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* Department of Microbiology, Brain Korea 21 Project for Medical Science,
Institute for Immunology and Immunological Diseases, and
National Core Research Center for Nanomedical Technology, Yonsei University College of Medicine, Seoul, Korea;
Diagnostic Division, LG Life Sciences, Seoul, Korea; and
¶ Department of Pediatrics, Seoul National University Borame Hospital, Seoul, Korea
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-
B 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-B, p38, and ERK pathways.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Korean Health 21 Research & Development Project (A050260) of the Ministry of Health & Welfare, a Korea Research Foundation grant funded by the Korean government (MOEHRD) (KRF-E00154), a KOSEF grant through the NCRC for Nanomedical Technology (R15-2004-024-00000-0), and a grant (F104AB010006-07A0201-00610) from the Korea Biotech Research & Development Group of the Next-Generation Growth Engine Project of the Ministry of Education, Science and Technology and the Brain Korea 21 Project for Medical Sciences, Republic of Korea.
2 Y.J.O. and J.H.Y. contributed equally.
3 Address correspondence and reprint requests to Dr. Jeon-Soo Shin, Department of Microbiology, Yonsei University College of Medicine, 250 Seongsanno Seodaemoongu, Seoul 120-752, Korea. E-mail address: jsshin6203{at}yuhs.ac
4 Abbreviations used in this paper: HMGB1, high-mobility group box 1 protein; CaM, calmodulin; CaMK, CaM kinase; CK II, casein kinase II; DAG, diacylglycerol; PBMo, peripheral blood monocyte; PDK1, phosphoinositide-dependent kinase 1; PKC, protein kinase C; cPKC, classical PKC; nPKC, novel PKC; pSer, phospho-Ser; NLS, nuclear localization signal; WT, wild type; RT, room temperature; mTOR, mammalian target of rapamycin; BAY-11, BAY 11-7082.
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