Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C

Background & Aims: 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 Pam₃CysSK4, 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 (PKCα and PKCδ). 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.