The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

LC3 is widely used marker for macroautophagy assays. After translation pro-LC3 is processed by Atg4 to expose C-terminal glycine residue for downstream conjugation reactions to accomplish the conversion of LC3-I to LC3-II. SDS-PAGE based Western blot (Wb) is generally utilized to quantify LC3-II levels where the LC3-I band migrates slower than LC3-II. We found that pro-human LC3B migrated at similar rate as LC3B-II in SDS-PAGE. The carboxyl-terminal five amino acids, particularly Lysine122 and Leucine123 of human LC3B play a major role in the faster migration of unprocessed LC3B, rendering it indistinguishable from LC3B-II in Wb assays. The unique faster migration of unprocessed LC3B than LC3B-I is also revealed in mouse LC3B, rat LC3B and rat LC3 but not in human LC3C. Our findings for the first time define pro-LC3 migration patterns for LC3 family member from human, mouse and rat species in SDS-PAGE. These findings provide a reference for pro-LC3 band patterns when Atg4 function is inhibited. © 2013 Wang et al

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression

Effect of rejection on electrophysiologic function of canine intestinal grafts: Correlation with histopathology and na-k-ATPase activity

To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na-K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18-24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were snitched between a pair of dogs (allograft). Animals receiving intestinal autotransplantation were used as controls. AIIograji recipients were sacrificed 3, 4, 5, 7, or 9 days after transplantation, and autograft recipients were sacrificed 3, 7, or 14 days afier transplantation. Immunosuppression was not used. Electrophysiologic measurements were done with an Ussing chamber. Histological analysis was performed blindly using whole thickness sections. Na-K-ATPase activity in the mucosal tissue, which is said to regulate the potential difference, was also measured. Potential difference, resistance, and Na-K-ATPase activity of the allografi intestine decreased with time and were significantly lower 7 and 9 days after transplantation compared to host intestine, normul intestine, and graft intestine of controls (autograft). Potential difference, resistance, and Na-K-ATPase activity of the native intestinal tissue and the autografts did not decrease with time. Detection of histologically mild rejection of the intestine, which is important for appropriate immunosup-pressive treatment in clinical cases, could not be achieved based on electrophysiology or Na-K-ATPase activity. Deterioration of electrophysiologic function during rejection correlated with the histological rejection process and Na-K-ATPase activity; however, electrophysiology my not be a reliable tool for monitoring grafrs, since it cannot detect early intestinal rejection. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis

Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high-mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1 alpha (HIF1 alpha) and activates the HIF-1 alpha-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1 alpha and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels, and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases

The HIV protease inhibitor saquinavir inhibits HMGB1 driven inflammation by targeting the interaction of cathepsin V with TLR4/MyD88

Extracellular HMGB1 (disulfide form), via activation of Toll-Like-Receptor (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report, that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1 induced TLR4 activation, as assayed by the production of TNF-alpha by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334, or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver I/R, models both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4 driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside

The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion

High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH2-terminal kinase and higher nuclear factor–κB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor kappaB, degradation of inhibitor of kappaB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor kappaB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage

Combined In Silico, In Vivo, and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO2-/NO3- data from "middle-aged" (6-8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for "young" (2-3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging. © 2013 Namas et al