PKCζ participates in activation of inflammatory response induced by enteropathogenic E-coli

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