title: Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling.
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.
authors.affiliation: INSERM, UMR756 «Signalisation et Physiopathologie des Cellules Epithéliales», Châtenay-Malabry, France Université Paris-Sud 11, Faculté de Pharmacie, Châtenay-Malabry, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Liévin-Le Moal
authors.forename: Isabelle
authors.initials: I
authors.lastname: Beau
authors.forename: Clémence
authors.initials: C
authors.lastname: Rougeaux
authors.forename: Imad
authors.initials: I
authors.lastname: Kansau
authors.forename: Sylvie
authors.initials: S
authors.lastname: Fabrega
authors.forename: Cédric
authors.initials: C
authors.lastname: Brice
authors.forename: Natalia
authors.initials: N
authors.lastname: Korotkova
authors.forename: Steve L
authors.initials: SL
authors.lastname: Moseley
authors.forename: Alain L
authors.initials: AL
authors.lastname: Servin
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D004926:Escherichia coli / Q000737:chemistry; D005818:Genetic Engineering; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008050:Lipid A; D008070:Lipopolysaccharides / Q000276:immunology; D020935:MAP Kinase Signaling System; D021962:Membrane Microdomains / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009092:Mucous Membrane / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D020349:Surface Plasmon Resonance; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D008050:Lipid A; D008070:Lipopolysaccharides; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D051197:Toll-Like Receptor 4; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases