Nuclear High Mobility Group Box Research Articles

PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication.

Porcine circovirus type 2 (PCV2) is an important swine pathogen that causes significant economic losses to the pig industry. PCV2 interacts with host cellular factors to regulate its replication. High-mobility-group box 1 (HMGB1) protein, a major nonhistone protein in the nucleus, was recently discovered to participate in viral infections. Here, we demonstrate that nuclear HMGB1 negatively regulated PCV2 replication as shown by overexpression of HMGB1 or blockage of its nucleocytoplasmic translocation with ethyl pyruvate. The B box domain was essential in restricting PCV2 replication. Nuclear HMGB1 restricted PCV2 replication by sequestering the viral genome via binding to the Ori region. However, PCV2 infection induced translocation of HMGB1 from cell nuclei to the cytoplasmic compartment. Elevation of reactive oxygen species (ROS) induced by PCV2 infection was closely associated with cytosolic translocation of nuclear HMGB1. Treatment of PCV2-infected cells with ethyl pyruvate or N-acetylcysteine downregulated PCV2-induced ROS production, suppressed nucleocytoplasmic HMGB1 translocation, and decreased PCV2 replication. Collectively, these findings offer new insight into the mechanism of the PCV2 evasion strategy: PCV2 manages to escape restriction of its replication by nuclear HMGB1 by inducing ROS to trigger the nuclear-to-cytoplasmic translocation of HMGB1.IMPORTANCE Porcine circovirus type 2 (PCV2) is a small DNA virus that depends heavily on host cells for its infection. This study reports the close relationship between subcellular localization of host high-mobility-group box 1 (HMGB1) protein and viral replication during PCV2 infection. Restriction of PCV2 replication by nuclear HMGB1 is the early step of host defense at the host-pathogen interface. PCV2 then upregulates host reactive oxygen species (ROS) to prevent sequestration of its genome by expelling nuclear HMGB1 into the cytosol. It will be interesting to study if a similar evasion strategy is employed by other circoviruses such as beak and feather disease virus, recently discovered PCV3, and geminiviruses in plants. This study also provides insight into the justification and pharmacological basis of antioxidants as an adjunct therapy in PCV2 infection or possibly other diseases caused by the viruses that deploy the ROS-HMGB1 interaction favoring their replication.

Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms.

A major risk factor for major depressive disorder (MDD) is stress. Stress leads to the release of high-mobility group box-1 (HMGB1), which in turn leads to neuroinflammation, a potential pathophysiological basis of MDD. The mechanism underlying stress-induced HMGB1 release is not known, but stress-associated glucocorticoids could be involved. To test this, rat primary cultured cortical astrocytes, the most abundant cell type in the central nervous system (CNS), were treated with corticosterone and HMGB1 release was assessed by Western blotting and ELISA. Significant HMGB1 was released with treatment with either corticosterone or dexamethasone, a synthetic glucocorticoid. HMGB1 translocated from the nucleus to the cytoplasm following corticosterone treatment. HMGB1 release was significantly attenuated with glucocorticoid receptor blocking. In addition, inhibition of pannexin-1, and P2X7 receptors led to a significant decrease in corticosterone-induced HMGB1 release. Taken together, corticosterone stimulates astrocytic glucocorticoid receptors and triggers cytoplasmic translocation and extracellular release of nuclear HMGB1 through a mechanism involving pannexin-1 and P2X7 receptors. Thus, under conditions of stress, glucocorticoids induce astrocytic HMGB1 release, leading to a neuroinflammatory state that could mediate neurological disorders such as MDD.

Exploring the role of high-mobility group box 1 (HMGB1) protein in the pathogenesis of Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by an increased and unstable CAG DNA expansion in the Huntingtin (HTT) gene, resulting in an elongated polyglutamine tract in huntingtin protein. Despite its monogenic cause, HD pathogenesis remains elusive and without any approved disease-modifying therapy as yet. A growing body of evidence highlights the emerging role of high-mobility group box 1 (HMGB1) protein in HD pathology. HMGB1, being a nuclear protein, is primarily implicated in DNA repair, but it can also translocate to the cytoplasm and participate into numerous cellular functions. Cytoplasmic HMGB1 was shown to directly interact with huntingtin under oxidative stress conditions and induce its nuclear translocation, a key process in the HD pathogenic cascade. Nuclear HMGB1 acting as a co-factor of ataxia telangiectasia mutated and base excision repair (BER) complexes can exert dual roles in CAG repeat instability and affect the final DNA repair outcome. HMGB1 can inhibit mutant huntingtin aggregation, protecting against polyglutamine-induced neurotoxicity and acting as a chaperon-like molecule, possibly via autophagy regulation. In addition, HMGB1 being a RAGE and TLR-2, TLR-3, and TLR-4 ligand may further contribute to HD pathogenesis by triggering neuroinflammation and apoptosis. Furthermore, HMGB1 participates at the unfolded protein response (UPR) system and can induce protein degradation and apoptosis associated with HD. In this review, we discuss the multiple role of HMGB1 in HD pathology, providing mechanistic insights that could direct future studies towards the development of targeted therapeutic approaches.

Uterine deficiency of high-mobility group box-1 (HMGB1) protein causes implantation defects and adverse pregnancy outcomes.

A reciprocal communication between the implantation-competent blastocyst and the receptive uterus is essential to successful implantation and pregnancy success. Progesterone (P4) signaling via nuclear progesterone receptor (PR) is absolutely critical for pregnancy initiation and its success in most eutherian mammals. Here we show that a nuclear protein high-mobility group box-1 (HMGB1) plays a critical role in implantation in mice by preserving P4-PR signaling. Conditional deletion of uterine Hmgb1 by a Pgr-Cre driver shows implantation defects accompanied by decreased stromal cell Hoxa10 expression and cell proliferation, two known signatures of inefficient responsiveness of stromal cells to PR signaling in implantation. These mice evoke inflammatory conditions with sustained macrophage accumulation in the stromal compartment on day 4 of pregnancy with elevated levels of macrophage attractants Csf1 and Ccl2. The results are consistent with the failure of exogenous P4 administration to rescue implantation deficiency in the mutant females. These early defects are propagated throughout the course of pregnancy and ultimately result in substantial subfertility. Collectively, the present study provides evidence that nuclear HMGB1 contributes to successful blastocyst implantation by sustaining P4-PR signaling and restricting macrophage accumulation to attenuate harmful inflammatory responses.

Peroxiredoxin-mediated disulfide bond formation is required for nucleocytoplasmic translocation and secretion of HMGB1 in response to inflammatory stimuli

The nuclear protein HMGB1 (high mobility group box 1) is secreted by monocytes-macrophages in response to inflammatory stimuli and serves as a danger-associated molecular pattern. Acetylation and phosphorylation of HMGB1 are implicated in the regulation of its nucleocytoplasmic translocation for secretion, although inflammatory stimuli are known to induce H2O2 production. Here we show that H2O2-induced oxidation of HMGB1, which results in the formation of an intramolecular disulfide bond between Cys23 and Cys45, is necessary and sufficient for its nucleocytoplasmic translocation and secretion. The oxidation is catalyzed by peroxiredoxin I (PrxI) and PrxII, which are first oxidized by H2O2 and then transfer their disulfide oxidation state to HMGB1. The disulfide form of HMGB1 showed higher affinity for nuclear exportin CRM1 compared with the reduced form. Lipopolysaccharide (LPS)–induced HMGB1 secretion was greatly attenuated in macrophages derived from PrxI or PrxII knockout mice, as was the LPS-induced increase in serum HMGB1 levels.

Cardiac Nuclear High-Mobility Group Box 1 Ameliorates Pathological Cardiac Hypertrophy by Inhibiting DNA Damage Response

High-mobility group box 1 (HMGB1) is a deoxyribonucleic acid (DNA)-binding protein associated with DNA repair. Decreased nuclear HMGB1 expression and increased DNA damage response (DDR) were observed in human failing hearts. DNA damage and DDR as well as cardiac remodeling were suppressed in cardiac-specific HMGB1 overexpression transgenic mice after angiotensin II stimulation as compared with wild-type mice. Invitro, inhibition of HMGB1 increased phosphorylation of extracellular signal-related kinase 1/2 and nuclearfactor kappa B, which was rescued by DDR inhibitor treatment. DDR inhibitor treatment provided a cardioprotective effect on angiotensin II-induced cardiac remodeling in mice.

Role of toll-like receptor 4 in the regulation of the cell death pathway and neuroinflammation

Septic brain injury is a severe disease of the central nervous system and involves alteration of the cell death pathway due to sepsis. Increasing evidence indicates that Toll-like receptor 4 (TLR4) plays a key role in the development of sepsis. Cell death is also thought to contribute to septic brain injury. However, the mechanism that regulates cell death pathways in response to TLR4 in neurons exposed to septic brain injury is unclear. Here, we established a rat model of septic brain injury to evaluate the effect of TLR4 inhibition on cell death pathways. Furthermore, primary neurons with and those without TLR4 inhibition were exposed to lipopolysaccharide (LPS). We found that septic brain injury induction by cecum ligation and puncture evoked autophagy, induced high-mobility group box-1(HMGB1) translocation and increased TLR4 expression. Inhibition of TLR4 also enhanced autophagy and increased nuclear HMGB1 levels in the sepsis-injured rat brain cortex. After LPS treatment in vitro, HMGB1 was released from neurons. This release of HMGB1 could be inhibited using a TLR4 inhibitor. Collectively, these results indicate that modulation of TLR4 may result in the regulation of neuron cell death pathways by regulating autophagy in cortical tissues. Thus, it may represent a potential therapeutic strategy for neuronal protection in septic brain injury.

Serum and blister-fluid elevation and decreased epidermal content of high-mobility group box 1 protein in drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis.

SummaryBackgroundHigh‐mobility group box 1 (HMGB1) is a damage‐associated molecular‐pattern protein. Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) are serious, immune‐mediated skin‐blistering conditions.ObjectivesTo determine serum and/or blister‐fluid total HMGB1 levels in SJS/TEN cohorts, and HMGB1 expression in formalin‐fixed, paraffin‐embedded (FFPE) SJS/TEN skin vs. healthy and maculopapular exanthema (MPE) skin. Methods Serum HMGB1 was quantified in Malawian nevirapine‐induced hypersensitivity, Taiwanese SJS/TEN and Spanish SJS/TEN cohorts. FFPE skin (healthy skin, MPE, SJS/TEN) was stained and assessed for HMGB1 expression.ResultsSerum total HMGB1 was not significantly elevated in patients with nevirapine‐induced SJS/TEN (3·98 ± 2·17 ng mL −1), MPE (3·92 ± 2·75 ng mL −1) or drug reaction with eosinophilia and systemic symptoms (4·73 ± 3·00 ng mL −1) vs. tolerant controls (2·97 ± 3·00 ng mL −1). HMGB1 was significantly elevated in Taiwanese patients with SJS/TEN, highest during the acute phase (32·6 ± 26·6 ng mL −1) vs. the maximal (19·7 ± 23·2 ng mL −1; P = 0·007) and recovery (24·6 ± 25·3 ng mL −1; P = 0·027) phases. In blister fluid from Spanish patients with SJS/TEN, HMGB1 (486·8 ± 687·9 ng mL −1) was significantly higher than in serum (8·8 ± 7·6 ng mL −1; P <0·001). Preblistered SJS/TEN skin showed decreased epidermal nuclear HMGB1 expression in upper epidermis vs. healthy or MPE skin but retained basal/suprabasal expression.ConclusionsEpidermal HMGB1 expression was decreased in SJS/TEN skin. Retained basal/suprabasal epidermal HMGB1 expression may exacerbate localized injury in SJS/TEN.

Effects of remote ischaemic preconditioning on intraportal islet transplantation in a rat model.

Remote ischaemic preconditioning (RIPC), which is the intermittent interruption of blood flow to a site distant from the target organ, is known to improve solid organ resistance to ischaemia-reperfusion injury. This procedure could be of interest in islet transplantation to mitigate hypoxia-related loss of islet mass after isolation and transplantation. Islets isolated from control or RIPC donors were analyzed for yield, metabolic activity, gene expression and high mobility group box-1 (HMGB1) content. Syngeneic marginal mass transplantation was performed in four streptozotocin-induced diabetic groups: control, RIPC in donor only, RIPC in recipient only, and RIPC in donor and recipient. Islets isolated from RIPC donors had an increased yield of 20% after 24h of culture compared to control donors (P=0.007), linked to less cell death (P=0.08), decreased expression of hypoxia-related genes (Hif1a P=0.04; IRP94 P=0.008), and increased intra-cellular (P=0.04) and nuclear HMGB1. The use of RIPC in recipients only did not allow for reversal of diabetes, with increased serum HMGB1 at day 1; the three other groups demonstrated significantly better outcomes. Performing RIPC in the donors increases islet yield and resistance to hypoxia. Validation is needed, but this strategy could help to decrease the number of donors per islet recipient.

A novel disulfide bond-mediated cleavable RGD-modified PAMAM nanocomplex containing nuclear localization signal HMGB1 for enhancing gene transfection efficiency.

BackgroundPolyamidoamine (PAMAM) dendrimers modified by polyethylene glycol (PEG) have frequently been investigated as a delivery carrier for gene therapy. However, modification of PAMAM with PEG using covalent linkage significantly reduces the cellular uptake rate and the transfection efficiency. How to conquer these barriers becomes a burning question in gene delivery.Materials and methodsThe present study constructed an effective disulfide bond-mediated cleavable RGD modified gene delivery system to overcome the aforementioned limitations. The disulfide bond was introduced between PAMAM dendrimers and PEG chains to realize the cleavage of PEG from the carrier system, whereas the arginine-glycine-aspartate (RGD) peptide was expected to promote the cellular uptake rate. A high mobility group Box 1 (HMGB1) protein containing nuclear localization signal (NLS) was simultaneously introduced to further promote gene expression efficiency. A pDNA/HMGB1/PAMAM-SS-PEG-RGD (DHP) nanocomplex was prepared via electrostatic interaction and characterized.ResultsThe results showed that DHP generated small particles and was able to condense and protect pDNA against degradation. In addition, the RGD peptide could significantly promote the cellular uptake of a nanocomplex. Intracellular trafficking and in vitro expression study indicated that the DHP nanocomplex escaped from lysosomes and the disulfide bonds between PAMAM and PEG cleaved due to the high concentration of GSH in the cytoplasm, pDNA consequently became exclusively located in the nucleus under the guidance of HMGB1, thereby promoting the red fluorescence protein (RFP) expression. Importantly, an in vivo antitumor activity study demonstrated that the DHP nanocomplex had higher antitumor activity than any other reference preparation.ConclusionAll these results confirm that DHP could be a new strategy for improving the transfection and expression efficiency in gene delivery.

N-(2'-Hydroxyphenyl)-2-propylpentanamide (OH-VPA), a histone deacetylase inhibitor, induces the release of nuclear HMGB1 and modifies ROS levels in HeLa cells.

N-(2′-Hydroxyphenyl)-2-propylpentanamide (OH-VPA) is a valproic acid (VPA) derivative with improved antiproliferative activity toward breast cancer (MCF-7, MDA-MB-231, and SKBr3) and human cervical cancer cell lines (HeLa) compared to that of VPA. However, the pharmacological mechanism of OH-VPA activity remains unknown. High-mobility group box 1 (HMGB1) is an important enzyme that is highly expressed in tumor cells and has a subcellular localization that is dependent on its acetylation or oxidative state. Therefore, in this study, we analyzed changes in HMGB1 sub-cellular localization and reactive oxygen species (ROS) as well as changes in HeLa cell viability in response to treatment with various concentrations of OH-VPA. This compound is formed by the covalent bond coupling VPA to a phenol group, which is capable of acting as a free radical scavenger due to its chemical similarities to quercetin. Our results show that OH-VPA induces nuclear to cytoplasmic translocation of HMGB1, as demonstrated by confocal microscopy observations and infrared spectra that revealed high quantities of acetylated HMGB1 in HeLa cells. Cells treated with 0.8 mM OH-VA exhibited decreased viability and increased ROS levels compared with the lower OH-VPA concentrations tested. Therefore, the antiproliferative mechanism of OH-VPA may be related to histone deacetylase (HDAC) inhibition, as is the case for VPA, which promotes high HMBG1 acetylation, which alters its subcellular localization. In addition, OH-VPA generates an imbalance in cellular ROS levels due to its biochemical activity.

Paclitaxel-induced HMGB1 release from macrophages and its implication for peripheral neuropathy in mice: Evidence for a neuroimmune crosstalk

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

P2824Cardiac nuclear high-mobility group box 1 attenuates angiotensin II induced pathological cardiac hypertrophy by inhibiting DNA damage response pathway

P2824Cardiac nuclear high-mobility group box 1 attenuates angiotensin II induced pathological cardiac hypertrophy by inhibiting DNA damage response pathway

PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy.

High-mobility group box 1 (HMGB1) exhibits various functions according to its subcellular location, which is finely conditioned by diverse post-translational modifications, such as acetylation. The nuclear HMGB1 may prevent from cardiac hypertrophy, whereas its exogenous protein is proven to induce hypertrophic response. This present study sought to investigate the regulatory relationships between poly(ADP-ribose) polymerase 1 (PARP1) and HMGB1 in the process of pathological myocardial hypertrophy. Primary-cultured neonatal rat cardiomyocytes (NRCMs) were respectively incubated with three cardiac hypertrophic stimulants, including angiotensin II (Ang II), phenylephrine (PE), and isoproterenol (ISO), and cell surface area and the mRNA expression of hypertrophic biomarkers were measured. the catalytic activity of PARP1 was remarkably enhanced, meanwhile HMGB1 excluded from the nucleus. PARP1 overexpression by infecting with adenovirus PARP1 (Ad-PARP1) promoted the nuclear export of HMGB1, facilitated its secretion outside the cell, aggravated cardiomyocyte hypertrophy, which could be alleviated by HMGB1 overexpression. PE treatment led to the similar results, while that effect was widely depressed by PARP1 silencing or its specific inhibitor AG14361. Moreover, SD rats were intraperitoneally injected with 3-aminobenzamide (3AB, 20 mg/kg every day, a well-established PARP1 inhibitor) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks, echocardiography and morphometry of the hearts were measured. Pre-treatment of 3AB relieved AAC-caused the translocation of nuclear HMGB1 protein, cardiac hypertrophy, and heart dysfunction. Our research offers a novel evidence that PARP1 combines with HMGB1 and accelerates its translocation from nucleus to cytoplasm, and the course finally causes cardiac hypertrophy.

Evaluation of Graft Effluent High Mobility Group Box-1 (HMGB-1) for Prediction of Outcome After Liver Transplantation.

BackgroundPre-transplant assessment of the graft for liver transplantation is crucial. Based on experimental data, this study was designed to assess both nuclear high mobility group box-1 (HMGB-1) protein and arginine-specific proteolytic activity (ASPA) in the graft effluent.Material/MethodsIn a non-interventional trial, both HMGB-1 and ASPA were measured in the effluent of 30 liver grafts after cold storage before transplantation. Values of HMGB-1 and ASPA levels were compared with established prognostic parameters such as the donor risk index, balance of risk score, and Donor-Model for End-Stage Liver Disease.ResultsThe early allograft dysfunction (EAD) was best predicted by recipient age (p=0.026) and HMGB-1 (p=0.031). HMGB −1 thresholds indicated the likelihood for initial non-function (1608 ng/ml, p=0.004) and EAD (580 ng/ml, p=0.017). The multivariate binary regression analysis showed a 21-fold higher (95% CI: 1.6–284.5, p=0.022) risk for EAD in cases with levels exceeding 580 ng/ml. The ASPA was lower in cases of initial non-function (p=0.028) but did not correlate with the rate of EAD (p=0.4).ConclusionsThis study demonstrates the feasibility of HMGB-1 detection in the graft effluent after cold storage. Along with conventional prognostic scores, it may be helpful to predict the early fate of a graft in human liver transplantation.

Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

High-mobility group box 1 (HMGB1) protein acts as a DNA chaperone for nuclear homeostasis. It translocates into the cytosol and is secreted into extracellular spaces, triggering proinflammatory cytokines and acting as a mediator in fibrosis. We determined whether HMGB1 plays a role in normal dermal fibrosis and keloid, and is involved with transforming growth factor β. We investigated the translocation and active release of HMGB1 from normal dermal fibroblasts under lipopolysaccharide stimuli, and the redistribution of nuclear HMGB1 into the cytoplasm of keloid fibroblasts. HMGB1 and its effector toll-like receptors and receptors for advanced glycation end product proteins are actively expressed in keloid tissues. Exogenous HMGB1 can induce the proliferation of human dermal fibroblasts, and could act as a profibrogenic molecule to produce collagen, decrease MMP-1, and increase TIMP-1 mRNA expression. Moreover, administration of HMGB1 increased the expression level of TGF-β1 and internal signaling molecules, such as Smad 2 and 3, phosphorylated Smad 2/3 complex, Erk 1/2, Akt, and NF-κB. Collectively, we demonstrate that HMGB1 treatment increases the expression level of collagen types I and III, elastin, and fibronectin in dermal spheroid cultures, thus making HMGB1 a promising therapeutic target for treatment of profibrogenic diseases.

Spinal PKC activation — Induced neuronal HMGB1 translocation contributes to hyperalgesia in a bone cancer pain model in rats

Bone cancer pain (BCP) remains a serious complication of malignancy, which is an intractable clinical problem due to the gap in knowledge of its underlying mechanisms. Recent studies have demonstrated that the major involvement of neuroinflammation, particularly high-mobility group box 1 (HMGB1), which was identified as a late mediator of inflammation, in a number of pain conditions. However, the underlying mechanisms and functions of HMGB1 release in spinal cord, and its contributions to the development of BCP as well, are poorly understood. In the present study, we examined the theory that PKC activation lead to nuclear translocation and cytosolic HMGB1 secretion, which subsequently induces spinal neuro inflammatory responses (cytokine release) causing hyperalgesia. Our results showed that PKC activation and HMGB1 release in spinal neurons as well as mechanical allodynia in BCP rats, were all attenuated by intrathecal administration of the PKC inhibitor Gö6983 and aggravated by its activator PMA. Intrathecal administration of anti-HMGB1 antibody also alleviated hypersensitivity caused by BCP. Meanwhile, phospho-PKC and cellular HMGB1 were found co-localized in neurons, but not in microglia and astrocytes, of the spinal dorsal horns of tumor-bearing rats. Additionally, we found that HMGB1 translocation from nuclei to cytoplasm may be the consequence of PKC translocation into the nuclei, which occurred 9 days after tumor inoculation. Total p-HMGB1 as well as nuclear and cytoplasmic HMGB1 expression levels were tested in response to broad-spectrum PKC inhibitor Gö6983 or activator PMA in BCP rats. Together, these findings suggest that bone cancer related hyperalgesia is driven by PKC induced phosphorylation of HMGB1, which results in its translocation from the nucleus, and releasing from the cytosol of the dorsal horn, and the activation of spinal pro-inflammatory mediators.

HMGB1 silencing in macrophages prevented their functional skewing and ameliorated EAM development: Nuclear HMGB1 may be a checkpoint molecule of macrophage reprogramming

High-mobility group box 1 (HMGB1), an important inflammatory factor, plays significant roles in CD4+T cell differentiation, cancer and autoimmune disease development. Our previous data have demonstrated that HMGB1 contributes to macrophage reprogramming and is involved in experimental autoimmune myocarditis (EAM) development. In contrast to the well-explored function of HMGB1, little is known about the nuclear function. Whether HMGB1 can serve as an architectural factor and control functional skewing of macrophages remains unclear. Therefore, the present work was performed to address the above speculation. The adenovirus-mediated shRNA (Ad-shRNA) was employed to knock down HMGB1 in RAW264.7 and monocytes/macrophages of EAM mice. Our data showed that in vitro HMGB1 silencing limited functional skewing of macrophages and down-regulated inflammatory factors secretion, which can't be reversed by the exogenous HMGB1. In M1 polarization system, the phosphorylations of NF-κB, p38 and Erk1/2 were inhibited following HMGB1 silencing. In vivo, HMGB1 silencing could effectively ameliorate EAM development. Our data suggest that HMGB1 may be a checkpoint nuclear factor of macrophage reprogramming. Our findings also provide an exciting therapeutic method for inflammatory disorders.

High mobility group box-1 (HMGB1) is increased in injured mouse spinal cord and can elicit neurotoxic inflammation

Inflammation is a ubiquitous but poorly understood consequence of spinal cord injury (SCI). The mechanisms controlling this response are unclear but culminate in the sequential activation of resident and recruited immune cells. Collectively, these cells can exert divergent effects on cell survival and tissue repair. HMGB1 is a ubiquitously expressed DNA binding protein and also a potent inflammatory stimulus. Necrotic cells release HGMB1, but HMGB1 also is actively secreted by inflammatory macrophages. A goal of this study was to quantify spatio-temporal patterns of cellular HMGB1 expression in a controlled mouse model of experimental SCI then determine the effects of HMGB1 on post-SCI neuroinflammation and recovery of function. We documented SCI-induced changes in nuclear and cytoplasmic distribution of HMGB1 in various cell types after SCI. The data reveal a time-dependent increase in HMGB1 mRNA and protein with protein reaching maximal levels 24–72 h post-injury then declining toward baseline 14–28 days post-SCI. Although most cells expressed nuclear HMGB1, reduced nuclear labeling with increased cytoplasmic expression was found in a subset of CNS macrophages suggesting that those cells begin to secrete HMGB1 at the injury site. In vitro data indicate that extracelluar HMGB1 helps promote the development of macrophages with a neurotoxic phenotype. The ability of HMGB1 to elicit neurotoxic macrophage functions was confirmed in vivo; 72 h after injecting 500 ng of recombinant HMGB1 into intact spinal cord ventral horn, inflammatory CNS macrophages co-localized with focal areas of neuronal killing. However, attempts to confer neuroprotection after SCI by blocking HMGB1 with a neutralizing antibody were unsuccessful. Collectively, these data implicate HMGB1 as a novel regulator of post-SCI inflammation and suggest that inhibition of HMGB1 could be a novel therapeutic target after SCI. Future studies will need to identify better methods to deliver optimal concentrations of HMGB1 antagonists to the injured spinal cord.

AG490 ameliorates early brain injury via inhibition of JAK2/STAT3‑mediated regulation of HMGB1 in subarachnoid hemorrhage

High mobility group box 1 (HMGB1) is a classic damage-associated molecular pattern that has an important role in the pathological inflammatory response. In vitro studies have demonstrated that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the regulation of HMGB1 expression, mediating the inflammatory response. Therefore, the purpose of the present study was to evaluate JAK2/STAT3 pathway involvement in the subarachnoid hemorrhage (SAH)-dependent regulation of HMGB1, using an in vivo rat model. A SAH model was established by endovascular perforation. Western blotting, immunohistochemistry and immunofluorescence were used to analyze HMGB1 expression after SAH. In addition, the effects of AG490 after SAH on JAK2/STAT3 phosphorylation, HMGB1 expression and brain damage were evaluated. The results of the present study demonstrated that JAK2/STAT3 was significantly phosphorylated (P<0.05) and the total HMGB1 protein level was significantly increased (P<0.05) after SAH. In addition, the cytosolic HMGB1 level after SAH demonstrated an initial increase followed by a decrease to the control level, while the nuclear HMGB1 level after SAH demonstrated the opposite trend, with an initial decrease and subsequent increase. AG490 administration after SAH significantly inhibited JAK2/STAT3 phosphorylation (P<0.05), suppressed the expression and translocation of HMGB1, reduced cortical apoptosis, brain edema and neurological deficits. These results demonstrated the involvement of the JAK2/STAT3 pathway in HMGB1 regulation after SAH.