PKC-zeta is essential for endotoxin-induced macrophage activation.

BACKGROUND

A critical element in sepsis-induced tissue injury is the release of pro-inflammatory mediators from LPS-activated macrophages. The cellular mechanisms involved in this process remain incompletely understood. The aim of the current study was to further clarify the mechanism of LPS activation through the TLR4 receptor complex by examining the roles of the various isoforms of PKC.

MATERIALS AND METHODS

Differentiated THP-1 cells were subjected to LPS stimulation. Selected cells were pretreated with various concentrations of Gö6983 to inhibit conventional, novel, and atypical PKC isoforms. Lipid raft, cellular, and nuclear proteins were then extracted and analyzed by Western blot and EMSA for components of the TLR4 pathway. Supernatants harvested under the various conditions were analyzed by ELISA for the production of TNF-alpha.

RESULTS

LPS stimulation led to the mobilization of TLR4 to lipid rafts followed by phosphorylation and activation of IRAK, ERK 1/2, p38, and JNK/SAPK. Subsequently, LPS induced the activation of NF-kappaB and AP-1. Activation of these TLR4-signaling components resulted in the production of TNF-alpha. Inhibition of conventional and novel PKC isoforms had no significant effect on macrophage activation. Inhibition of the atypical PKC, PKC-zeta, was associated with significant attenuation in the mobilization of TLR4 to lipid rafts, the activation of all TLR4-signaling components, and the production of TNF-alpha.

CONCLUSION

This study demonstrates that the atypical PKC isoform, PKC-zeta, is critical to regulation of LPS-induced TLR4 lipid raft mobilization within macrophages, TLR4-signaling, and TNF-alpha production. Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-zeta activity during Gram-negative infections may limit associated inflammatory-induced morbidity.