高速泳动族蛋白1的致炎症作用机制

高速泳动族蛋白1(high-mobility group box 1,HMGB1)是一种高度保守的DNA结合蛋白,具有维持核小体结构和调节基因转录的功能,近来发现它是炎性反应强有力的促炎因子。在大多炎性疾病,特别是脓毒症病例中,HMGB1的血清和组织水平均显著升高,而且它与其受体如糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、Toll样受体4(toll-like receptor,TLR4)、Toll样受体2(TLR2)等相互作用促进炎性疾病的发展。为了进一步了解HMGB1,本文就HMGB1的结构、生物学活性、与免疫细胞相互作用、细胞表面受体、以及拮抗HMGB1的药物等进行综述。     

The Saccharomyces cerevisiae high mobility group box protein HMO1 contains two functional DNA binding domains

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K(d) = 39.6 +/- 5.0 nm and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 +/- 2.2 nm, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 +/- 1.1 nm). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure.     

High mobility group box transcription factor 1 (HBP1) from Lampetra japonica affects cell cycle regulation

High mobility group (HMG) box‐containing protein 1 (HBP1) is a member of the HMG family of chromosomal proteins. Previous studies have shown that human HBP1 exhibits tumor‐suppressor activity. Here, we identified a homologue of HBP1, L‐hbp1, in Lampetra japonica. The L‐hbp1 gene shared high sequence similarity with its homologues in jawed vertebrates, as shown by bioinformatics analyses. L‐hbp1 contains a 1,584‐bp open reading frame that encodes 527 amino acids. A pAdenox‐L‐HBP1 plasmid was constructed and transfected successfully in Raji cells, as revealed by real‐time PCR. The overexpression of L‐HBP1 reduced cell growth rates, inhibited G1 phase progression, decreased cyclin D1 and c‐Myc protein expression, and increased p53 protein expression. Western blot and immunohistochemical assays showed that L‐HBP1 was primarily distributed in the heart, kidney, gill and liver of lamprey. Cell cycle analysis revealed that decreased L‐HBP1 expression in HBP1 morpholino oligonucleotide‐transfected lamprey cells resulted in a decreased fraction of cells in the G1 phase and corresponding increases in the S and G2/M phases. Additionally, treatment of lamprey cardiac cells with pharmacological inhibitors of p38 MAP kinase released the cells from G1 arrest. Together, these results indicated that HBP1 expression in lamprey was correlated with the onset of mitotic arrest in these cells, which have implications for cell cycle regulation.     

Structural basis for the proinflammatory cytokine activity of high mobility group box 1

High mobility group box 1 (HMGB), a ubiquitous DNA-binding protein, has been implicated as a proinflammatory cytokine and late mediator of lethal endotoxemia. HMGB1 is released by activated macrophages. It amplifies and extends the inflammatory response by inducing cytokine release and mediating acute lung injury, anorexia, and the inflammatory response to tissue necrosis. The kinetics of HMGB1 release provide a wide therapeutic window for endotoxemia because extracellular levels of HMGB1 begin to increase 12 to 24 h after exposure to inflammatory stimuli. Here, we demonstrate that a DNA-binding domain of HMGB1, the B box, recapitulates the cytokine activity of full length HMGB1 and efficiently activates macrophages to release tumor necrosis factor (TNF) and other proinflammatory cytokines. Truncation of the B box revealed that the TNF-stimulating activity localizes to 20 amino acids (HMGB1 amino acids 89 to 108). Passive immunization of mice with antibodies raised against B box conferred significant protection against lethal endotoxemia or sepsis, induced by cecal perforation. These results indicate that a proinflammatory domain of HMGB1 maps to the highly conserved DNA-binding B box, making this primary sequence a suitable target in the design of therapeutics.     

Hyperlipidemia stimulates the extracellular release of the nuclear high mobility group box 1 protein

Our aim was to evaluate the effect of hyperlipidemia on the activation of endogenous alarmin, the high mobility group box 1 (HMGB1) protein, related to systemic inflammation associated with the progression of experimental atherosclerosis and to establish whether statin treatment regulates the HMGB1 signaling pathway. Hyperlipidemia was induced in vivo in golden Syrian hamsters and in monocyte cell culture (U937) by feeding the animals with a high-fat Western diet and by exposing the cells to hyperlipidemic serum. Blood samples, heart, lung and cells were harvested for biochemical, morphological, Western blot, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses. The data revealed that, in the atherosclerotic animal model, the protein HMGB1 and its gene expression were increased and that fluvastatin treatment significantly reduced the release of HMGB1 into the extracellular space. The cell culture experiments demonstrated the relocation of HMGB1 protein from the nucleus to cytoplasm under hyperlipidemic stress. The high level of detected HMGB1 correlated positively with the up-regulation of the advanced glycation end product receptors (RAGE) in the lung tissue from hyperlipidemic animals. During hyperlipidemic stress, the AKT signaling pathway could be activated by HMGB1-RAGE interaction. These results support the existence of a direct correlation between experimentally induced hyperlipidemia and the extracellular release of HMGB1 protein; this might be controlled by statin treatment. Moreover, the data suggest new potentials for statin therapy, with improved effects on patients with systemic inflammation induced by hyperlipidemia.     

Induction of arthritis by high mobility group box chromosomal protein 1 is independent of tumour necrosis factor signalling

Introduction

TNFα and high mobility group box chromosomal protein 1 (HMGB1) are two potent proinflammatory cytokines implicated as important mediators of arthritis. Increased levels of these cytokines are found in the joints of rheumatoid arthritis patients, and the cytokines trigger arthritis when applied into the joints of naïve mice. HMGB1 is actively released from immune cells in response to TNFα; once released, HMGB1 in turn induces production of several proinflammatory cytokines – including IL-6 and TNFα – by macrophages. Whether HMGB1-induced arthritis is mediated via the TNFα pathway, however, is unknown. The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFα expression in vivo and to assess whether TNFα deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.

Methods

TNFα knockout mice and backcrossed control animals on a C57Bl6 background were injected intraarticularly with 5 μg HMGB1. Joints were dissected 3 days after intraarticular injection and were evaluated histologically by scoring the frequency and severity of arthritis. For in vitro studies, mouse spleen cultures from TNFα knockout mice and from control mice were incubated with different doses of HMGB1, and cell culture supernatants were collected at different time points for analysis of IL-6.

Results

Intraarticular injection of HMGB1 into healthy mouse joints resulted in an overall frequency of 32% to 39% arthritic animals. No significant differences were found with respect to the severity and incidence of synovitis between mice deficient for TNFα (seven out of 18 mice with arthritis) in comparison with control TNFα^+/+ animals (six out of 19). No significant differences were detected between spleen cells from TNFα^+/+ mice versus TNFα^-/- mice regarding IL-6 production upon stimulation with highly purified HMGB1 after 24 hours and 48 hours. Upon stimulation with a suboptimal dose of recombinant HMGB1, however, the splenocytes from TNFα^+/+ animals released significantly more IL-6 than cells from the knockout mice (602 ± 112 pg/ml and 304 ± 50 pg/ml, respectively; P < 0.05).

Conclusion

Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway. Combined with our previous study, we suggest that HMGB1-triggered arthritis is probably mediated through IL-1 activation.     

Systemic release of high mobility group box 1 protein during severe murine influenza

Hypercytokinemia is gaining recognition as the mechanism of fatality from influenza. No work to date has addressed the role of high mobility group box 1 protein (HMGB1) in influenza, the parallel being that in other severe proinflammatory cytokine syndromes (e.g., sepsis and malaria) levels of circulating HMGB1 are elevated and may correlate with death. Using a commercially available ELISA for HMGB1, we found that HMGB1 was not increased in the plasma of influenza virus-infected mice (A/Japan/305/57) on day 7 post infection, about the time of peak mortality, and peak levels of HMGB1 in the plasma did not occur until relatively late in infection, on day 9 post infection. In keeping with the late peak of HMGB1 being unassociated with mortality, administration of ethyl pyruvate, which inhibits active secretion but not passive release of HMGB1, to influenza virus-infected mice, did not affect their survival. Further work is required to determine whether influenza virus infection induces passive release of HMGB1, and whether HMGB1 neutralization with a specific Ab would improve survival.