Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C.
OBJECTIVE
Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria. Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites. The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs.


METHODS
TLR2 agonist (synthetic bacterial lipopeptide Pam(3)CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays-combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2. Transepithelial electrical resistance characterized intestinal epithelial barrier function.


RESULTS
Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCalpha and PKCdelta). Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant. Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists. This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident. Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation.


CONCLUSIONS
The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation.
Cario
Elke
E
Division of Gastroenterology and Hepatology, University Hospital of Essen, Germany. elke.cario@uni-essen.de
Gerken
Guido
G
Podolsky
Daniel K
DK
D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D062826:Zonula Occludens-1 Protein
D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; C569042:TJP1 protein, human; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D062826:Zonula Occludens-1 Protein; D011493:Protein Kinase C