Calcium/Calmodulin-Dependent Protein Kinase (CaMK) IV Mediates Nucleocytoplasmic Shuttling and Release of HMGB1 during Lipopolysaccharide Stimulation of Macrophages

Abstract

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mφ) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mφ were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mφ isolated from CaMKIV+/+ and CaMKIV−/− mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mφ treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV+/+ Mφ showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV−/− Mφ. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.

Bacterial Endotoxin Induces the Release of High Mobility Group Box 1 via the IFN-β Signaling Pathway

“…Recently, it was published that calcium/calmodulin-dependent protein kinase (CaMK) mediated HMGB1 release from macrophages treated with LPS (41). By using various methods to prevent CaMK activation, such as chemical inhibitors (STO609 and KN93), RNA interference, as well as gene knockout mice, the authors showed that CaMK promotes the extracellular release of HMGB1.…”

Messengers without Borders

“…[44][45][46] Recent studies suggest the possible role of elevated cytosolic calcium concentration and calcium/calmodulin-dependent and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent regulatory pathways in the nucleocytoplasmic shuttling and release of HMGB1. 39,40 Oh et al showed in monocytes that HMGB1 secretion is induced by a calcium ionophore and inhibited by calcium chelators. 39 These authors found that calcium-dependent protein kinase C phosphorylates HMGB1 in LPS-activated cells.…”

“…For instance, Youn et al have shown in monocytes that phosphorylation of HMGB1 modulates its nucleocytoplasmic shuttling. [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.…”

Protective effect of olmesartan against cardiac ischemia/reperfusion injury in spontaneously hypertensive rats

“…I/R injury is characterized by marked oxidative stress and intracellular calcium overload (38), which activates the release of HMGB1 (32,39). HMGB1 then acts via its receptor (RAGE/TL4) to stimulate the release of proinflammatory cytokines, induce NFκB and enhance myocardial injury (8,9).…”