Abstract
CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.