were lysed in a buffer containing 10 mm NEM to prevent oxidation of cysteines during sample preparation. DUSP3 oxidation was monitored by changes in electrophoretic mobility. Ox, oxidized DUSP3. myc, reduced DUSP3.

FIGURE 5.

Antioxidant pretreatment inhibits LGG- or fMLF-induced phosphorylation of ERK in murine enterocytes. A, immunofluorescence of phospho-ERK within intestinal whole mount preparations (as described under “Experimental Procedures”) in either media- or NAC (20 μm)-pretreated intestinal mucosa 30 min prior to treatment with 100 μl of LGG (10⁷ cfu/ml) or fMLF (500 nm) for 7 min. B, immunoblot analysis for phospho-ERK in mouse colonic epithelial cell scrapings pretreated with NAC (20 μm) 30 min prior to in vivo treatment with 100 μl LGG (10⁷ cfu/ml) or fMLF (500 nm) for 7 min.

FIGURE 6.

Model for commensal bacteria-induced ERK pathway signaling via FPR-dependent redox modulation of DUSP3.