Human HMGB1 Research Articles

HMBG-1: A Biomarker in the Management of Diabetic Retinopathy

Introduction: High mobility group box (HMGB)-1 protein is a promising prognostic marker for predicting the course of retinopathy and the prognosis in patients with diabetic retinopathy (DR). The aim of this study is to investigate whether serum HMGB-1 protein levels can be used as a biomarker about the severity of the disease in patients with diabetic retinopathy. Material and methods: This study was carried out in the internal medicine and ophthalmology departments of the Gaziantep Medicalpark Hospital. The records of 50 patients diagnosed with type 2 diabetes mellitus with diabetic retinopathy and 47 healthy subjects who applied to our clinic between January 2016 and December 2018 were retrospectively scanned. Five cc of venous blood was taken from the patients, and control groups were included in the study for routine laboratory tests and serum HMGB-1 levels to be studied under appropriate conditions. Human HMGB-1 levels were measured using the ELISA method. Results: There was a significant difference among the groups in terms of HMGB-1 levels (p<0.001) (Table-1). There was a positive correlation between blood glucose and HMGB-1 in the diabetic retinopathy group (Spearman's rho (ρ)=0.07). Similarly, there was a positive correlation between HbA1c and HMGB-1 levels in the diabetic retinopathy group. (Spearman's rho (ρ)=0.06). Conclusions: Our study suggests that HMGB-1 may be a useful biomarker in managing DR, aiding in the prediction and treatment of microvascular complications.

FDA-Approved Excipient N, N-Dimethylacetamide Attenuates Inflammatory Bowel Disease in In Vitro and In Vivo Models.

Inflammatory bowel disease (IBD) affects almost 7 million people worldwide and is increasing in incidence. While the precise pathogenesis of IBD remains unknown, the production of inflammatory cytokines and chemokines play a central role. We have previously found that N, N-dimethylacetamide (DMA), a widely used non-toxic drug excipient, suppresses cytokine and chemokine secretion in vitro and prevents inflammation-induced preterm birth in vivo. Using sandwich enzyme-linked immunosorbent assays (ELISAs), we tested whether DMA attenuates cytokine and chemokine secretion from LPS- or TNFα-stimulated human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. To test our hypothesis that the mechanism of DMA's effects in in vitro and in vivo models of IBD is inhibition of the NF-κB pathway, we used western blotting to track levels of the nuclear factor kappa B (NF-κB) inhibitory molecule I kappa B alpha (IκBα) in THP-1 human monocytes in the absence or presence of DMA. Finally, we induced colitis in C57Bl/6 mice with dextran sodium sulfate (DSS) and then tested whether i.p injections of DMA at 2.1 g/kg/day attenuates clinical and histopathologic signs of colitis. DMA attenuated cytokine and chemokine release from human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. Importantly, DMA prevented degradation of IκBα in THP-1 cells, thereby suggesting one mechanism for DMA's effects. Finally, we show here, for the first time, that DMA attenuates clinical and histologic features of DSS-induced colitis. Based on these data, DMA should be further explored in preclinical and clinical trials for its potential as novel drug therapy for IBD.

Effects of HMGB1 on Proliferation and Secretion of Human Bone Marrow Mesenchymal Stem Cells

To investigate the effect of high mobility group protein 1(HMGB1) on the proliferation and cytokine expression of human bone marrow mesenchymal stem cells (MSC). Different concentrations of recombinant human HMGB1 protein (100, 200, 400, 800 and 1000 ng/ml) were incubated with MSC for 24, 48, 72 h and the proliferation of MSC were detected respectively by using the CCK-8 method and flow cytometry. The best concentrations of HMGB1 incubated with MSC was determined (200 ng/ml, 1000 ng/ml), and the flow cytomerty was used to determine the effect of HMGB1 on the proliferation of MSC. The mRNA expression levels of IL-10, TGF- β1, TSG-6 and IFN-γ in MSC incubated with HMGB1 protein were detected by real-time quantitative PCR and ELISA. The result of MSC identification and flow cytometry showed that the CD105, CD73 and CD90 were expressed, but did not expression CD45, CD34, CD11b, CD19 and HLA-DR; CCK-8 showed that HMGB1 at the concentrations of 100 ng/ml, 200 ng/ml and 400 ng/ml could promote the proliferation of MSC incubated for 24, 48 and 72 h as compared with the control group (P<0.05), and the most effective concentration was 200 ng/ml; flow cytometry showed that the compared with the control group, HMGB1 200 ng/ml could induce MSC from G1 phase to S phase to promote the proliferation of MSC; QPCR showed that the mRNA expression of MSC cytokines IL-10, TGF-β1, TSG-6 increased while IFN-γ decreased at the concentration of 200 ng/ml HMGB1 as compared with the control group. ELISA experiments showed that the HMGB1 200 ng/ml acting on MSC for 48 h could significantly promoted the secretion of IL-10, TGF-β 1 and TSG-6(P<0．05), while IFN-γ showed no significant difference as compared with control group. Recombinant human HMGB1 can promote the proliferation and secretion of MSC in healthy people.

The Viability and Cytotoxic Effect of High Mobility Group Box Protein 1 on MC3T3-E1 Pre-osteoblast Cells: an In Vitro Study

Osteoblast proliferation and migration are significantly involved in bone healing, regeneration and embryonic skeletal development. Recently, reports suggest anabolic properties of High Mobility Group Box Protein 1 (HMGB1) within the subsequent phase of tissue regeneration as well as HMGB1 has been shown to be highly expressed during bone fracture and regulates osteochondral ossification. In this study, the effect of various concentrations of Recombinant Human HMGB-1 (rhHMGB-1) (50, 100,150 and 200 µg/L) on the viability and proliferation of Pre-osteoblast cells MC3T3-E1 were examined over 24, 48 and 72 hours intervals in comparison with control, untreated cells. A cell count kit (CCk-8) was used to assess osteoblast viability and proliferation, results were quantified using a microplate reader and the viability %( survival rate) was calculated. All experiments were done in triplicate and outcomes were statistically analyzed. A direct positive impact of HMGB-1 on the viability and proliferation of osteoblast cells was in a dose dependent manner. Lower concentration of HMGB-1 (50 and 100 µg/L) showed a non-significance difference in the viability and proliferation of osteoblast cells over time, however, a higher concentration of HMGB-1 (150 and 200 µg/L) showed a significant increase in the proliferation and survival rate by 1.7 fold compared to control group in the 2nd and 3rd day of treatment. No significant difference was found between the 150 and 200 µg/L concentrations which suggested the appropriate dose will be 150 µg/L. Moreover, further in vivo and in vitro investigations will be required.

N-glycosylation of High Mobility Group Box 1 protein (HMGB1) modulates the interaction with glycyrrhizin: A molecular modeling study

High Mobility Group Box 1 protein (HMGB1) is an abundant protein with multiple functions in cells, acting as a DNA chaperone and damage-associated molecular pattern molecule. It represents an attractive target for the treatment of inflammatory diseases and cancers. The plant natural product glycyrrhizin (GLR) is a well-characterized ligand of HMGB1 and a drug used to treat diverse liver and skin diseases. The drug is known to bind to each of the two adjacent HMG boxes of the non-glycosylated protein. In cells, HMGB1 is N-glycosylated at three asparagine residues located in boxes A and B, and these N-glycans are essential for the nucleocytoplasmic transport of the protein. But the impact of the N-glycans on drug binding is unknown. Here we have investigated the effect of the N-glycosylation of HMGB1 on its interaction with GLR using molecular modelling, after incorporation of three N-glycans on a Human HMGB1 structure (PDB code 2YRQ). Sialylated bi-antennary N-glycans were introduced on the protein and exposed in a folded or an extended conformation for the drug binding study. The docking of the drug was performed using both 18α- and 18β-epimers of GLR and the conformations and potential energy of interaction (ΔE) of the different drug-protein complexes were compared. The N-glycans do not shield the drug binding sites on boxes A and B but can modulate the drug-protein interaction, via both direct and indirect effects. The calculations indicate that binding of 18α/β-GLR to the HMG box is generally reduced when the protein is N-glycosylated vs. the non-glycosylated protein. In particular, the N-glycans in an extended configuration significantly weaken the binding of GLR to box-B. The effects of the N-glycans are mostly indirect, but in one case a direct contact with the drug, via a carbohydrate-carbohydrate interaction, was observed with 18β-GLR bound to Box-B of glycosylated HMGB1. For the first time, it is shown (at least in silico) that N-glycosylation, one of the many post-translational modifications of HMGB1, can affect drug binding.

Analysis of Single Nucleotide Polymorphisms in HLA-DRA, IL2RA , and HMGB1 Genes in Multiple Sclerosis.

Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen (HLA) complex, IL2RA , and HMGB1 genes with the pathophysiology of MS. In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls. Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from HLA-DRA gene, and rs12722489 and rs12722490 variants from IL2RA gene (p< 0.05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the HLA-DRA gene. This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.

Glycyrrhizin improves inflammation and apoptosis via suppressing HMGB1 and PI3K/mTOR pathway in lipopolysaccharide-induced acute liver injury.

Acute liver injury (ALI) is mainly characterized by the symptom of metabolic disorders, homeostasis unbalance, and loss of liver function. There are no effective treatment methods at present stage except the liver transplantation. Effective treatment for early ALI is of great significance for the treatment of liver injury thereof. Glycyrrhizin (GL) is a promising inhibitor of the high-mobility group box-1 gene (HMGB1) which is expressed much higher in an inflammatory injury. However, it is not clear whether GL improves ALI via the inhibition of HMGB1. The present study is to probe the function and mechanism of glycyrrhizin on acute liver injury. The expression of HMGB1 and inflammation in liver macrophages were analyzed. Lipopolysaccharide (LPS) was used in stimulating the macrophages to activate inflammatory response and recombined human HMGB1 was used to resist the function of GL to explore whether GL acted via the target of HMGB1. Then, LPS injection was utilized to induce ALI in mice, and then we evaluated GL treatment in ALI model. The results showed that the expressions of HMGB1 and inflammatory factors were markedly increased in LPS-activated liver macrophages. GL inhibited the progress of macrophages inflammation by restraining HMGB1, and the administration of GL could reverse the effects of LPS-induced ALI in mice. Moreover, PI3K/mTOR pathway was significantly suppressed by GL application. These results suggest that GL prevents inflammation in liver macrophages via inhibition of HMGB1. GL restrains inflammation and cell apoptosis by inhibiting HMGB1 via PI3K/mTOR signaling pathway in ALI. GL may become a novel drug for the therapy of ALI in the future.

New insights in acetaminophen toxicity: HMGB1 contributes by itself to amplify hepatocyte necrosis in vitro through the TLR4-TRIF-RIPK3 axis

Extracellular release of HMGB1 contributes to acetaminophen-induced liver injury. HMGB1 acts as a danger-associated molecular patterns during this toxic process but the mechanisms of action and targeted cells are incompletely defined. Here we studied, in vitro, the role of HMGB1 in amplifying the acetaminophen-induced hepatocyte necrosis process. Using cultured HepaRG cells, primary human hepatocytes and selective chemical inhibitors we evaluated acetaminophen-induced toxicity. We confirmed that addition of acetaminophen induced HepaRG cell death and HMGB1 release. We showed that inhibition of HMGB1 decreased acetaminophen-induced HepaRG cell death, suggesting a feedforward effect. We provide the first evidence that exposure of HepaRG cells to recombinant human HMGB1 (rhHMGB1) also resulted in cell death. Moreover, we found that both acetaminophen and rhHMGB1 induced programmed HepaRG cell necrosis through a RIPK3-dependent mechanism. By using TLR4 blocking antibody, we demonstrated the reduction of the HepaRG cell death induced by acetaminophen and rhHMGB1. Furthermore, inhibition of TRIF, known to induce a RIPK3-dependent cell death, reduced rhHMGB1-induced HepaRG cell death. Our data support that released HMGB1 from acetaminophen-stressed hepatocytes induced necrosis of neighboring hepatocytes by TLR4-TRIF-RIPK3- pathway. This in vitro study gives new insights in the role of HMGB1 in the amplification of acetaminophen-induced toxicity.

Regulatory role of high mobility group box-1 protein in the balance of Th17/Treg in peripheral blood of immune thrombocytopenia patients

Objective To explore the effect of high mobility group box-1 protein (HMGB1) on the balance of Th17/Treg in patients with immune thrombocytopenia (ITP). Methods A total of 30 patients who were first diagnosed as ITP in the Fifth People's Hospital of Datong from July 2017 to April 2018 were selected as the case group, and another 30 healthy volunteers in the corresponding period were taken as the control group. The proportion of Th17 and Treg cells was detected by using flow cytometry, and the concentration of HMGB1, interleukin (IL)-17 and transforming growth factor β (TGF-β) in plasma was tested by using enzyme-linked immunosorbent assay (ELISA). Isolated peripheral blood mononuclear cells (PBMC) were cultured in vitro. After the treatment with recombinant human HMGB1 (rhHMGB1), real-time polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression changes in Treg cell transcription factor intracellular forkhead helix transcription factor 3 (Foxp3) and Th17 cell transcription factor retinoid related orphan receptor γt (RORγt). The differences of indicators in Treg cell transcription factor peripheral blood between the case group and the control group were compared, and the balance correlation between HMGB1 and Th17/Treg was analyzed. Results Compared with the healthy control group, the proportion of Th17 cells and the expression level of HMGB1 and IL-17 in peripheral blood of ITP patients were increased (all P < 0.01), while the proportion of Treg cells and the level of TGF-β were decreased (all P < 0.01). The proportion of Th17 cells and the expression level of IL-17 and HMGB1 in peripheral blood of ITP patients were positively correlated with the concentration of HMGB1 (all P < 0.01); the proportion of Treg cells and the level of TGF-β were negatively correlated with the expression level of HMGB1 (all P < 0.01). In vitro experiments, the expression of intracellular RORγt mRNA was increased compared with the negative control group (1.50±0.24 vs. 0.93±0.22, t = 9.612, P < 0.01), and the expression of Foxp3 mRNA was decreased compared with the negative control group after the stimulation of PBMC by rhHMGB1 (0.72±0.19 vs. 1.08±0.18, t = 7.387, P < 0.01). Conclusion The high level of HMGB1 in the peripheral blood of ITP patients induces Th17/Treg imbalance and aggravates inflammatory reactions, which may be an important cause of ITP. Key words: Immune thrombocytopenia; High mobility group box 1 protein; Th17/Treg balance; In vitro culture

High-mobility group box-1 increases epithelial sodium channel activity and inflammation via the receptor for advanced glycation end products.

Cystic fibrosis (CF) lung disease persists and remains life-limiting for many patients. Elevated high-mobility group box-1 protein (HMGB-1) levels and epithelial sodium channel hyperactivity (ENaC) are hallmark features of the CF lung. The objective of this study was to better understand the pathogenic role of HMGB-1 signaling and ENaC in CF airway cells. We hypothesize that HMGB-1 links airway inflammation [via signaling to the receptor for advanced glycation end products (RAGE)] and airway surface liquid dehydration (via upregulation of ENaC) in the CF lung. We calculated equivalent short-current (Isc) and single-channel ENaC open probability (Po) in normal and CF human small airway epithelial cells (SAEC) in the presence and absence of human HMGB-1 peptide (0.5 μg/mL). In normal SAECs, HMGB-1 increased amiloride-sensitive Isc and elevated ENaC Po from 0.15 ± 0.03 to 0.28 ± 0.04 (P < 0.01). In CF SAECs, ENaC Po increased from 0.45 ± 0.06 to 0.73 ± 0.04 (P < 0.01). Pretreatment with 1 μM FPS-ZM1 (a RAGE inhibitor) attenuated all HMGB-1 effects on ENaC current in normal and CF SAECs. Confocal analysis of SAECs indicates that nuclear size and HMBG-1 localization can be impacted by ENaC dysfunction. Masson's trichrome labeling of mouse lung showed that intraperitoneally injected HMGB-1 significantly increased pulmonary fibrosis. Bronchoalveolar lavage fluid from HMGB-1-treated mice showed significant increases in IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and granulocyte-macrophage colony-stimulating factor compared with vehicle-injected mice (P < 0.05). These studies put forth a new model in which HMGB-1 signaling to RAGE plays an important role in perpetuating ENaC dysfunction and inflammation in the CF lung.

The pathogenicity, structural and functional exploration of human HMGB1 single nucleotide polymorphisms using in silico study

The human HMGB1 gene mutations have a major impact on several immune-related diseases and cancer. The detrimental effect of non-synonymous mutations of HMGB1 has not been investigated yet, hence the present study aims to examine single nucleotide polymorphisms and their implications on the structure-function of human HMGB1. The multifaceted HMGB1 protein acts as pleiotropic cytokine and regulates essential genes for coordinated cellular functions. The mutational effect on HMGB1 was analyzed by sequence-based homology methods, supervised learning methods, and structure-based methods. The study identified 58 non-synonymous mutations in human HMGB1, out of which only 2 mutations; R10T (rs61742222) and F103C (rs61733675) were classified as the SNPs with highest deleterious and disease-causing mutants. The effect of these mutations in structure of HMGB1 was scrutinized and the R10T mutant found to have a distinct structural behaviour in the B-box domain. In addition, R10T mutant predicted that it affects the MoRF function of HMGB1 and it could disrupt the DNA binding or/and protein partner interaction activity by HMGB1. F103C mutation takes place at the TLR binding and cytokine inducing region of HMGB1, hence it could affect the protein binding activity which involves in many cellular signaling. The study identified potent mutations R10T (a cancer-causing somatic mutation) and F103C (a novel mutation) and these mutations either directly or indirectly hinder DNA binding activity and TLR and cytokine binding of HMGB1. These findings will help in understanding the molecular basis of these promising mutations and functional role of human HMGB1 in cancer and immunological diseases.AbbreviationsAGERAdvanced glycosylation end product-specific receptorCXCLChemokine (C-X-C motif) liganddbSNPThe single nucleotide polymorphism databaseHMGB1High mobility group box 1LINCSLINear Constraint SolverMDSMolecular dynamics simulationMoRFMolecular recognition featuresNPTNumber of particle, Pressure and TemperatureNVTNumber of particle, Volume and TemperaturensSNPNon-synonymous SNPPBCPartial boundary conditionPCAPrincipal component analysisPMEPartial mesh EwaldRMSDRoot mean square deviationRMSFRoot mean square fluctuationSNPSingle nucleotide polymorphismSPCSingle-point chargeTLRToll-like receptorUTRUn-translated RegionCommunicated by Ramaswamy H. Sarma

In silico investigation of extracellular domain of RAGE receptor interaction with A-box and B-box of HMGB1 protein

HMGB1 protein which is a non-histone chromosomal protein with two functional domains named A-box and B-box can also act as a signaling molecule after releasing from the cell and binding to the cell surface receptors such as RAGE. HMGB1 through its B-box domain binds to extracellular domain of RAGE and activates the signaling pathways involved in various pathological conditions like sepsis and tumor growth and metastasis. Interaction of recombinant HMGB1 A-box with RAGEantagonizes the RAGE activation by HMGB1. In the present study, interaction of human RAGE (hRAGE) extracellular domain (VC1C2) and B-box and A-box of human HMGB1 (hHMGB1) was investigated using a protein-protein docking software, HADDOCK. The results obtained were analyzed by PyMOL and LigPlot softwares. The results show B-box and A-box bind to different sites on the VC1domain of RAGE and one of the B-box binding points is a positively charged groove located on the V domain surface which is also a major binding site for another RAGE ligand, Advanced Glycation Endproducts (AGEs). The obtained results can be utilized to design new potent drugs for treatment of HMGB1-RAGE-related diseases such as cancer and sepsis.&#x0D;

Different concentration of human cord blood HMGB1 according to delivery and labour: A pilot study

ObjectiveOxidative stress is involved in several maternal conditions characterized both by an increase in free radicals synthesis and a parallel decrease in the antioxidant activity. Parturition induces considerable oxidative stress and many inflammatory mediators, among which HMGB1, are involved from the beginning of pregnancy to the birth of the infant. We evaluated serum cord blood HMGB1 levels in a population of neonates to investigate correlation with mode of delivery, as well as the influence of labour. Setting and patientsThe study subjects were 325 neonates delivered at University Hospital “G. Martino” of Messina over an 18-month period. Following cord separation, venous blood sampling was performed on umbelical cords. ResultsIn the cord venous blood, we found HMGB1 values significantly more elevated in spontaneous vaginal group when compared to elective or emergency caesarean section group. Regarding labour, umbilical cord venous blood HMGB1 levels were significantly higher in the spontaneous and induced labour group, compared to non-labouring women. ConclusionThese results could highlight a possible role of HMGB1 during birth time related to mode of delivery and labour.

HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity

HMGB proteins are involved in structural rearrangements caused by regulatory chromatin remodeling factors. Particular interest is attracted to a DNA chaperone mechanism, suggesting that the HMGB proteins introduce bends into the double helix, thus rendering DNA accessible to effector proteins and facilitating their activity. The review discusses the role that the HMBG proteins play in key intranuclear processes, including assembly of the preinitiation complex during transcription of ribosomal genes; transcription by RNA polymerases I, II, and III; recruitment of the SWI/SNF complex during transcription of nonribosomal genes; DNA repair; etc. The functions of the HMGB proteins are considered in detail with the examples of yeast HMO1 and NHP6. The two proteins possess unique features in adition to properties characteristic of the HMGB proteins. Thus, NHP6 stimulates a large-scale ATP-independent unwrapping of nucleosomal DNA by the FACT complex, while in its absence FACT stabilizes the nucleosome. HMO1 acts as an alternative linker histone. Both HMO1 and NHP6 are of applied interest primarly because they are homologs of human HMGB1, an important therapeutic target of anticancer and anti-inflammatory treatments.

NF-κB signaling pathway mediates respiratory syncytial virus-induced HMGB1 release in airway epithelial cells to promote an inflammatory response

Abstract Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in young children, the elderly and immunocompromised individuals. In addition to acute morbidity, RSV infections have been linked to both the development and severity of asthma. No effective treatment or vaccine is currently available for RSV. High mobility group box 1 protein (HMGB1) is a multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. Although HMGB1 is implicated in the pathogenesis of many inflammatory diseases, its involvement in RSV-induced lung inflammation is not yet investigated. The goal of this study is to investigate the molecular mechanisms that induce the release of HMGB1 and the involvement of the NF-κB and TLR4 pathway in RSV pathogenesis. Human lung epithelial cells were infected with RSV in the presence or absence of human recombinant HMGB1 and harvested over time to analyze HMGB1, NF-κB and TLR4 expression by Western blot, immunohistochemistry and qRT-PCR. Our studies with airway epithelial cells clearly show that RSV-induces a significant release of HMGB1 from cells as a result of its translocation from the nucleus to the cytoplasm and subsequently to the extracellular space; that secreted HMGB1 triggers immune responses by activating primary human monocytes via NF-κB-TLR4 signaling pathway. Blockage of one of these molecules could represent a novel therapeutic strategy in the treatment not only for RSV infection, but also for other respiratory diseases that affect human population at any age.

378 - The role of High-Mobility Group Box 1 protein in mesenchymal–epithelial signalling in the tumour microenvironment

Introduction Glucose depravation, hypoxia, and acidosis are characteristic features of the central core most solid tumours. Myofibroblasts are stromal cells present in many such solid tumours including those of the colon, and they are known to be involved in all stages of tumour progression. HMGB1 is a nuclear protein that plays an important role in nucleosome stabilisation and gene transcription. HMGB1 is also released from immune cells and is involved in the inflammatory process. This work reports a novel finding that the microenvironmental condition of glucose depravation is responsible for the active release of HMGB1 from different types of cancer cell lines (HT-29, MCF-7 and A549) under normoxic conditions. Materials and method Human colonic myofibroblasts cells CCD18COCo, and cancer cells, colon HT-29, lung A549 and breast MCF7 were obtained from the ATCC and bladder cancer cells EJ138 from the ECACC. The human recombinant HMGB1, anti-HMGB1 antibody, anti-RAGE primary antibody and anti-TLR4 antibody were purchased from R&D systems. MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signalling Technology (USA). Medium was collected from HT-29, MCF-7, EJ138 and A549 cells after 48h treatment with various conditioned or fresh medium and analysed by western blotting for the presence of HMGB1. The MTT assay was used measure proliferation of myofibroblasts after treatment with conditioned medium with or without an MEK1/2 inhibitor or a PI3K inhibitor. Migration of CCD18Co cells in response to medium previously conditioned on HT-29 cells for 20h was studied using 8µm pore Boyden chamber inserts in 24-well plate system (BD Bioscience, California, USA). In addition, invasion assays of myofibroblast cells were performed using 8µm pore matrigel matrix Biocoat® inserts according to the manufacturer’s instruction (Becton Dickinson). Results and discussion Recombinant HMGB1 (10ng/ml) was shown to trigger proliferation in myofibroblasts cells via activation of PI3K and MEK1/2. Conditioned medium collected from glucose deprived HT-29 colon cancer cells was shown to stimulate migration and invasion of colonic myofibroblasts, and these processes were significantly inhibited by immunoneutralising antibodies to HMGB1, RAGE and TLR4, along with specific inhibitors of PI3K and MEK1/2. Conclusion Together these data suggests that HMGB1 released from the cancer cells under glucose depravation is involved in stimulating colonic myofibroblast migration and invasion and that this was through activation of RAGE and TLR4, resulting in activation of the MAPK and PI3K signalling pathways. Thus, it is proposed that HMGB1 may be released by cancer cells in areas of low glucose in solid tumours with the resulting activation of myofibroblasts. Therefore, HMGB1 may be considered as potential therapeutic target to inhibit solid tumour growth.

High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway.

Recent studies have indicated that high-mobility group box 1 protein (HMGB1) and the receptor for advanced glycation end-products (RAGE) contribute to the pathogenesis of asthma. However, whether the activation of the HMGB1/RAGE axis mediates airway epithelial barrier dysfunction remains unknown. Thus, the aim of this study was to examine the effects of HMGB1 and its synergistic action with interleukin (IL)-1β on airway epithelial barrier properties. We evaluated the effects of recombinant human HMGB1 alone or in combination with IL-1β on ionic and macromolecular barrier permeability, by culturing air-liquid interface 16HBE cells with HMGB1 to mimic the differentiated epithelium. Western blot analysis and immunofluorescence staining were utilized to examine the level and structure of major junction proteins, namely E-cadherin, β-catenin, occludin and claudin-1. Furthermore, we examined the effects of RAGE neutralizing antibodies and mitogen-activated protein kinase (MAPK) inhibitors on epithelial barrier properties in order to elucidate the mechanisms involved. HMGB1 increased FITC-dextran permeability, but suppressed epithelial resistance in a dose-and time-dependent manner. HMGB1-mediated barrier hyperpermeability was accompanied by a disruption of cell-cell contacts, the selective downregulation of occludin and claudin-1, and the redistribution of E-cadherin and β-catenin. HMGB1 in synergy with IL-1β induced a similar, but greater barrier hyperpermeability and induced the disruption of junction proteins. Furthermore, HMGB1 elicited the activation of the RAGE/extracellular signal-related kinase (ERK)1/2 signaling pathway, which correlated with barrier dysfunction in the 16HBE cells. Anti-RAGE antibody and the ERK1/2 inhibitor, U0126, attenuated the HMGB1-mediated changes in barrier permeability, restored the expression levels of occludin and claudin-1 and pevented the redistribution of E-cadherin and β-catenin. Taken together, the findings of our study demonstrate that HMGB1 is capable of inducing potent effects on epithelial barrier function and that RAGE/ERK1/2 is a key signaling pathway involved in the crosstalk between formations of junction proteins and epithelial barrier dysfunction.

RhHMGB1 drives osteoblast migration in a TLR2/TLR4- and NF-κB-dependent manner.

Osteoblast migration is significant in skeletal development. Recently, high mobility group box 1 protein (HMGB1) has been shown to highly expressed in cartilage to regulate endochondral ossification. Nevertheless, whether HMGB1 can modulate osteoblast proliferation and migration is poorly understood, as well as the intracellular signalling pathways that are involved in this process. Herein, we examined the effects of recombinant human HMGB1 (rhHMGB1) on the proliferation and migration of rat osteoblasts and investigated whether Toll-like receptor 2 (TLR2)- and TLR4-dependent signalling pathways are involved in the regulation of intracellular signalling. A transwell chamber assay was used to evaluate the migration of osteoblasts and the MTT assay was used to assess osteoblast proliferation. rhHMGB1 could significantly promote the migration of osteoblasts without inhibiting their proliferation. Meanwhile, rhHMGB1 can increase the nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Specific siRNA constructs that target TLR2 or TLR4 could markedly inhibit HMGB1-induced migration of osteoblasts and HMGB1-enhanced activation of NF-κB. Collectively, HMGB1 could significantly enhance the migration of osteoblasts invitro, and TLR2/TLR4-dependent NF-κB pathways are involved in HMGB1-induced osteoblast migration.

High-mobility group box-1 was released actively and involved in LPS induced depressive-like behavior

Depression disorder is a common mental illness, of which the pathogenesis is not well understood. Studies suggest that immunity imbalance and up-regulation of pro-inflammatory cytokines may be associated with the pathogenesis of depression. High-mobility group box 1 protein (HMGB1) has gained much attention as an important player in innate immune responses and an modulating factor in several inflammatory diseases. Here we sought to explore the role of HMGB1 in the development of depression. Depression model was established with low dose of lipopolysaccharide (LPS) administration. Depressive behavior was reflected with increased immobility time in tail suspension test. Accompanying with depressive-like behavior, translocation of HMGB1 from nuclei to cytoplasm was observed by immunofluorescence assays. Meanwhile, no significant necrosis was observed evaluated by hematoxylin-eosin staining. These data indicated that HMGB1 was released actively in the central nervous system. In addition, treating the mice with human recombinant HMGB1 (rHMGB1) could induce the development of depressive-like behavior. Blockage of HMGB1 with GZA abrogated the depressive-like behavior induced by LPS or rHMGB1. These results implicated that HMGB1 was involved in LPS-induced depressive-like behavior.

Genomic Analysis and Differential Expression of HMG and S100A Family in Human Arthritis: Upregulated Expression of Chemokines, IL-8 and Nitric Oxide by HMGB1

We applied global gene expression arrays, quantitative real-time PCR, immunostaining, and functional assays to untangle the role of High Mobility Groups proteins (HMGs) in human osteoarthritis (OA)-affected cartilage. Bioinformatics analysis showed increased mRNA expression of Damage-Associated Molecular Patterns (DAMPs): HMGA, HMGB, HMGN, SRY, LEF1, HMGB1, MMPs, and HMG/RAGE-interacting molecules (spondins and S100A4, S100A10, and S100A11) in human OA-affected cartilage as compared with normal cartilage. HMGB2 was down-regulated in human OA-affected cartilage. Immunohistological staining identified HMGB1 in chondrocytes in the superficial cartilage. Cells of the deep cartilage and subchondral bone showed increased expression of HMGB1 in OA-affected cartilage. HMGB1 was expressed in the nucleus, cytosol, and extracellular milieu of chondrocytes in cartilage. Furthermore, HMGB1 was spontaneously released from human OA-affected cartilage in ex vivo conditions. The effects of recombinant HMGB1 was tested on human cartilage and chondrocytes in vitro. HMGB1 stimulated mRNA of 2 NFκB gene enhancers (NFκB1 and NFκB2), 16 CC and CXC chemokines (IL-8, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2, CCL5, CCL8, CXCL1, CXCL10, CXCL2, CXCL3, and CXCL6) by ≥10-fold. Furthermore, HMGB1 and IL-1β and/or tumor necrosis factor α (but not HMGI/Y) also significantly induced inducible nitric oxide synthase, NO, and interleukin (IL)-8 production in human cartilage and chondrocytes. The recombinant HMGB1 utilized in this study shows properties that are similar to disulfide-HMGB1. The differential, stage and/or tissue-specific expression of HMGB1, HMGB2, and S100A in cartilage was associated with regions of pathology and/or cartilage homeostasis in human OA-affected cartilage. Noteworthy similarities in the expression of mouse and human HMGB1 and HMGB2 were conserved in normal and arthritis-affected cartilage. The multifunctional forms of HMGB1 and S100A could perpetuate damage-induced cartilage inflammation in late-stage OA-affected joints similar to sterile inflammation. The paracrine effects of HMGB1 can induce chemokines and NO that are perceived to change cartilage homeostasis in human OA-affected cartilage.