John R. Klune

While foreign pathogens and their products have long been known to activate the innate immune system, the recent recognition of a group of endogenous molecules that serve a similar function has provided a framework for understanding the overlap between the inflammatory responses activated by pathogens and injury. These endogenous molecules, termed alarmins, are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. One nuclear protein, High mobility group box-1 (HMGB1), has received particular attention as fulfilling the functions of an alarmin by being involved in both infectious and non-infectious inflammatory conditions. Once released, HMGB1 signals through various receptors to activate immune cells involved in the immune process. Although initial studies demonstrated HMGB1 as a late mediator of sepsis, recent findings indicate HMGB1 to have an important role in models of non-infectious inflammation, such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. Furthermore, in contrast to its pro-inflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration. The complex functions of HMGB1 as an archetypical alarmin are outlined here to review our current understanding of a molecule that holds the potential for treatment in many important human conditions.

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1. The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.