Mechanisms Regulating Expression Of Human B7-H1 Gene-Role Of LPS Stimulated Signaling Pathway In The Transcriptional Control

Lipopolysaccharide (LPS), or endotoxin, is the major component of the outer surface of Gram-negative bacteria. LPS is a potent activator of cells of the immune and inflammatory systems, including macrophages, monocytes, and endothelial cells, and contributes to the systemic changes seen in septic shock . The basic paradigm of septic shock is that microbial antigens such as Gram-negative bacterial LPS initiate an uncontrolled network of host-derived pro-inflammatory mediators, which ultimately lead to cardiovascular shock and death. As soon as the bacteria in the body fluid is killed and LPS released, as the first barrier the mononuclear macrophage system encounts LPS firstly. To activate the mononuclear macrophage system, circulating LBP recognizes LPS in the plasma and brings it to CD14. This aids the loading of LPS onto the LPS receptor complex on cell surface, which is composed of dimerized TLR4 receptors and two molecules of the extracellular adapter MD-2. Subsequent a serial of signal events activated by TLR4 are passed through MyD88, IRAK, TRAF6/ECSIT and NIK/IKK, at last lead to the translocation of transcription factors especially NF-kB to nucelus, then induces or inhibits serials of genes to transcript, and inflammatory reaction occurs. The popular cell model to study the differentiation and function of the mononuclear macrophage system is U937 cell strain, and LPS is the most effective stimulator to induce an inflammatory reaction. It was reported that LPS could enhanced the expression of negative costimulator B7-H1 on the surface of U937 cells. In our study, U937 cells was cultured with 1 μ,g/ml LPS for 24 hours, then through flourescence semiquantitative real-time PCR assay and FCS assay, we found LPS can upregulate the constitutive expression of B7-H1, at the level of mRNA and protein.Now that the expression of B7-H1 in U937 cells can be upregulated by LPS, and this effect is mediated by the TFs, which unite the enhancer and coactivators to bind thepromoter region of B7-H1, and we know the downstream signal event of LPS stimuli is mainly through the translocation of NF-kB to nucleus to regulate the transcription of genes, then we have several questions to answer: Where and what structure is the promoter of B7-H1? Which part is the core promoter region that needed by fundamental transcription? Which region is needed by activitated transcription? Where is the TSS of B7-H1 gene? Are there several binding sites for NF-kB within the promoter region? Aren't these sites very important? If not, which other binding sites are important for the fundamental or activitated transcription of B7-H1 gene especially after LPS stimuli? We can't find any paper to answer above questions.To elucidate mechanisms regulating expression of human B7-H1 gene and the role of LPS stimulated signaling pathway in the transcriptional control, we do three part of research works as following:1. Utilized U937 cells, we observed the expression regulation mode of B7-H1 gene after LPS stimulated, through flourescence semiquantitative real-time PCR assay and FCS assay, we found LPS can upregulate the constitutive expression of B7-H1, not only at the level of mRNA ,but also of protein, so it shown that U937 cells was a suitable cell model for studying the mechanisms regulating transcription of human B7-H1 gene and the role of LPS stimulated signaling pathway in the transcriptional control;2. To identify the transcription initiation site of human B7-H1, we used a 5' -RACE approach, a single PCR product was amplified. Sequence analysis revealed that the major transcriptional start site maps 60 bp upstream to the AUG. Then we analysised the probably promoter region(-2150~+65) which is adjacent to TSS through bioinformatics, we found 8 Spl binding sites, 4 NF-kB binding sites, but not the traditional promoter elements such as TATA box and CAAT box, so the promoter of B7-H1 gene is a TATA-less promoter;3. To evaluate promoter activity of the human B7-H1 gene, the 2215 bp of genomic DNA immediately 5 ' to the transcriptional start site(-2150~+65) were cloned into pGL3-Basic vector. To map the minimal promoter region in the B7-H1 gene required for initiation and induction of gene transcription, luciferase reporter constructs containing progressive deletions of the 2215 bp genomic DNA fragment were generated. Each construct as well as the control vector pGL3-Basic were transiently transfected into U937 cells and assayed for reporter activity. Our results show that the -1537-+65 bp fragmentinduces a large increase in basal luciferase activity ,then the -2150-+65 bp, the -501—1-65 bp, the -896~+65 bp and the -137-+65 bp(from large to small),this implies the -137-+65 bp fragment has the activity of minimal core promoter, is essential for basal transcription, and the - 2150- -1537 bp and the - 896- -501 bp fragment maybe have the inhibitory c/s-regulatory elements; After LPS stimuli, the -1537-+65 bp fragment induces a large increase in activated luciferase activity ,then the -2150-+65 bp, the -896~+65 bp, the -501-+65 bp and the -137-+65 bp(from large to small), this implies the NF-kB binding sites upstream -137 bp are important for LPS effect.Thus, the present study establishes a molecular basis to further understand the mechanisms governing human B7-H1 gene expression, and consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in B7-H1 production in conditions characterized by immunopathologic activation of the mononuclear macrophage system such as inflammatory reaction.