Ikuro Maruyama

The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation-induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature.

Anandamide induces apoptosis of PC‐12 cells: involvement of superoxide and caspase‐3

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC‐12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub‐G0/G1 and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32‐like protease activity in PC‐12 cells markedly. Furthermore, antioxidant N‐acetyl cysteine prevented anandamide‐induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide‐induced apoptosis of PC‐12 cells.

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum ‐associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin‐null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Proteolytic Cleavage of High Mobility Group Box 1 Protein by Thrombin-Thrombomodulin Complexes

Objective—High mobility group box 1 protein (HMGB1) was identified as a mediator of endotoxin lethality. We previously reported that thrombomodulin (TM), an endothelial thrombin-binding protein, bound to HMGB1, thereby protecting mice from lethal endotoxemia. However, the fate of HMGB1 bound to TM remains to be elucidated. Methods and Results—TM enhanced thrombin-mediated cleavage of HMGB1. N-terminal amino acid sequence analysis of the HMGB1 degradation product demonstrated that thrombin cleaved HMGB1 at the Arg10-Gly11 bond. Concomitant with the cleavage of the N-terminal domain of HMGB1, proinflammatory activity of HMGB1 was significantly decreased (P<0.01). HMGB1 degradation products were detected in the serum of endotoxemic mice and in the plasma of septic patients with disseminated intravascular coagulation (DIC), indicating that HMGB1 could be degraded under conditions in which proteases were activated in the systemic circulation. Conclusions—TM not only binds to HMGB1 but also aids the proteolytic cleavage of HMGB1 by thrombin. These findings highlight the novel antiinflammatory role of TM, in which thrombin–TM complexes degrade HMGB1 to a less proinflammatory form.

High-mobility group box 1 protein promotes development of microvascular thrombosis in rats.

Summary.  Background: Sepsis is a life‐threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High‐mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. Objectives: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. Methods: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. Results: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin–thrombomodulin complex, and stimulated tissue factor expression on monocytes. Conclusions: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

High Mobility Group Protein 1 (HMGB1) Quantified by ELISA with a Monoclonal Antibody That Does Not Cross-React with HMGB2

High mobility group protein 1 (HMGB1) has been implicated in diverse cellular functions, including determination of nucleosomal structure and stability and binding of transcription factors to their cognate DNA sequences (1)(2)(3)(4). HMGB1 is also present in a membrane-associated form, termed amphoterin, that mediates neurite outgrowth (5). Amphoterin can interact with macrophage cell surface receptors for advanced glycation end products to enhance expression of tissue-type plasminogen activator (6). Recently, HMGB1 was identified as a late mediator of endotoxin lethality (7). Mice had increased serum HMGB1 concentrations after exposure to endotoxin, and sepsis patients who succumbed to infection also had increased serum HMGB1. It would therefore be useful to develop an easy and highly sensitive method to measure serum HMGB1. However, this study revealed that HMGB1 and HMGB2, with extremely high homology (81%) to HMGB1, coexist in the serum. We report an ELISA method we have developed that measures only HMGB1 without simultaneous determination of HMGB2. To prepare an anti-peptide monoclonal antibody reacting only with HMGB1, we selected a peptide sequence (peptide 1; GKGDPKKPRGK) with high antigenicity and different from that of HMGB2. The monoclonal anti-calf HMGB1 antibody was prepared against calf thymus-derived HMGB1, which was 98% homologous to human HMGB1. Each protein sample used for the analysis was prepared as follows. The peptides were purified and separated by HPLC. The peptide synthesized was added to maleimidobenzoyl- N -hydroxysuccinimide ester (Pierce Chemical Co.)-labeled keyhole limpet hemocyanin (Calbiochem) or maleimidobenzoyl- …

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)‐immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

Thrombomodulin: protectorate God of the vasculature in thrombosis and inflammation

Summary.  Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin‐mediated activation of protein C. Recently, we conducted a multicentre, double‐blind, randomized trial to evaluate the efficacy and safety of recombinant human soluble thrombomodulin (rhsTM, also known as ART‐123) for the treatment of disseminated intravascular coagulation (DIC), and found that rhsTM therapy is more effective and safer than low‐dose heparin therapy. Thus, in 2008, rhsTM (Recomodulin) was approved for the treatment of DIC in Japan. Here we re‐evaluate the therapeutic basis of this drug from the view of its anticoagulant, anti‐inflammatory, and cytoprotective properties. Structurally, the extracellular portion of TM is composed of three domains: an N‐terminal lectin‐like domain (TM‐D1), followed by an epidermal growth factor (EGF)‐like domain (TM‐D2), and an O‐glycosylation–rich domain (TM‐D3). TM‐D2 and TM‐D3 are important for the protein’s anticoagulant cofactor activities, i.e. inhibition of thrombin and activation of protein C. TM‐D1 plays an important role in attenuation of inflammatory responses, through inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, neutralization of lipopolysaccharide (LPS), and sequestration and degradation of pro‐inflammatory high‐mobility group box 1 protein (HMGB1). Thus, TM on the surface of endothelial cells prevents dissemination of pro‐coagulant and pro‐inflammatory molecules, and by doing so, allows these molecules to act locally at the site of injury. In patients with sepsis and DIC, TM expression is down‐regulated, which may result in dissemination of pro‐coagulant and pro‐inflammatory molecules throughout the systemic circulation. Replacement with rhsTM may offer therapeutic value in such conditions.

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.

Abstract: Anandamide triggers various cellular activities by binding to cannabinboid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-β cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.