Identification Of Differential Proteins Binding To S100A8 Promoter In Endotoxemic Mice

S100 proteins are low-molecular-weight (10-14 kD) acidic proteins with two EF domains that bind Ca2+ ions selectively and with high to low affinity. Most of S100 gene form a cluster of linked genes in the mouse (chromosome 3) and human (1 q21) genomes. To date, more than 20 different S100 proteins have been identified, which are expressed in a controlled tissue-or cell-type-specific manner. Although the S100 proteins have no structural sequences required for secretion by the classical endoplasmic reticulum (ER)/Golgi pathway, extracellular functions of a number of them are well accepted. The S100 family is considered to be involved in various biological processes, such as regulation of protein phosphorylation, cell cycle regulation, cell differentiation, cell motility, and formation of cell-cell and cell-matrix contacts.Murine S100A8(S100 calcium-binding protein A8, S100A8), also known as CP-10 (chemotactic protein,10 kD), was isolated as a soluble product of activated murine spleen cells. Of the human (h) S100 family, human S100A8 (also known as MRP8, calgranulin A, L1 light chain, cystic fibrosis antigen) is the most closely related to mS100A8, although the level of homology is low (69% at the DNA level; 58% at the amino acid level). Some study suggest important roles for mS100A8 in embryogenesis, innate inflam matory responses, and in chronic inflammation. The cDNA sequence of S100A8 comprises an ORF of 267 bp with a 5’flanking sequence of 42 bp and a 3’ flanking sequence of 67 bp (plus poly A tail). In contrast to the human proteins, mS100A8/A9 is not constitutively expressed in monocytes or Mac and S100A9 is not co-induced with mS100A8 in these cells with any stimulant tested. It is up-regulated by bacterial lipopolysaccharide (LPS), tumor necrosis factorα(TNFα), Interferon(IFN)and interleukin-1(IL-1) in macrophages. Induction of mS100A8 by IFN in Mac is rapid and peaks at 12 h, whereas the response to TNF is slower and more prolonged. A number of other proinflammatory mediators failed to stimulate the mS100A8 gene, and interleukin-4 (IL-4) and interleukin-13(IL-13) suppressed activation in both cell types. Some studies showed that S100A8 expression in murine macrophages induced by LPS is a late-phase response that is neutralized by anti-IL-10 Abs, although IL-10 does not directly induce the gene. S100A8/S100A9-positive macrophages are found in numerous autoimmune diseases and in atherosclerotic plaque. They are also inducible in other cell types such as epithelial cells and fibroblasts by appropriate stimulants and their expression is associated with several tumors. S100A8 and S100A9 have intracellular and extracellular functions and although the S100A8-S100A9 complex is considered the major functional form, separate functions for the individual proteins are reported. LPS-induced mRNA in Mac is dependent on new protein synthesis, whereas induction by IFN and TNF is apparently not, indicating different types of regulation. These proteins are proposed to function as novel damage-associated molecular pattern molecules. Induction of S100A8 through activation of TLR4 by LPS is well studied in murine macrophages, which is both IL-10 and COX-2 dependent. Induction by LPS was dependent on p38 and ERK MAPK. The complex is antimicrobial, anti-apoptotic for some cells, and inhibits matrix metall proteinase (MMP) activities. S100A8 and S100A8/S100A9 were recently shown to induce MMP-2,3,9, and particularly 13, in murine bone marrow-derived macrophages, suggesting that the S100 proteins mediate macrophage migration and matrix degradation via MMP production. Although there is increasing interest in S100A8 in inflammatory disease, the specific regulatory mechanism about LPS-induced expression of S100A8 gene has not been clarified.Gene expression in eukaryotes is a complicated and strictly regulated process, and it can be regulated and controlled at different levels, such as replication transcription activation, post-transcription modification, translation and post-translation modification. Initiation of transcription is the most important step in gene expression, which is actually an integration mechanism involving cis-acting sequences and trans-acting factors. Cis-acting sequence usually lies 5’of the transcriptional start site. These sequences are the substrate for trans-acting factors. It’s same way to defined by the interaction between the promoter of a specific gene and regulatory proteins with spatial and temporal specificity. In fact, many factors but not just a single one, are involved in the transcription initiation of a specific gene, and those factors would interact reciprocally to form a regulatory network. So far, there are mainly two modes of promoter DNA binding protein research, the in-cell method, which utilizes the known promoter DNA sequence to screen out the coding gene of its binding protein and confirm it with bio-informatic study. The out-cell method, which exploit the known recombinant protein binding to the promter DNA.Our study, mainly based on the out-cell method, is the combination of out-cell method and the in-cell method. The purpose of this study is to discover the unknown regulatory proteins, further to find out its binding site to DNA and to prove the specificity of their interaction.In order to identify novel proteins involved in transcriptional regulation of S100A8 gene and decipher the mechanism of LPS-induced expression of S100A8, based on DNA-protein interaction principal and using isolation technique by magnetic beads and mass spectrum technique, we had identified differential proteins binding to the promoter of S100A8 gene in endotoxemic mice. Our researches are as follows: 1. Bioinformatics analysis of S100A8 promoterThe transcriptional start site of S100A8 gene was determined and the nucleotide sequence of -826 bp to +468 bp around the transcriptional start site was obtained by TranscriptionalRegulatory Element Database(http://rulai.cshl.edu/cgi-bin/TRED). The nucleotide sequence was aligned with NCBI/BLAST and Ensembl database. The possible binding sites of transcription factors in S100A8 gene promoter were also predicted by Consite, a promoter analysis web system. The S100A8 promoter sequence of -826 bp to +468 bp around the transcriptional start site was determined as the object of study. 2. Cloning of S100A8 promoter and identification of its transcriptional activity.Genomic DNA was extracted from mouse liver, and the promoter sequence of S100A8(-826～+468) was amplified by polymerase chain reaction (PCR). The red fluorescent protein reporter gene vector driven by S100A8 promoter was constructed. After the promoter sequence were identified right by sequencing, the recombinant vectors were transiently transfected into murine macrophage RAW264.7, then the expression of the red fluorescent protein in RAW264.7 cell was observed using Axiovert 200M Fluorescence/Live cell Imaging Microscope at rest or stimulated by NaAsO2, LPS or PolyI:C, by which activity of S100A8 promoter was detected. The results showed that the promoter of S100A8(-826～+468) presented the transcriptional activity at rest, and the S100A8 expression had been enhanced after the cells were stimulated by inflammatory substance or oxidative stress. The successful construction of reporter gene vector driven by S100A8 promoter will provide an experimental base for following experiment studing the transcriptional factor of S100A8.3. Screening of differential proteins binding to S100A8 promoter in endotoxemic mice.DNA probes of S100A8 promoter labeled with biotin were amplified by PCR. Nuclear extracts of endotoxemic mouse liver were prepared. DNA pull-down assay was performed using a magnetic beads conjugated with streptavidin. Proteins binding to the beads were eluted by sample lysis buffer, and the elution proteins were separated by 2-D DIGE. Analysing by DeCyder difference 2-D analysis software, twenty-nine differential protein spots had been identified. twenty-five proteins were up-regulated, and four proteins were down-regulated.4. Identification of the differential protein binding to S100A8 promoter by mass spectrum.The differential proteins were further identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF). Nineteen proteins were identified. Among them, four proteins have been reported to be involved in transcriptional regulation, such as core histone marco-H2A, transcription cofactor HES-6, methyltransferase -like protein 4 and heterogeneous nuclear ribonucleoprotein hnRNP A2/B1.5. Bioinformatics analysis of differential protein.The protein function and subcellular location annotations were from Swiss-Prot and TrEMBL protein database. In cases where Swiss-Prot annotations were not available, NCBI was searched for informations on the function and subcellular location of the novel proteins. Subcellular localization of differential proteins was also predicted using subcellular localization prediction software WoLF PSORT (http://wolfpsort.org/).The results showed that six proteins specifically localized in nuclei; eight proteins were distributed in nuclei or other cell organelles; five proteins were only located in other subcellular structures but not in nuclei. These results indicated that some of differential proteins were located in nuclei. At physiological or pathological conditions these proteins located in nuclei are more likely to participate in transcriptional regulation of S100A8 gene.Taken together the above data, we draw the following conclusions:First, the promoter sequence of S100A8(-826～+468) presented the transcriptional activity at rest, and the transcriptional expression of S100A8 had been enhanced after the cells were stimulated by inflammatory substance or oxidative stress.Second, four proteins in the liver nuclear extracts were found probably to be involved in the transcriptional regulation of S100A8 gene in endotoximic mice, such as core histone marco-H2A, transcription cofactor HES-6, methyltransferase-like protein 4(MT-4) and heterogeneous nuclear ribonucleoprotein A2/B1(hnRNPA2/B1).In general, based on DNA-protein interaction principal, we had screened out the differential proteins binding to S100A8 promoter in endotoxemic mouse liver by biotin-streptavidin technology, which created a novel way to study the transcriptional regulation mechanisms of gene expression from the view of "transcriptional regulationomics". At the same time, this experiment, combined biotin-streptavidin technology with DIGE and mass spectrometry technology, realized the docking of research technique between transcriptional regulation study and the latest differential proteomics study. It provided a new approach to the successful efficient screening and identification of DNA binding proteins. Screening and identifying the differential proteins binding to S100A8 promoter in endotoxemic mice are important for understanding the LPS-inducible expression and regulation mechanism of S100A8 gene.