The role of HSP27 in RACK1-mediated PKC activation in THP-1 cells.

Receptor for Activated C Kinase 1 (RACK1) pseudosubstrate is a commercially available peptide that directly activates protein kinase C-β (PKCβ). We have recently shown that RACK1 pseudosubstrate, alone or in combination with classical immune activators, results in increased cytokine production and CD86 upregulation in primary leukocytes. Furthermore, we demonstrated a role of PKCβ and RACK1 in chemical allergen-induced CD86 expression and IL-8 production in both THP-1 cells and primary human dendritic cells. Aim of this study was to shed light on the mechanisms underlying RACK1 pseudosubstrate-induced immune activation and to compare it to lipopolysaccharide (LPS). The human promyelocytic cell line THP-1 was used throughout the study. RACK1 pseudosubstrate induced rapid (5 min) and dose-related PKCβ activation as assessed by its membrane translocation. Among the proteins phosphorylated, we identified Hsp27. Both RACK1 pseudosubstrate and LPS induce its phosphorylation and release in culture medium. The release of Hsp27 induced by RACK1 pseudosubstrate was also confirmed in peripheral blood mononuclear cells. To evaluate the role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, we conducted Hsp27 silencing and neutralization experiments. Both strategies confirmed the central role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, as assessed by IL-8 production and upregulation of CD86.