High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.
cerebral ischemia; HMGB1; inflammation; RAGE; TLR
The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.
High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. To function as an alarmin, HMGB1 translocates from the nucleus of the cell to the extra-cellular milieu, a process that can take place with cell activation as well as cell death. HMGB1 can interact with receptors that include RAGE (receptor for advanced glycation endproducts) as well as Toll-like receptor-2 (TLR-2) and TLR-4 and function in a synergistic fashion with other proinflammatory mediators to induce responses. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. New approaches to therapy for these diseases may involve strategies to inhibit HMGB1 release from cells, its interaction with receptors, and downstream signaling.
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 functional relationship and cross-regulation between autophagy and apoptosis is complex. Here we show that high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anti-cancer agents enhanced autophagy and apoptosis as well as HMGB1 release. HMGB1 release may be a pro-survival signal for residual cells following various cytotoxic cancer treatments. Diminished HMGB1 by shRNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led to predominantly apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGE) but not Toll-like receptor 4 (TLR4), induces Beclin1-dependent autophagy, and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA-intercalators (oxaliplatin or adriamycin). Oxidized HMGB1 conversely increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release as well as its redox state thus link autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.
High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced glycation end products (RAGE), leading to tissue injury. Although HMGB1’s ability to induce inflammation has been well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1’s effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα, whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses.
High-mobility group box-1 (HMGB1) is a nuclear protein with cytokine-type functions upon its extracellular release. HMGB1 activates inflammatory pathways by stimulating multiple receptors, chiefly toll-like receptor 4 (TLR4) and Receptor for Advanced Glycation End Products (RAGE). TLR4 and RAGE activation has been implicated in memory impairments, although the endogenous ligand subserving these effects is unknown. We examined whether HMGB1 induced memory deficits using novel object recognition test, and which of the two receptor pathways was involved in these effects. Non-spatial long-term memory was examined in wild type, TLR4 knockout, and RAGE knockout mice. Recombinant HMGB1 (10 μg, intracerebroventricularly, i.c.v.) disrupted memory encoding equipotently in wild type, TLR4 knockout and RAGE knockout animals, but affected neither memory consolidation, nor retrieval. Neither TLR4 knockout nor RAGE knockout mice per se, exhibited memory deficits. Blockade of TLR4 in RAGE knockout mice using Rhodobacter sphaeroides lipopolysaccharide (LPS-Rs; 20 μg, i.c.v.) prevented the detrimental effect of HMGB1 on memory. These data show that elevated brain levels of HMGB1 induce memory abnormalities which may be mediated by either TLR4, or RAGE. This mechanism may contribute to memory deficits under various neurological and psychiatric conditions associated with the increased HMGB1 levels, such as epilepsy, Alzeheimer’s disease and stroke.
Inflammation; High-mobility group box-1; toll-like receptor 4; Receptor for Advanced Glycation End Products; memory; novel object recognition test
A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE), or Toll-like receptor 4 (TLR4), HMGB1 activates macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of observations that many viruses (e.g., West Nile virus, Salmon anemia virus) can induce passive HMGB1 release, we propose a potential pathogenic role of HMGB1 in viral infectious diseases.
High mobility group box chromosomal protein 1 (HMGB1) is a DNA binding protein that exhibits pro-inflammatory properties when present in the extracellular compartment. Putative receptors for HMGB1 include Toll-like receptor (TLR) 4, TLR2 and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce tolerance to the bacterial product, lipoteichoic acid (LTA). Pretreatment of human monocyte-like THP-1 cells with 1 μg/ml HMGB1 18 h before exposure to LTA (10 μg/ml) decreased secretion of TNF, NF-κB DNA-binding, and degradation of IκBα. Denaturation of HMGB1 with boiling water or co-incubation with anti-HMGB1 antibody abrogated the induction of tolerance to LTA. In contrast, co-incubation with polymyxin B failed to diminish HMGB1-induced tolerance to LTA. These findings support the view that the induction of LTA tolerance by HMGB1 was not due to LPS contamination. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type and RAGE-deficient mice became LTA-tolerant following HMGB1 exposure ex vivo. We were unable to demonstrate LTA tolerance in TLR2 and TLR4-deficient macrophages, as they are hyporesponsive to LTA. These findings suggest that extracellular HMGB1 induces LTA tolerance, and RAGE receptor is not required for this induction.
High mobility group box chromosomal protein 1 (HMGB1); Lipoteichoic acid; tolerance; signal transduction; Toll-like receptor -2
A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells, and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE) or toll-like receptor 4 (TLR4), HMGB1 activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cytokines, chemokines and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of the pathogenic role of inflammation in cardiovascular diseases, we propose that HMGB1, a proinflammatory cytokine derived from both injured endothelium and activated macrophages/monocytes, could contribute to the progression of atherosclerosis and other cardiovascular diseases.
High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23–C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4−/−, CD14−/−, TIR domain-containing adapter-inducing interferon-β (TRIF)−/− or myeloid differentiation primary response protein 88 (MyD88)−/− macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma–induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.
High mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear binding protein that has recently been shown to be an early trigger of sterile inflammation in animal models of trauma-hemorrhage via the activation of the Toll-like-receptor 4 (TLR4) and the receptor for the advanced glycation endproducts (RAGE). However, whether HMGB1 is released early after trauma hemorrhage in humans and is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of the present study.
One hundred sixty eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level 1 Trauma center. Blood was drawn within 10 minutes of arrival to the emergency room before the administration of any fluid resuscitation. HMGB1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, von Willebrand Factor (vWF), angiopoietin-2 (Ang-2), Prothrombin time (PT), prothrombin fragments 1+2 (PF1+2), soluble thrombomodulin (sTM), protein C (PC), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and D-Dimers were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared to outcome measures obtained from the electronic medical record and trauma registry.
Plasma levels of HMGB1 were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, tissue hypoperfusion, early posttraumatic coagulopathy and hyperfibrinolysis as well with a systemic inflammatory response and activation of complement. Non-survivors had significantly higher plasma levels of HMGB1 than survivors. Finally, patients who later developed organ injury, (acute lung injury and acute renal failure) had also significantly higher plasma levels of HMGB1 early after trauma.
The results of this study demonstrate for the first time that HMGB1 is released into the bloodstream early after severe trauma in humans. The release of HMGB1 requires severe injury and tissue hypoperfusion, and is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.
Recent studies have found that overexpression of the High-mobility group box-1 (HMGB1) protein, in conjunction with its receptors for advanced glycation end products (RAGEs) and toll-like receptors (TLRs), is associated with proliferation of various cancer types, including that of the breast and pancreatic.
We have developed a rule-based model of crosstalk between the HMGB1 signaling pathway and other key cancer signaling pathways. The model has been simulated using both ordinary differential equations (ODEs) and discrete stochastic simulation. We have applied an automated verification technique, Statistical Model Checking, to validate interesting temporal properties of our model.
Our simulations show that, if HMGB1 is overexpressed, then the oncoproteins CyclinD/E, which regulate cell proliferation, are overexpressed, while tumor suppressor proteins that regulate cell apoptosis (programmed cell death), such as p53, are repressed. Discrete, stochastic simulations show that p53 and MDM2 oscillations continue even after 10 hours, as observed by experiments. This property is not exhibited by the deterministic ODE simulation, for the chosen parameters. Moreover, the models also predict that mutations of RAS, ARF and P21 in the context of HMGB1 signaling can influence the cancer cell's fate - apoptosis or survival - through the crosstalk of different pathways.
Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process.
Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo.
Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro.
HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.
Type 1 diabetes mellitus is caused by the autoimmune destruction of β cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were β cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on β cells and that HMGB1 may signal via TLR4 to selectively damage β cells rather than α cells during the development of type 1 diabetes mellitus.
diabetes mellitus, type 1; HMGB1 protein; islets of Langerhans; mice, inbred NOD; toll-like receptor 4
The high mobility group box 1 (HMGB1) protein is an abundant non-histone component of chromatin well known for its two DNA binding domains, HMG box A and HMG box B. The main characteristics of the HMGB1 protein as an ‘architectural’ factor are its ability to recognize and bind with high affinity to distorted DNA and its ability to induce kinks in linear DNA fragments. The HMGB1 protein has been correlated to cancer progression. An elevated expression of HMGB1 occurred in certain types of primary tumor, including melanoma and colon, prostate, pancreatic and breast cancers, and in the majority of cases HMGB1 is associated with invasion and metastasis. The main signaling pathway is activated through the interaction of HMGB1 with its Receptor for Advanced Glycation End products (RAGE). Certain data indicate that an elevated expression of RAGE and HMGB1 is not always a prerequisite of poor prognosis of tumor development. The cellular localization of the ligand/receptor pair also requires consideration. The data concerning the expression of HMGB1 protein and its receptor RAGE in various tissues and tumor cells reflect the overall production of the proteins. However, they do not refer to their cellular localization and there is no direct evidence for the formation of a stable complex between them. In the present study, we investigated the subcellular distribution of HMGB1 and its receptor RAGE in various rat organs compared to Guerin ascites tumor cells. In the normal tissues the proteins exist in their soluble form, whereas in the tumor cells they are insoluble and membrane-bound. HMGB1 forms a stable complex with RAGE only in the protein extract derived from the cancer cells predominantly in the membrane fraction.
HMGB1 protein; RAGE; tumorigenesis; subcellular localization
In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes.
Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA.
Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively.
HMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.
CXCL12 forms a complex with HMGB1 that binds to the chemokine receptor CXCR4 and increases inflammatory cell migration.
After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1-induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1–CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms.
High-mobility group box 1 protein (HMGB1), an abundant nuclear protein, was recently established as a proinflammatory mediator of experimental sepsis. Although extracellular HMGB1 has been found in atherosclerotic plaques, its potential role in the pathogenesis of atherothrombosis remains elusive. In the present study, we determined whether HMGB1 induces tissue factor (TF) expression in vascular endothelial cells (ECs) and macrophages. Our data showed that HMGB1 stimulated ECs to express TF (but not TF pathway inhibitor) mRNA and protein in a concentration- and time-dependent manner. Blockade of cell surface receptors (including TLR4, TLR2, and RAGE) with specific neutralizing antibodies partially reduced HMGB1-induced TF expression. Moreover, HMGB1 increased expression of Egr-1 and nuclear translocation of NF-κB (c-Rel/p65) in ECs. Taken together, our data suggest that HMGB1 induces TF expression in vascular endothelial cells via cell surface receptors (TLR4, TLR2, and RAGE), and through activation of transcription factors (NF-κB and Egr-1).
tissue factor; HMGB1; thrombosis; atherosclerosis
High-mobility group protein box-1 (HMGB1) has recently been recognized as a novel candidate in a specific upstream pathway promoting inflammation after brain ischemia. However, its downstream pathway and underlying mechanism have yet to be elucidated. The HMGB1 level in the acute cerebral infarct (ACI) group was significantly increased compared with that of control group, and correlated with the severity of neurologic impairment of ACI patients. Further, recombinant human HMGB1 (rhHMGB1) had no effect on microglia derived from mice lacking the Toll-like receptor 4 (TLR4−/−). Intracerebroventricular injection of rhHMGB1 in TLR4+/+ mice cause significantly more injury after cerebral ischemia–reperfusion than control group. But, TLR4−/− mice administered with rhHMGB1 showed moderate impairment after ischemia–reperfusion than TLR4+/+ mice. To determine the potential downstream signaling of HMGB1/TLR4 in cerebral ischemic injury, we used the ischemic–reperfusion model with Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β knockout mice (TRIF−/−) and evaluated the activity and expression of TRIF pathway-related kinases. The results suggest that the TRIF pathway is not likely to be involved in TLR4-mediated ischemia brain injury. Finally, we found that TLR4 expressed by immigrant macrophages was involved in the development of ischemic brain damage. These results suggest that HMBG1 mediates ischemia–reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. The TLR4 expressed by immigrant macrophages may be involved in the development of ischemic brain damage.
acute cerebral infarct; HMGB1; ischemia–reperfusion injury; TLR4
Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4−/− mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.
The migration of hepatic stellate cells (HSCs) is essential to the hepatic fibrotic response, and recently High-mobility group box 1 (HMGB1) has been shown up-regulated during liver fibrosis. Nevertheless, whether HMGB1 can modulate the proliferation and migration of HSCs is poorly understood, as well as the involved intracellular signaling. In this study, we examined the effect of HMGB1 on proliferation, migration, pro-fibrotic function of HSCs and investigated whether toll-like family of receptor 4 (TLR4) dependent signal pathway is involved in the intracellular signaling regulation.
Modified transwell chamber system to mimic the space of Disse was used to evaluate the migration of human primary HSCs, and the protein expressions of related signal factors were evaluated by western blot. Cell proliferation was analyzed by MTT assay, the pro-fibrotic functions of HSCs by qRT-PCR and ELISA respectively. Recombinant human HMGB1 could significantly promote migration of HSCs under both haptotactic and chemotactic stimulation, especially the latter. Human TLR4 neutralizing antibody could markedly inhibit HMGB1-induced migration of HSCs. HMGB1 could enhance the phosphorylation of JNK and PI3K/Akt, and TLR4 neutralizing antibody inhibited HMGB1-enhanced phosphorylation of JNK and PI3K/Akt and activation of NF-κB. JNK inhibitor (SP600125) and PI3K inhibitor (LY 294002) significantly inhibited HMGB1-induced proliferation and migration of HSCs, and also reduced HMGB1-enhanced related collagen expressions and pro-fibrotic cytokines production.
HMGB1 could significantly enhance migration of HSCs in vitro, and TLR4-dependent JNK and PI3K/Akt signal pathways are involved in the HMGB1-induced proliferation, migration and pro-fibrotic effects of HSCs, which indicates HMGB1 might be an effective target to treat liver fibrosis.
Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.
High mobility group protein box 1 (HMGB1) is a DNA binding protein located in nucleus. It is released into extracellular fluid where it acts as a novel proinflammatory cytokine which interacts with Toll like receptor 4 (TLR4) to activate nuclear factor-κB (NF-κB). This sequence of events is involved in tumor growth and progression. However, the effects of HMGB1, TLR4 and NF-κB on epidermal tumors remain unclear.
Human epidermal tumor specimens were obtained from 96 patients. Immunohistochemistry was used to detect expression of HMGB1, TLR4 and NF-κB p65 in human epidermal tumor and normal skin specimens. Western blot analysis was used to detect the expression of NF-κB p65 in epithelial cell nuclei in human epidermal tumor and normal tissues.
Immunohistochemistry and western blot analysis indicated a progressive but statistically significant increase in p65 expression in epithelial nuclei in benign seborrheic keratosis (SK), precancerous lesions (PCL), low malignancy basal cell carcinoma (BCC) and high malignancy squamous cell carcinoma (SCC) (P <0.01). The level of extracellular HMGB1 in SK was significantly higher than in normal skin (NS) (P <0.01), and was higher than in SCC but without statistical significance. The level of TLR4 on epithelial membranes of SCC cells was significantly higher than in SK, PCL, BCC and NS (P <0.01). There was a significant positive correlation between p65 expression in the epithelial nuclei and TLR4 expression on the epithelial cell membranes (r = 0.3212, P <0.01).
These findings indicate that inflammation is intensified in parallel with increasing malignancy. They also indicate that the TLR4 signaling pathway, rather than HMGB1, may be the principal mediator of inflammation in high-grade malignant epidermal tumors. Combined detection of p65 in the epithelial nuclei and TLR4 on the epithelial membranes may assist the accurate diagnosis of malignant epidermal tumors.
HMGB1; TLR4; NF-κB; Seborrheic keratosis; Precancerous lesions; Squamous cell carcinoma
High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells.
Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data.
These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection.