HMGB1 is phosphorylated by classical protein kinase C and is secreted by a calcium-dependent mechanism.
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-kappaB 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 Ca(2+) chelators BAPTA-AM and EGTA. These findings support a role for Ca(2+)-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-kappaB, p38, and ERK pathways.
Oh
Young Joo
YJ
Department of Microbiology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Youn
Ju Ho
JH
Ji
Yeounjung
Y
Lee
Sang Eun
SE
Lim
Kook Jin
KJ
Choi
Ji Eun
JE
Shin
Jeon-Soo
JS
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D015536:Down-Regulation / Q000276:immunology; D024243:HMGB1 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000502:physiology; D051379:Mice; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology
D024243:HMGB1 Protein; D008070:Lipopolysaccharides; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D002118:Calcium