Volume 127, Issue 5, Pages 1401-1409 (November 2004) hPepT1 transports muramyl dipeptide, activating NF-κB and stimulating IL-8 secretion in human colonic Caco2/bbe cells  Stephan R. Vavricka, Mark W. Musch, Jonathan E. Chang, Yasushi Nakagawa, Kittiporn Phanvijhitsiri, Tonya S. Waypa, Didier Merlin, Olaf Schneewind, Eugene B. Chang  Gastroenterology  Volume 127, Issue 5, Pages 1401-1409 (November 2004) DOI: 10.1053/j.gastro.2004.07.024 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 C2 cell uptake of [3H]Gly-Sar is stereospecifically and competitively inhibited by MDP. Uptake of Gly-Sar (100 μmol/L) was measured in C2 cell monolayers as described in the Materials and Methods section. (A) Time dependence of uptake is shown as means ± SE (n = 6). (B) Varying concentrations of MDP or its inactive sterioisomers were used to inhibit Gly-Sar uptake at 15 minutes (means ± SE, n = 6). Addition of 1 μmol/L or 600 μmol/L MDP or LL or DD to the apical side of C2 cells in media containing 100 μmol/L Gly-Sar concentration decreased Gly-Sar uptake to 73% (*P < .05) and 56% (**P < .01), respectively. This effect is stereospecific because addition to the apical side of MDP isoforms LL and DD does not decrease Gly-Sar uptake. ■, No addition; □, 1 μm; ▩, 600 μm. (C) Dixon plot and (D) Cornish-Bowden analysis of inhibition of hPepT1-mediated Gly-Sar uptake by MDP in C2 cells. To compare the inhibition kinetics of MDP on C2 cells in more detail, Gly-Sar uptake in C2 cells was measured during the linear uptake phase (15 min) at the concentrations of Gly-Sar 10 μmol/L, 15 μmol/L, and 60 μmol/L in the presence and absence of the indicated MDP concentrations. MDP yielded a Ki value of Ki ∼83 ± 2 μmol/L for hPepT1. The pattern was compatible with competitive inhibition. The image is representative of those from 3 separate experiments. ○, 10 μmol/L; ▴, 15 μmol/L; ■, 60 μmol/L. Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Uptake of [3H]MDP of C2 monolayers. (A) Time-dependent uptake of labeled MDP was measured as described in the Materials and Methods section (means ± SE, n = 6). (B) Affinity of MDP was determined by varying [MDP] and measuring at 15 minutes (means ± SE, n = 3). The line was obtained by fitting a Michaelis-Menton equation by nonlinear regression using GraphPad software and yielding an apparent Michaelis constant ∼4.3 ± 0.4 mmol/L. Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Specific induction of hPepT1 increases MDP uptake. (A) C2 cells were infected with either or both packaged virus (tet operator, hPepT1) 24 hours before uptake measurements. Tetracycline was added when appropriate to keep the tet operator inactive. (B) hPepT1 protein expression was confirmed by Western blotting of total cell protein and surface biotinylatable expression. Means ± SE (n = 5) are presented below the respective panel. Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 siRNA of hPepT1 decreases MDP transport. C2 cells were treated with Lipofectamine 2000 alone or with d-siRNA for hPepT1 or NOD2/CARD15. Cells were treated twice with 200 nmol/L d-siRNA, harvested for hPepT1 expression, and biotinylated on the apical surface for brush-border protein expression. Uptake of labeled Gly-Sar or MDP then was performed and results were expressed as means ± SE for 3 separate experiments. The image shown is representative of those experiments. □, Gly-Sar uptake; ■, MDP uptake. Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 MDP activates NF-κB and stimulates cytokine release. C2 cell monolayers were treated with 100 μmol/L MDP and harvested either for 30 minutes for nuclear translocation of NF-κB (B), or medium was harvested for 24 hours for cytokine releases (A), both described in the Materials and Methods section. Values are means ± SE for at least 3 experiments. Basal release of IL-8 and MCP-1 were 1055 ± 329 and 978 ± 365 pg/mL, respectively. *P < .05, **P < .01, ***P < 0.001. Positive control was TNF-α (50 ng/mL) stimulation of C2 cells for 24 hours (IL-8 and MCP-1) and for 30 minutes (NF-κB). Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 MDP-, but not TNF-α–stimulated, IL-8 release is dependent on NOD2/CARD15 expression. C2 cells were treated with d-siRNA for NOD2/CARD15 or hPepT1 and, when appropriate, stimulated with either MDP (100 μmol/L) or TNF-α (100 ng/mL). Values shown are means ± SE for 3 separate experiments. Statistical comparisons were made by analysis of variance using a Bonferroni correction using Instat software (GraphPad). Significance for presented comparisons is *P < .05, +P < .01. Gastroenterology 2004 127, 1401-1409DOI: (10.1053/j.gastro.2004.07.024) Copyright © 2004 American Gastroenterological Association Terms and Conditions