PKC-zeta is essential toward endotoxin-induced macrophage activation

Abstract

A critical feature of sepsis-induced adult respiratory distress syndrome (ARDS) is the release of cytokines, such as TNF-α, from LPS-activated macrophages. However, the cellular mechanisms involved are incompletely understood. The aim of this study is further clarify the mechanism of LPS activation through the Toll-4 receptor (TLR4) by examining the roles of the various isoforms of PKC within the tissue-fixed macrophage. Methods: RAW 246.7 cells were subjected to LPS stimulation. Selected cells were pretreated with 10 nM Gö6983 to inhibit calcium dependent and novel PKC isoforms, or 60 nM Gö6983 to inhibit atypical PKC isoforms. Following stimulation, cellular and nuclear protein were extracted and analyzed by Western blot and EMS for components of the TLR4 pathway. Supernatants obtained from cells under the various conditions were analyzed by ELISA for the production of TNF-α. Statistical analysis was performed by student t-tests, with a p < 0.05 being significant. Results: LPS stimulation led to the phosphorylation and activation of IRAK, which was followed by activation of JNK, ERK 1/2, and p38. Subsequently, LPS induced the activation of NF-κB and AP-1. Activation of these TLR4-signaling factors was followed by the production of TNF-α. Inhibition of calcium dependent and novel PKC isoforms had no significant effect on any component of TLR4 mediated signaling or TNF-α production. However, inhibition of the atypical PKC, PKC-ζ, was associated with a significant attenuation in the activation of IRAK-1, JNK, ERK 1/2, p38, NF-κB and AP-1 (p<0.05 vs. control for each). This modulation in TLR4 signaling was associated with a significant attenuation of TNF-α (p = 0.001 vs. control). Based on these findings, specific blockade of PKC-ζ was performed using a cell permeable myristoylated PKC-ζ pseudopeptide resulting in similar effects. Conclusion: This study demonstrates that within the macrophage the atypical PKC isoform PKC-ζ is critical to regulation of LPS-induced TLR4-signaling and TNF-α production. Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-ζ activity during gram-negative infections may limit associated inflammatory-induced morbidity, such as ARDS.