ORIGINAL ARTICLE The role of HSP27 in RACK1-mediated PKC activation in THP-1 cells Emanuela Corsini 1 • Valentina Galbiati 1 • Angela Papale 1 • Elena Kummer 1 • Antonella Pinto 2 • Antonio Guaita 3 • Marco Racchi 2 Ó Springer Science+Business Media New York 2016 Abstract Receptor for Activated C Kinase 1 (RACK1) pseudosubstrate is a commercially available peptide that directly activates protein kinase C-b (PKCb). 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 PKCb 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 pseu- dosubstrate-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 PKCb activation as assessed by its membrane transloca- tion. 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 produc- tion and upregulation of CD86. Keywords RACK1 Á PKC Á Cytokines Á CD86 Á mRNA stability Introduction In early studies aimed to characterize the molecular mechanisms underlying immunosenescence, we identified a defective protein kinase C (PKC) activation central to the reduced response to immune stimulation. In particular, we demonstrated that in the absence of a defect in total PKC expression, the failing element in its activation was the reduced expression of the Receptor for Activated C Kinase 1 (RACK1), which underlies functional impairment asso- ciated with aging, including cytokine production, cell proliferation [1–4], and response to influenza vaccination [5]. More recently, we showed that the direct activation of PKCb using RACK1 pseudosubstrate, alone or in combi- nation with classical immune activators, results in increased cytokine production and CD86 upregulation, and in reversion of some of the age-associated immune dys- functions [6]. RACK1 pseudosubstrate alone induced IL-6, IL-8, and CD86 expression in both young and old donors, and IFN-c in old donors. Using the pseudosubstrate in combination with LPS, we observed an increase in IL-8, IL-10, and TNF-a, resulting in restoration of age-associ- ated defective production, supporting a central, although not exclusive role of PKCb, in immune cell activation and immunosenescence. To further support its role in immunity, we also demonstrated that PKCb activation is a key component of the signal transduction pathways that induce dendritic cells & Marco Racchi racchi@unipv.it 1 Laboratory of Toxicology, DiSFeB, Universita ` degli Studi di Milano, Milan, Italy 2 Department of Drug Sciences - Pharmacology, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy 3 ‘‘Golgi Cenci’’ Foundation, Abbiategrasso, Italy 123 Immunol Res DOI 10.1007/s12026-016-8802-1