High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-B and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Tolllike receptor ( HMGB11 (formerly HMG1) was originally described as a non-histone, chromatin-associated nuclear protein (1-4).HMGB1 has a highly conserved sequence among species, with murine HMGB1 differing from the human form by only two amino acids. HMGB1-deficient mice die within a few hours of birth, demonstrating the crucial role of this protein in cellular function. HMGB1 consists of two tandem L-shaped domains, HMGB boxes A and B, each ϳ75 amino acids in length, and a highly acidic carboxyl terminus of 30 amino acids in length.HMGB1 appears to have two distinct functions in cellular systems. First, it has been shown to be an intracellular regulator of transcription, and, second, HMGB1 can occupy an extracellular role in which it promotes tumor metastasis and inflammation (2-9). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (10, 11). Monocytes and macrophages stimulated by lipopolysaccharide (LPS), tumor necrosis factor (TNF)-␣, or interleukin-1 (IL-1) secrete HMGB1 (5, 11). Culture of monocytes with HMGB1 results in the release of TNF-␣, IL-1␣, IL-1␤, IL-1Ra, IL-6, IL-8, macrophage inflammatory protein-1␣, macrophage inflammatory protein-1␤, but not IL-10 or IL-12 (5, 11). Production of proinflammatory cytokines after exposure to HMGB1 occurs with delayed kinetics as compared with LPS-induced stimulation. For example, culture of macrophages with LPS results in increases in TNF-␣ that are apparent within less than 1 h, whereas TNF-␣ synthesis following HMGB1 exposure only begins to occur after 2 h and then persists for as long as 8 h (8, 11). The signaling mechanisms responsible for the delayed expression of proinflammatory cytokines by HMGB1-stimulated cells remain incompletely explained but appear to involve the p38, ERK, JNK, and Akt kinases and to lead to enhanced nuclear translocation of .In recent studies (13), we found that the magnitude and kinetics of cytokine expression and nuclear translocation of NF-B after culture of neutrophils with HMGB1 or LPS were similar, suggesting that overlapping mechanisms of cellular activation might be involved. Comparison of gene expression arrays also demonstrated substantial but not complete homology in response to HMGB1 and LPS. Signaling via the TLR 4 receptor is responsible for LPS-induced activation of the IKK kinase complex, including IKK...

Activation of microglia by classical inflammatory mediators can convert astrocytes to a neurotoxic A1 phenotype in a variety of neurological diseases1,2. Development of agents that could inhibit the formation of A1 reactive astrocytes could be used to treat these diseases for which there are no disease modifying therapies. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been touted as potential neuroprotective agents for neurologic disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD)3-13. The mechanisms by which GLP-1R agonists are neuroprotective are not known. Here we show that a potent, brain penetrant long acting GLP-1R agonist NLY01 protects against the loss of dopamine neurons and behavioral deficits in the α-synuclein preformed fibril (α-syn PFF) model of sporadic PD14,15. NLY01 also prolongs the life and reduces the behavioral deficits and neuropathological abnormalities in the human A53T α-synuclein (hA53T) transgenic (Tg) model of α-synucleinopathy induced neurodegeneration16. We found that NLY01 is a potent GLP-1R agonist with favorable properties that is neuroprotective via the direct prevention of microglial mediated conversion of astrocytes to an A1 neurotoxic phenotype. In light of NLY01 favorable properties it should be evaluated in the treatment of PD and related neurologic disorders characterized by microglial activation.

High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-Bdependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS. fluorescence resonance energy transfer; receptor of advanced glycation end products HIGH MOBILITY GROUP BOX 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly by monocytes and macrophages stimulated by LPS, TNF-␣, or IL-1 (2, 44). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (1,44,46), and also appears to be involved in pathophysiological processes associated with cellular necrosis, such as acetaminophen-induced liver injury (34).Although HMGB1 and LPS appear to initiate similar intracellular events, including activation of kinases such as p38, ERK1/2, and Akt and transcriptional factors including NF-B, that lead to production of proinflammatory cytokines, gene arrays demonstrated differences in expression profiles with each of these stimuli (12, 30). Unlike LPS, which primarily increased the activity of IKK-␤, HMGB1 exposure resulted in activation of both IKK-␣ and IKK-␤ (31). In addition, culture of neutrophils lacking Toll-like receptor (TLR)4 with HMGB1, but not with LPS, still resulted in enhanced nuclear translocation of NF-B (31). Such results suggest that the receptors interacting with HMGB1 and leading to cellular activation and gene transcription are likely to be distinct from TLR4, which is responsible for LPS-induced responses (40). Recent data indicate that HMGB1 interacts not only with TLR4 but also with TLR2 and the receptor for advanced glycation end products (RAGE) (31, 46). In particular, a decrease in NF-B-dependent reporter gene expression after transfection with dominantnegative constructs to TLR2, TLR4, or both, demonstr...

High mobility group box 1 (HMGB1) protein, a DNA binding protein that stabilizes nucleosomes and facilitates transcription, was recently identified as a late mediator of endotoxin lethality. High serum HMGB1 levels in patients with sepsis are associated with increased mortality, and administration of HMGB1 produces acute inflammation in animal models of lung injury and endotoxemia. Neutrophils occupy a critical role in mediating the development of endotoxemia-associated acute lung injury, but previously it was not known whether HMGB1 could influence neutrophil activation. In the present experiments, we demonstrate that HMGB1 increases the nuclear translocation of NF-kappaB and enhances the expression of proinflammatory cytokines in human neutrophils. These proinflammatory effects of HMGB1 in neutrophils appear to involve the p38 MAPK, phosphatidylinositol 3-kinase/Akt, and ERK1/2 pathways. The mechanisms of HMGB1-induced neutrophil activation are distinct from endotoxin-induced signals, because HMGB1 leads to a different profile of gene expression, pattern of cytokine expression, and kinetics of p38 activation compared with LPS. These findings indicate that HMGB1 is an effective stimulus of neutrophil activation that can contribute to development of a proinflammatory phenotype in diseases characterized by excessively high levels of HMGB1.

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0 -10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90 -240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor ␣ receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor ␣ (TGF␣) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGF␣ cleavage 120 -180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGF␣. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGF␣. Neutralization of TGF␣ function by an anti-TGF␣ antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGF␣-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.© 1999 by The American Society for Cell Biology 2493 INTRODUCTIONIonizing radiation has been shown to activate multiple signaling pathways within cells in vitro, which can lead to either increased cell death or increased proliferation depending on the cell type, the radiation dose, and the culture conditions (Xia et al., 1995;Rosette and Karin, 1996;Santana et al., 1996;Chmura et al., 1997;Schmidt-Ullrich et al., 1997;Carter et al., 1998;Haimovitz-Friedman, 1998;Kavanagh et al., 1998). Recently, a novel cellular target for ionizing radiation has been shown to be the ...

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-κB, and expression of the proinflammatory cytokines IL-1β and TNF-α through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110γ catalytic subunit of PI3-K. In PI3-Kγ−/− mice, lung edema, neutrophil recruitment, nuclear translocation of NF-κB, and pulmonary levels of IL-1β and TNF-α were significantly lower after endotoxemia as compared with PI3-Kγ+/+ controls. Among neutrophils that did accumulate in the lungs of the PI3-Kγ−/− mice after endotoxin administration, activation of NF-κB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kγ+/+ mice. These results show that PI3-K, and particularly PI3-Kγ, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.

Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signalregulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun