Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism

Abstract

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.

Activation of TLR2 Enhances Tight Junction Barrier in Epidermal Keratinocytes

“…Until now, the relationship between innate immune response and TJ functions was investigated using intestinal epithelial cells and cholangiocytes (34,35). In intestinal epithelial cells, stimulation with TLR2 ligands led to enhanced TER via PKCa and PKCd activation; however, involvement of atypical PKC had not been examined.…”

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

“…However, effective delivery of siRNA duplex and exogenous genes into confluent T84 monolayers is difficult to date and is still challenging to those who use T84 monolayers (48) although some researchers have applied siRNA duplexes to subconfluent T84 cells that were grown on plastic (49 -51). One report indicated that siRNA was introduced into 70 -75% confluent rat cholangiocytes that were then trypsinized and seeded onto Transwell supports (52). This approach is problematic in T84 cells because the duration required for T84 cells to reach full confluence and functional differentiation is typically around 7 days, and it is beyond the time frame for studying functions of protein kinases by knockdown.…”

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

“…Measurement of Trans-epithelial Electrical Resistance (TEER)-TEER was measured using Millicell electrical resistance system (Millipore) as reported (14,26) and expressed as ohms/ cm 2 , where ohm is the observed value of resistance, and cm 2 is the surface area of the membrane. The background resistance of the trans-well membrane (ϳ30 ohms/cm 2 ) was subtracted from the observed resistance values.…”