摘要
高迁移率族蛋白B1(high mobility group protein B1,HMGB1)是高迁移率族(high mobility group,HMG)蛋白家族成员之一,1973年英国科学家Goodwin在牛胸腺细胞中首次发现,因其在聚丙烯酰胺电泳中的高迁移率得名,它是一种含量丰富、分子量小、高度保守的染色质相关非组蛋白。HMGB1分布十分广泛,多种器官细胞(如淋巴组织、脑、肝、肺、心、脾、肾等)中均有表达,并位于大多数细胞的胞核和胞浆中。最初发现其作为核因子直接参与基因表达调控,近年陆续发现其具有丰富的生物学功能,包括参与脑的发育和神经细胞生长、纤维蛋白溶酶原的活化、肿瘤细胞转移、免疫细胞分化以及广泛参与全身的炎症反应和组织损伤等。
脓毒症目前仍然是临床危重患者的重要死因之一,其死亡率高达30～70%。研究表明,HMGB1作为重要的炎症介质在全身炎症反应过程中起重要作用。致炎刺激脂多糖(lipopolysaccharide,LPS)等激活的单核/巨噬细胞和内皮细胞经8～32 h延迟后释放HMGB1,同时坏死组织细胞崩解也释放出大量的HMGB1。释放到细胞外的HMGB1分子通过进一步激活单核或内皮等细胞,引起大量炎症因子和黏附分子的表达和释放,导致并参与了包括脑组织、肺、胃肠道、关节、心脏等多种脏器的炎症损伤以及广泛的全身性炎症反应甚至死亡。由此可见,HMGB1在炎症反应过程中起着重要的中枢作用。
HMGB1在大多数细胞和组织中都控制在一个基础的表达水平,但HMGB1并不是组成型表达,而是被严格调控的。在增殖组织和活化的分裂细胞,其表达水平可以提高2倍,在雌激素刺激的乳癌细胞中其表达量也可提高2倍以上;在用顺氯氨铂治疗的癌症中,其表达水平也升高,且能够增强顺氯氨铂的抗癌效果。不同刺激在不同细胞、不同种系可能通过不同的信号通路诱导HMGB1的表达,相关的调控机制也不尽相同。
Lum等人克隆并分析了人HMGB1基因的的上游区域和第一个内含子,发现在乳癌(human breast adenocarcinoma cell line,MCF-7)细胞中人HMGB1基因的转录起始于第一个内含子—外显含子交界处上游57个核苷酸的位点。人HMGB1基因的表达受TATA框缺失的启动子的调控,这一启动子的活性比猴空泡病毒40(simian vacuolating virus 40,SV40)强18倍之多。其中的沉默子能抑制启动子的活性至原来的六分之一,基因中的第一个内含子包含有增强子,能使此启动子的活性增强2～3倍,因此我们设想HMGB1在此增强子的作用下其表达水平能够明显的提高,而我们在大多数细胞中观察到HMGB1的基础表达量可能是由于沉默子抑制作用。
参与内毒素休克中HMGB1表达的信号调控通路也仅仅有一些初步的研究。Gardella研究发现脂多糖结合蛋白(lipopolysaccharide-binding protein,LBP)/白细胞分化抗原14(cluster of differentiation 14,CD14)作为LPS的结合受体,与HMGB1的基因表达密切相关,提示LPS可能通过LBP/CD14及其下游的信号激活单核/巨噬细胞,HMGB1从核内移至胞浆中的细胞器,然后通过溶酶体的自溶、胞吐作用将HMGB1释放出来。乙基丙酮酸通过抑制细胞外信号调控激酶1/2(extracellular signal-regulated kinases 1/2,ERK1/2)磷酸化和抑制巨噬细胞的活性也可以抑制HMGB1的释放。Wang等人发现通过抑制蛋白酪氨酸激酶(Janusprotein tyrosine kinase,JAK)/信号转导子和转录激活子(signal transducer andactivator of transcription,STATs)通路活化可明显下调肝脏组织HMGB1 mRNA表达,从而有助于减轻脓毒症大鼠的急性肝损伤。胥彩林等人则发现核因子κB(nuclear factor-kappaB,NF-κB)抑制剂能显著抑制内毒素休克动物组织(肝、肺、肾)HMGB1 mRNA的表达,使各脏器损伤和功能得到明显改善。由此可知上述信号分子所在信号通路可能参与了HMGB1的表达调控,但确切的机制目前仍不清楚。
基因表达的调控是细胞对外部或内部的刺激发生应答的方式,这是一个高度复杂,精确调控的过程,基因表达调控可以在复制、转录、转录后、翻译和翻译后多等级水平上进行,但DNA转录起始调控是基因表达调控的关键控制点,即在一定时间、空间上,多种调节蛋白作用于特异的基因启动子的复杂过程,其本质是DNA—蛋白质/蛋白质—蛋白质间广泛的相互作用。另外,人们发现,在基因表达调控过程中,往往不是单个因素起作用,而是多种调节因素相互交织,相互影响,形成复杂的调控网络,最终使基因表达调控呈现出一种高度的有序性。鉴于这种广泛的蛋白质与蛋白质、蛋白质与DNA相互作用网络参与其中,相关研究策略也层出不穷。其中,启动子DNA结合蛋白的研究是从转录水平上研究基因表达的调节,以寻找新的蛋白质为手段,以了解其转录调节作用为目的,从而最终为阐明某些疾病的发病机制及基因治疗奠定基础。目前,对启动子DNA结合蛋白的研究方法可分为两大类:第一类是细胞内法,以已知启动子DNA序列筛选出与其相结合的蛋白,通过生物信息分析来确定该蛋白质;第二类是细胞外法,即在体外用重组的已知蛋白质与启动子DNA结合。二者各有其自身的优势,又有各自的缺陷。我们将以上二者结合应用,以期达到既能找到未知调控蛋白,又能找到该蛋白质的精确的结合位点并证实二者结合特异性的目的。
由此,我们以小鼠肝脏基因组DNA为模板,扩增小鼠HMGB1基因5'非编码区(noncoding region,NCR)1378 bp长的调控序列,将其插入pDsRed1-1红色荧光素酶报告基因载体上。应用脂质体介导基因共转染技术将质粒pDsRed1-1/HMGB1p导入NIH/3T3细胞,给予TNF-α持续刺激,观察红色荧光蛋白表达情况,证明所得到的启动子序列具有转录活性,为进一步研究HMGB1基因表达信号调控机制提供一个方便、可靠的工具。
同时,从“组学”的角度和活体组织水平,根据DNA—蛋白质相互作用的原理,基于生物素—链亲和素系统、磁珠分离μMACS~(TM)系统,结合生物质谱技术筛选了内毒素休克小鼠的肝脏组织中与HMGB1启动子发生相互作用的结合蛋白。利用末端生物素(biotin)标记的HMGB1启动子区引物,PCR扩增制备带biotin标记的HMGB1启动子探针HMGB1p-biotin。制备BALB/c小鼠的内毒素休克模型,提取肝脏组织核蛋白,与扩增的HMGB1p-biotin探针行结合反应,再以标记有链亲和素(streptavidin)的磁珠分离HMGB1p-biotin—蛋白反应复合物,不同盐浓度缓冲液洗脱结合的蛋白,使用聚丙稀酰胺凝胶电泳分离洗脱的样品蛋白,并对凝胶行改良考染显色,比较内毒素休克组与正常对照组的差异条带(DNA pull-down assay)。结果表明,在肝脏组织胞核中,调出11个差异蛋白,这些蛋白可能直接或间接参与了内毒素休克小鼠肝脏中HMGB1的表达调控。其中有10个蛋白在LPS作用后与HMGB1启动子结合增加,提示这些蛋白可能参与了LPS作用后HMGB1的转录调控,多次重复均得到相同的结果。
对结果中出现的11个差异条带进行质谱(mass spectrometry,MS)鉴定,根据得到的肽指纹图谱,利用Mascot查询软件搜索NCBI数据库,综合分析后得到7个与转录调控有关的蛋白分子,分别为组蛋白2A(histone 2A,H2A)、组蛋白2B(histone 2B,H2B)、组蛋白3(histone 3,H3)、组蛋白4(histone 4,H4)、S100A9蛋白(myeloid-related protein 14,MRP14)、双功能过氧化物酶(peroxisomalbifunctional enzyme,Ehhadh)、乙酰辅酶A酰基转移酶2(acetyl-Coenzyme Aacyltransferase 2,ACox2)。其中,H3作为染色体结构中高度保守的一类核心组蛋白,主要通过其乙酰化水平的调节控制基因转录的“开”和“关”;乙酰辅酶A酰基转移酶则参与氨基酸残基乙酰基化反应、MS鉴定得到的这些结果充分说明内毒素休克小鼠中HMGB1基因的表达调控可能受到了多种因素的影响,这些因素相互协调,共同作用从而影响HMGB1基因的表达调控。
通过以上研究,我们得出:一、克隆出具有启动基因表达活性的HMGB1启动子DNA片段;二、构建了红色荧光蛋白报告基因载体pDsRed1-1/HMGB1p,并在NIH/3T3细胞内证实该启动子具有启动红色荧光蛋白表达的活性,可有效用于HMGB1基因表达信号调控机制的研究;三、利用生物素-链亲和素、磁珠分离μMACS~(TM)系统成功筛选到参与HMGB1基因调控的转录因子,在胞核蛋白中至少有11个蛋白可能参与了这一过程,并利用生物质谱技术鉴定相关蛋白分别为H2A、H2B、H3、H4,S100A9,Ehhadh,ACox2。
本研究利用DNA—蛋白质相互作用的原理,为从“转录组学”的角度分析基因的转录表达调控过程及其机制开创了新的思路。同时差异条带的出现说明在内毒素休克小鼠HMGB1的转录调控发生了较为复杂的变化,可能有多种核蛋白或转录因子参与了这一过程。这些蛋白的鉴定对于我们深入理解HMGB1基因表达调控有重要的意义,同时也将为临床治疗内毒素休克提供新的理论依据及可能的治疗靶点。
High mobility group protein B1 (HMGB1) is a member of high mobility group (HMG) protein family, which was first found in bovine thymus cells by British scientist Goodwin in 1973, and was named HMG for its high mobility rate in polyacrylamide gel electrophoresis. HMGB1 is a high abundance low molecular weight nonhistone protein with highly conserved sequence in cells. HMGB1 is widely expressed in various organ cells, including lymph tissue, brain, lung, liver, heart, spleen, kidney, etc., and it is commonly located in the nucleus and cytoplasm in most of cells. Recently, it was found that HMGB1 was a mutifunction protein, which can enhance neuronal growth, activate plasminogen, assist tumour invasion and metastasis, promote differentiation of immune cells, participate in the systemic inflammatory response and injury.
Sepsis remains to be one of the chief causes of death in intensive care units at present, with the mortality is between 30～70%. Several therapeutic agents that target tumor necrosis factor (TNF) and interleukin-1 (IL-1) have been tested in clinical trials of sepsis, and a significant survival advantage has not been observed. It is partly because of the rapid kinetics of the TNF and IL-1 response. It has been found that 8～32 h following stimulation with LPS, TNF or IL-1, HMGB1 is actively secreted from monocytes and macrophages. HMGB1 can also be passively released from necrotic cells. Once released, HMGB1 is able to activate many other cells including monocytes, macrophages, endothelial cells and epithelium to produce proinflammatory cytokine and adhesion molecules. HMGB1 causes inflammatory responses in various systems in vivo, including brain, lung, gastrointestinal tract and heart, leading to the systemic inflammatory response even death. This implicates HMGB1 plays a key role in inflammatory reactions.
The expressions of HMGB1 in most of cells and tissues are controlled in a basal expression level, but HMGB1 is not a constructive expressed protein and is strictly regulated. In proliferative tissues and active dividing cells, the expression level could be doubled, the same in mammary cancer cells stimulated by estrogen. In cancer patients which are treated with platinum dichloro-diamine, the expression level of HMGB1 is increased which associated with enhancement of the anti-cancer effect of platinum dichloro-diamine. The regulation mechanism of HMGB1 is different in different cells, species and signal pathways.
Lum et al cloned and analyzed the up-stream region and the first exon of human HMGB1 gene, and found in breast cancer cells MCF-7, the transcription of human HMGB1 gene was started at the -57 site of the first exon. The expression of human HMGB1 gene was regulated by a TATA box deletion promoter, and such promoter had 18 times activity than SV40. The silencer within the promoter could inhibit the promoter activity to 1/6, and the enhancer in the first intron of gene could enhance the promoter activity to 2 - 3 times, so we suspect that the expression level of HMGB1 could be greatly enhanced by such enhancer, and the basal expression level in most of the cells is mainly inhibited by the silencer.
At present, the study of signal transduction pathway of HMGB1 in endotoxic shock is still at very early stage. Gardella et al found LBP/CD14, which worked as the binding receptor of LPS, had close relationship with the expression of HMGB1, suggested that LPS may activate mononuclear phagocyte through LBP/CD14 and its down-stream signals. HMGB1 first transfered from nucleus to cytoplasmic organelle and then released by cytolysosome autolysis and exocytosis. Ethyl pyruvate could inhibit the release of HMGB-1 by ERK1/2 phosphorylation inhibition and inhibiting the activity of macrophage. Wang et al found the inhibition of activation of JAK/ATAT pathway could significantly up-regulate the expression of HMGB1 mRNA in liver tissue, so as to alleviate the acute liver injury of sepsis mouse. Xu et al. found NF-kappa B inhibitor could notably inhibit the expression of HMGB1 mRNA in various tissues of endotoxic shock animals, and therefore ameliorated the injury of those organs. Those signal molecules may participate the regulation process of HMGB1 expression, but the certain mechanism is still unknown.
Mice genomic DNA was used as template and the 5 flank noncoding region (NCR) of mice HMGB1 gene was amplified by PCR, which was consisted of 1378 bps. The amplication product was inserted into pDsRed1-1 red fluorescent protein (RFP) reporter gene vector to construct the RFP expression plasmid regulated by 5' NCR of HMGB1, and the new plasmid was named pDsRed1-1/HMGB1p. pDsRed1-1/HMGB1p was transfected into NIH/3T3 cells by liposome mediated gene cotransfect technology and give 50 ng/ml TNF stimulation after 24 hours of transfection. After 36 hours of stimulation, AxioVision system was used to observe the RFP signal intensity which provides us a reliable tool to further study the signal regulation mechanism of HMGB1.
Meanwhile, from the "omic" aspect and on the living tissue level, based on DNA-protein interacting principle, biotin-streptavidin system, magnetic bead separating technology and biological mass- spectrum technology, we had screened the binding proteins which were interacting with HMGB1 promoter in liver tissue of endotoxic shock mice. We use terminal biotin labeled primers to amplify the HMGB1 promoter probe (named HMGB1p-biotin) by PCR. Preparing endotoxic shock model of BALB/c mice and extracted nuclear protein from liver tissue. The HMGB1p-biotin-protein complex was adhered to streptavidin magnetic beads after the nuclear protein binded the HMGB1p-biotin prober. Then used different concentration buffers elute the protein and employed polyacrylamide gel electrophoresis (PAGE) to isolate the target protein. A refined Coomassie brilliant blue staining method was used to identify the different bands between endotoxic group and control group. The result suggested that 11 proteins may participate the regulatory process of HMGB1 gene expression in endotoxic mice liver. 10 proteins' binding affinities to HMGB1 promoter were increased after LPS stimulation, suggested that these proteins may take part in the post LPS stimulation regulation. The result could be stably repeated in different experiments.
These differentially expressed 11 proteins were identified by mass-spectrum and searched against the NCBI database using Mascot software. After bioinfomatic analysis, 7 proteins related to transcriptional regulation were obtained, they were histone H2A, H2B, H3, H4, S100A9, peroxisomal bifunctional enzyme and acetyl-coenzyme A acyltransferase 2 (ACAA2). Histone H3 was a highly conserved core histone in chromosome structure and controlled the on and off switch of gene transcription by regulating acetylate level, while ACAA2 could acetylate the amino residue. The result of MS clearly suggested that the regulation of HMGB1 gene expression in endotoxic mice were affected by various factors and these factors acted coordinately to affect the regulation of HMGB1 gene expression.
Following conclusions can be drown through above research: 1, HMGB1 promoter was cloned successfully which activity has been proved. 2, We constructed the mice HMGB1 promoter RFP reporter plasmid vector pDsRed1-1-HMGB1p and proved its ability to activate the protein RFP expression by transfected this plasmid into NIH/3T3 cells ,which provide us a convenient method to research regulatory mechanism of HMGB1 gene expression. 3, Some transcriptional factors were screened out by using biotin-streptavitin and magnetic beads technology, which may participate the regulation of HMGB1 gene expression. There were at least 11 proteins took part in the regulatory process which include histone H2A, H2B, H3, H4, S100A9, Ehhadh and ACox2 etc. identified by MS technology.
Based on the principle of DNA-protein interactions, our research created a new way for gene transcriptional regulation from the point of view of "transcriptomics ". In addition, the identified differential proteins illustrate that the expression of HMGB1 gene in endotoxic shock is a complex process, and these identified proteins could help us further understand the meaning of HMGB1 express regulation and also provide us new theoretical basis and possible therapy target of clinical trial of endoxic shock.
引文
1.Saluk-Juszczak J,Wachowicz B.The proinflammatory activity of lipopolysaccharide[J].Postepy Biochem,2005,51(3):280-287
2.Amersfoort ES,Van Berkel TJ,et al.Receptors,mediators,and mechanisms involved in bacterial sepsis and septic shock[J].Clin Microbiol Rev,2003,16(3):379-414
3.Ingalls RR,Heine H,et al.Lipopolysaccharide recognition,CD14,and lipopolysaccharide receptors[J].Infect Dis Clin North Am,1999,13(2):341-353
4.姜勇,Ulevitch RJ.LPS介导细胞激活的信号转导:从CD14到p38MAPK通路的研究[J].生理科学进展,1999,30(1):29-34
5.Ulevitch RJ,Tobias PS.Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin[J].Annu Rev Immunol,1995,13:437-457
6.Wright SD,Ramos RA,et al.CD14,a receptor for complexes of lipopolysaccharide(LPS)and LPS binding protein[J].Science,1990,249(4975):1431-1433
7.Muller S,Ronfani L,et al.Regulated expression and subcellular localization of HMGB-1,a chromatin protein with a cytokine function[J].J Intern Med,2004,255(3):332-343
8.Luscombe NM,Austin SE,et al.An overview of the structures of protein-DNA complexes [J].Genome Biol,2000,1(1):REVIEWS001
9.Luscombe NM,Thornton JM.Protein-DNA interactions:amino acid conservation and the effects of mutations on binding specificity[J].J.Mol.Biol,2002,320(5):991-1009
10.Sandaltzopoulos R,Becker PB.Solid-phase DNase I footprinting:quick and versatile[J].Nucleic Acids Research,1994,22(8):1511-1512
11.Sandaltzopoulos R,Quivy P,et al.Analysis of protein-DNA interactions by solid-phase footprinting methods.Molecular and Cellular Biology,1995,5(3):176-181
12.Nelson HC.Structure and function of DNA-binding proteins[J].Curr Opin Genet Dev,1995,5(2):180-189
13.Klug A.Gene regulatory proteins and their interaction with DNA[J].Ann N Y Acad Sci,1995,758:143-160
14.P(a|¨)hler A,Hendrickson WA,et al.Characterization and crystalization of core streptavidin[J].J.Biol.Chem,1987,262(29):13933-13937
15.Weber PC,Ohlendorf DH,et al.Structural origins of high-affinity biotin binding to streptavidin[J].Science,1989,243(4887):85-88
16.张晓春,陆燕蓉.生物素—亲和素技术的研究进展[J].现代预防医学,2001,28(4):485-494
17.Yaneva M,Tempst P.Isolation and mass spectrometry of specific DNA binding proteins[J].Methods Mol Biol,2006,338(21):291-303
18.Aneva M,Tempst P.Affinity capture of specific DNA-binding proteins for mass spectrometric identification[J].Anal Chem,2003,75(23):6437-6448
19.Zhang X,Scalf M,et al.Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass[J].J Am Soc Mass Spectrom,2006,17(4):490-499
20.Nordhoff E,Krogsdam AM,et al.Rapid identification of DNA-binding proteins by mass spectrometry[J].Nat Biotechnol,1999,17(9):884-888
21.黎永明,姜勇.脓毒症发生机制的新进展[J].感染.炎症.修复,2005,6(1):45-47
22.Baxevanis AD,Landsman D.The HMG-1 box protein family:classification and functional relationships[J].Nucleic Acids Res,1995,23(9):1604-1613
23.Harris HE,Andersson U.Mini-review:The nuclear protein HMGB1 as a proinflammatory mediator[J].Eur J Immunol,2004,34(6):1503-1512
24.Lotze MT,Tracey KJ.et al.High-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenal[J].Nat Rev Immunol,2005,5(4):331-342
25.Gardella S,Andrei C,et al.The nuclear protein HMGB-1 is secreted by monocytes via a non-classical,vesicle-mediated secretory pathway[J].EMBO Rep,2002,3(10):995-1001
26.Bonaldi T,Talamo F,et al.Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion[J].EMBO J.2003,22(20):5551-5560
27.Muller S,Ronfani L,et al.Regulated expression and subcellular localization of HMGB-1,a chromatin protein with a cytokine function[J].J Intern Med,2004,255(3):332-343
28.Lum HK,Lee KD,et al.The human HMGB-1 promoter is modulated by a silencer and an enhancer-containing intron.Biochimica et Biophysica Acta.2001,1520(1):79-84
29.王松柏,姚咏明,董宁,等.JAK/STAT通路介导脓毒症大鼠肝组织高迁移率族蛋白B1mRNA表达的研究[J].中国危重病急救医学,2003,15(3):147-149
30.胥彩林,姚咏明,于燕,等.NF-κB抑制剂对内毒素休克大鼠高迁移率族蛋白B1表达的影响[J].解放军医学杂志,2004,29(1):45-47
31.张立天,姚咏明,陆家齐,等.脓毒症大鼠高迁移率族蛋白-1基因表达及其与内毒素血症的关系[J].中华普通外科杂志,2002,17(9):556-558
32.Baird GS,Zacharias DA,et al.Biochemistry,mutagenesis,and oligomerization of DsRed,a red fluorescent protein from coral[J].Proc Natl Acad Sci,2000,7(22):1984-1989
33.刘志锋,姜勇.报告基因技术的理论基础及应用[J].生理科学进展,2002,33(4):361-363
34.郭爱华,姜勇.从全省炎症反应综合症到脓毒症休克[J].中国危重病急救医学,2002,14(8):500-503
35.Hessian PA,Fisher L.The heterodimeric complex ofMRP-8(S100A8) and MRP-14(S100A9).Antibody recog2nition,ep itope definition and the imp lications for structure[J].Eur J Biochem,2001,268(2):353-365
36.Hiroshi I,Min Y,et al.The crystal structure of human MRP14(S100A9),Ca~(2+) dependent regulator protein in inflammatory process[J].JMB,2002,316(5):265-267
37.37 Irmgard S,Thorey,et al.The Ca~(2+) binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes[J].J Biol Chem,2001;276(38):35818-35835
1.Wang,H.,Bloom,O.,Zhang,M.,et al.HMG-1 as a late mediator of endotoxin lethality in mice.Science 1999,285(5425),248-251.
2.Goodwin GH,Sanders C,Johns EW.A new group of chromatin associated proteins with a high content of acidic and basic amino acids.Eur J Biochem,1973,38(1):14-19.
3.Ferrari,S.,Finelli,P.,Rocchi,M.,et al.The active gene that encodes human high mobility group 1 protein(HMG1) contains introns and maps to chromosome 13.Genomics 1996,35(2),367-371.
4.Andersson U,Wang H,Palmblad K,et al.High mobility group 1 protein(HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.J Exp Med 2000,192(4):565-570.
5.Messmer D,Yang H,Telusma G,et al.High mobility group box protein 1:an endogenous signal for dendritic cell maturation and Thl polarization.J Immunol 2004,173(1):307-313.
6.Rendon-Mitchell B,Ochani M,Li J,et al.IFN-gamma induces high mobility group box 1protein release partly through a TNF-dependent mechanism.J Immunol 2003,170(7):3890-3897.
7.Liu S,Stolz DB,Sappington PL,et al.HMGB1 is secreted by immuno stimulated enterocytes and contributes to cytomix-induced hyperpermeability of caco-2 monolayers.Am J Physiol Cell Physiol 2006,294(4):990-999.
8.Semino C,Angelini G,Poggi A,et al.NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1.Blood 2005,106(2):609-616.
9.Bonaldi T,Talamo F,Scaffidi P,et al.Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion.EMBO J 2003,22(20):5551-5560.
10.Gardella S,Andrei C,Ferrera D,et al.The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep 2002,3(10):995-1001.
11. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002,418(6894):191-195.
12. Kokkola R, Andersson A, Mullins G, et al. RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scand J Immunol 2005; 61(l):l-9.
13. Huttunen, H. J., Fages, C, Kuja-Panula, J., et al. Receptor for advanced glycation end products-binding COOH terminal motif of amphoterin inhibits invasive migration and metastasis.Cancer Res. 2002, 62(16), 4805-4811.
14. Park, J. S., Svetkauskaite, D., He, H., et al. Strassheim, D., Ishizaka, A., Abraham, E.Involvement of TLR2 and TLR4 in cellular activation by high mobility group box 1 protein (HMGB1). J. Biol. Chem. 2004, 279(9): 7370-7376.
15. Man Yu,Hai chao Wang, Aihao Ding et al.HMGB1 singals through Toll-like receptor (TLR)4 and TLR2.Shock.2006 ,26(2),174-179.
16. Andersson, U., Wang, H., Palmblad, K., et al., HMG-1 stimulates proinflammatory cytokine synthesis in human monocytes. J. Exp. Med. 2000,192(4): 565-570.
17. Fiuza, C, Bustin, M., Talwar, S., et al .,Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood ,2003, 101(7): 2652-2660.
18. Messmer, D., Yang, H., Telusma, G., et al., High mobility group box 1 (HMGB1) is an endogenous Thl polarizing signal for dendritic cell maturation. J. Immunol 2004,173(1):307-313.
19. Rovere-Querini, P., Capobianco, A., Scaffidi, P., et al., .HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep. 2004,5(8):825-830.
20. Yang H, Ochani M, Li J, et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci U S A 2004; 101(1):296-301.
21. Sunden-Cullberg J, Norrby-Teglund A, Rouhiainen A, et al. Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 2005; 33(3):564-573.
22. Hatada T, Wada H, Nobori T, et al. Plasma concentrations and importance of High Mobility Group Box protein in the prognosis of organ failure in patients with disseminated intravascular coagulation. Thromb Haemost 2005;94(5):975-979.
23. Jonas Sunden-Cullberg,AnnaNorryby-Teglund ,et al.,The role of high mobility group box-1 protein in severe sepsis.Pathpgenesis and immune response 2006,19(3):231-236.
24. Li J, Wang H, Mason JM, et al. Recombinant HMGBl with cytokine-stimulating activity. J Immunol Methods 2004;289(l-2):211-223.
25. Ulloa L, Ochani M, Yang H, et al.,Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation.Proc Natl Acad Sci U S A. 2002,17;99(19):12351-12356.
26. Wang H, Liao H, Ochani M, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004;10(11):1216-1221.