HMGB1 Is Phosphorylated by Classical Protein Kinase C and Is Secreted by a Calcium-Dependent Mechanism

Abstract

High-mobility group box 1 protein (HMGB1) has been studied as a key mediator of inflammatory diseases, including sepsis. Regulating secretion is important in the control of HMGB1-mediated inflammation. Previously, it was shown that HMGB1 needs to be phosphorylated for secretion. In this study, we show that HMGB1 is phosphorylated by the classical protein kinase C (cPKC) and is secreted by a calcium-dependent mechanism. For this study, RAW264.7 cells and human peripheral blood monocytes were treated with PI3K inhibitors wortmannin, LY294002, and ZSTK474, resulting in inhibition of LPS-stimulated HMGB1 secretion, whereas inhibitors of NF-κB and MAPKs p38 and ERK showed no inhibition. Akt inhibitor IV and mammalian target of rapamycin inhibitor rapamycin did not inhibit HMGB1 secretion. However, the PKC inhibitors Gö6983 (broad-spectrum PKC), Gö6976 (cPKC), and Ro-31-7549 (cPKC) and phosphoinositide-dependent kinase 1 inhibitor, which results in protein kinase C (PKC) inhibition, inhibited LPS-stimulated HMGB1 secretion. PKC activators, PMA and bryostatin-1, enhanced HMGB1 secretion. In an in vitro kinase assay, HMGB1 was phosphorylated by recombinant cPKC and by purified nuclear cPKC from LPS-stimulated RAW264.7 cells, but not by casein kinase II or cdc2. HMGB1 secretion was also induced by the calcium ionophore A23187 and inhibited by the Ca2+ chelators BAPTA-AM and EGTA. These findings support a role for Ca2+-dependent PKC in HMGB1 secretion. Thus, we propose that cPKC is an effector kinase of HMGB1 phosphorylation in LPS-stimulated monocytes and PI3K-phosphoinositide-dependent kinase 1 may act in concert to control HMGB1 secretion independent of the NF-κB, p38, and ERK pathways.

Chaperone-like Activity of High-Mobility Group Box 1 Protein and Its Role in Reducing the Formation of Polyglutamine Aggregates

“…This translocation was decreased by treatment of ROS scavengers NAC and Mito-tempo, demonstrating the important role of ROS in HMGB1 translocation and binding to expanded polyQ. Our previous finding shows that posttranslational modification of phosphorylation is also responsible for the nucleocytoplasmic transport of HMGB1, and Ca 2+ -dependent classical protein kinase C is an effector kinase of HMGB1 phosphorylation (53). Considering that expanded polyQ expression induces Ca 2+ -dependent protein kinase C via metabotropic glutamate receptor-mediated cell signaling (54), it is possible that members of the classical protein kinase C enzyme (a, bI, bII, and g) family are the main kinases for nucleocytoplasmic transport of HMGB1 to interact with polyQ.…”

Messengers without Borders

“…[39][40][41] When HMGB1 is phosphorylated, its binding to the nuclear cargo carrier protein, karyopherin-a1, is reduced and HMGB1 is translocated to the cytoplasm and eventually secreted into the extracellular compartment. 42 Translocation of HMGB1 from the nucleus to the cytoplasm also involves its acetylation, 38,39,41 an effect possibly mediated by decreased deacetylase activity. 43 Furthermore, in the absence of calcium, HMGB1 DNA binding properties are enhanced.…”

p16INK4a Exerts an Anti-Inflammatory Effect through Accelerated IRAK1 Degradation in Macrophages

“…Human acute monocytic leukemia cell line THP-1 cells were cultured and differentiated to macrophages as described elsewhere (17). RAW264.7 cells were cultured as described elsewhere (18). For activation, cells were stimulated with the optimal doses of LPS, which were minimum dose to induce maximum IL-6 production in each cell type.…”