Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism.
Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayers. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time- and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, claudin-1, claudin-4, and zonula occludens (ZO)-1 from the intercellular junctions and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4, or LBP by specific small interfering RNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, a myosin light chain kinase inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1 were found to be highly phosphorylated on threonine residues in intact cell monolayers. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residues was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src-, TLR4-, LBP-, and myosin light chain kinase-dependent mechanism.
Sheth
P
P
Department of Physiology, University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, USA.
Delos Santos
N
N
Seth
A
A
LaRusso
N F
NF
Rao
R K
RK
D000209:Acute-Phase Proteins / Q000502:physiology*; D000818:Animals; D001652:Bile Ducts / Q000166:cytology* / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology*; D015209:Cholangitis, Sclerosing / Q000503:physiopathology; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D019833:Genistein / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D062793:Occludin; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D019108:Tight Junctions / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D062826:Zonula Occludens-1 Protein; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D019833:Genistein; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009219:Myosin-Light-Chain Kinase; D062793:Occludin; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D011743:Pyrimidines; D034741:RNA, Small Interfering; D051197:Toll-Like Receptor 4; D062826:Zonula Occludens-1 Protein; D000099231:Lipopolysaccharide-Binding Protein; C412373:AG 1879; C568987:Ocln protein, rat; C569044:Tjp1 protein, rat; C495347:Tlr4 protein, rat