Identification And Functional Study Of HMGB1 Promoter Binding Proteins In Mice With Endotoxic Shock

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.