PKCζ participates in activation of inflammatory response induced by enteropathogenicE. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-κB and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-κB activation but that other signaling molecules such as PKCζ may be involved. The aim of this study was to determine whether PKCζ is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKCζ from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKCζ by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKCζ pseudosubstrate (MYR-PKCζ-PS), or transient expression of a nonfunctional PKCζ significantly suppressed EPEC-induced IκBα phosphorylation. Although PKCζ can activate ERK, MYR-PKCζ-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKCζ can regulate NF-κB activation by interacting with and activating IκB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKCζ and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKCζ-IKK complexes from infected intestinal epithelial cells and recombinant IκBα as a substrate showed a 2.5-fold increase in IκBα phosphorylation. PKCζ can also regulate NF-κB by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKCζ-PS, suggesting that other signaling events may be involved in this particular arm of NF-κB regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKCζ and ERK that lead to NF-κB activation, thus ensuring the proinflammatory response.