hPepT1 transports muramyl dipeptide, activating NF-kappaB and stimulating IL-8 secretion in human colonic Caco2/bbe cells.
OBJECTIVE
Bacterial proteoglycan-derived muramyl dipeptide (MDP) activates the intracellular NOD2/CARD15 gene product. How intestinal epithelial cells take up MDP is poorly understood. We hypothesized that the intestinal apical di-/tripeptide transporter, hPepT1, transports MDP, thereby activating downstream pathways similar to nuclear factor kappa B (NF-kappaB).


METHODS
Time- and concentration-dependent (3)H-MDP uptakes were studied in Caco2/bbe (C2) cell monolayers where hPepT1 expression was either over- or underexpressed, using an inducible adenovirus system or silencing RNA (siRNA), respectively. NF-kappaB activation and interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) release were determined by enzyme-linked immunosorbent assay. NOD2/CARD15 expression was inhibited by siRNA. MDP in human duodenal, cecal, and stool samples was measured.


RESULTS
MDP, but not its isoforms, inhibited uptake of glycosylsarcosine in C2 cells, indicating stereoselective and competitive inhibition. Approximately 90% of the MDP was cytosolic, showing uptake rather than binding. The K m for MDP uptake was 4.3 mmol/L. Cells overexpressing hPepT1 showed increased Gly-Sar and MDP uptake, whereas decreased uptake was observed after hPepT1 siRNA-inhibition. MDP treatment activated NF-kappaB, resulting in IL-8 release, an effect blocked by siRNA-inhibited expression of NOD2/CARD15. MDP content in cecal and stool samples (in normal subjects) was 20-87 micromol/L, but undetectable in duodenal fluid.


CONCLUSIONS
In colonic epithelial cells, MDP is taken up by hPepT1 and activates NF-kappaB and chemokine production. Because hPepT1 expression in chronic colonic inflammation is increased, this may play an important role in promoting colonocyte participation in host defense and pathogen clearance through increased uptake of MDP.
Vavricka
Stephan R
SR
IBD Research Center, University of Chicago, Chicago, IL 60637, USA.
Musch
Mark W
MW
Chang
Jonathan E
JE
Nakagawa
Yasushi
Y
Phanvijhitsiri
Kittiporn
K
Waypa
Tonya S
TS
Merlin
Didier
D
Schneewind
Olaf
O
Chang
Eugene B
EB
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D045744:Cell Line, Tumor; D018932:Chemokine CCL2 / Q000378:metabolism; D003110:Colonic Neoplasms; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D007413:Intestinal Mucosa; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D000074011:Peptide Transporter 1; D034741:RNA, Small Interfering / Q000235:genetics; D027981:Symporters / Q000235:genetics* / Q000378:metabolism
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; D016328:NF-kappa B; D010446:Peptide Fragments; D000074011:Peptide Transporter 1; D034741:RNA, Small Interfering; C082186:SLC15A1 protein, human; D027981:Symporters; D000119:Acetylmuramyl-Alanyl-Isoglutamine