Protein Kinase C α Is Critically Involved in MyD88-Dependent Toll-Like Receptor 2-Mediated Activation of Dendritic Cells.

Abstract Introduction: Protein kinases C (PKC) isoforms are key regulators of several Toll-like receptor (TLR) signalling pathways. In the present study, we investigated the role of conventional PKCα in the MyD88-dependent TLR2-mediated activation of dendritic cells (DC). Methods: We compared the functional and molecular responses of DC derived from wild type and PKCa deficient mice to TLR2 stimulation. We next investigated the consequences of conventional PKC (cPKC) inhibition during TLR2 stimulation in human monocyte-derived DC using the cPKC pharmacological inhibitor Gö6976. Finally, reporter gene assays were performed in human embryonic kidney (HEK) cells stably expressing TLR2 in the presence of dominant negative (DN) PKCα. Results: Bone marrow-derived DC from PKCα deficient mice showed an attenuated response to the TLR2/1 ligand Pam3CSK4 characterized by a statistically significant reduction in interleukin (IL)-12p40 and TNFα production. IL-6 synthesis was also reduced albeit not significantly. In western blot analysis, PKCα−/− DC displayed an impaired phosphorylation of the mitogen activated protein (MAP) kinases p38 and c-Jun NH2-terminal kinase (JNK) in response to Pam3CSK4. TLR2-mediated activation of the IκB kinases (IKK) α and β and inhibitor of κB (IκB) degradation were abolished in PKC deficient DC, suggesting an impaired nuclear factor (NF)-κB activation. In human monocyte-derived DC, inhibition of conventional PKC activity by the isoform specific inhibitor Gö6976 severely decreased cytokines production in response to TLR2 ligands. At the concentration of 1μM, Gö6976-treated DC exhibited impaired p38 and JNK activation. The NF-κB activation pathway was repressed with higher concentrations of the pharmacological inhibitor. Finally, in reporter gene assays, DN PKCα but not DN PKC𝛉 repressed AP-1 and NF-κB mediated promoter activity upon TLR2 engagement. Conclusions: Our results demonstrate that PKCα is a key component of the MyD88-dependent TLR2 signalling pathway in dendritic cells and regulates the activation of NF-κB and MAP kinases pathways.