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Transcriptional Regulation of Pathways Activated by Lipopolysaccharide in RAW 264.7 Cells Sangdun Choi*, Yun Anna Cao*, Sun Young Lee*, Mi Sook Chang*, Xiaocui Zhu*, Meghdad Rahdar†, Robert Hsueh†, Paul Sternweis†, and Melvin Simon* *Molecular Biology Laboratory, Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, California †Cell Preparation and Analysis Laboratory, Alliance for Cellular Signaling, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas (interferon regulatory factor-3) TIR (Toll/interleukin-1 receptor) domain (TANK-binding kinase-1) TIR domain-containing adapter-inducing IFN-beta 2004, Semi in Immunol 16:3 MyD88 dependent pathway MyD88 independent pathway triacylated lipopeptide diacylated lipopeptide triacylated lipopeptide diacylated lipopeptide LPS Resiquimod (R-848) Imidazoquinoline dsRNA CpG DNA TLR (Toll-like receptor)-mediated Signaling Pathways Regulatory Elements analyzed by Ingenuity Pathway Analysis * * 2. Early Responses (1hr) 3. Intermediate Responses (4hr) 4. Late Responses (16hr) 5. Tumor Necrosis Factor-related Transcripts 6. Interleukin-related Transcripts 7. Interferon-related Transcripts 9. Bio-Plex Cytokine Antibody Bead Experiments (Performed by Ruth Levitz in the Antibody Lab) IL1b IL1a IL6 IL10 G-CSF IL12(p70) MIP-1a RANTES TNFa LPS Ingenuity Pathway Analysis 11. LPS Network IL1a, IL1b TNF TNFRSF5 TNFAIP3 Products: Ap2, Birc2, C3, CD83, Clecsf9, Csf3, Dusp2, Egr1, Ier2, Ier3, Ifnb, IL10, IL1b, Planh1, Ptpn16, Saa3, Scya4, Scyb10, Scyb2, Slfn2, Tnf, Tnfaip3, Tnfrsf5, Traf1, Zfp36, …… 10. LPS Stimulated NF-kB Pathway Summaries of LPS Signaling 8. Cytokines Targeted by Bio-Plex Introduction Microarray experiments using RAW 264.7 cells treated with LPS (Lipopolysaccharide, 100ng/ml) / LBP (LPS-binding protein, 100pM) (0, 1, 2, 4, 8, 16, and 48hr) were performed. Time matched untreated samples were used as controls. Dye-swapping was also carried out, in which controls were labeled with Cy3 (green dye) and test samples with Cy5 (red) in the 1st run, and vice versa in the 2nd run. This eliminates possible bias caused by variation in incorporation efficiency of the dyes. Samples were prepared in triplicate by The Cell Lab (Dallas) and hybridizations were performed on 16K Operon-Compugen Oligonucleotide Arrays. The transcription analysis of LPS/LBP treated RAW cells showed about 70 to 280 elements regulated more than 2 fold, peaking at 16 hours and providing a picture of the temporal pattern of gene expression responses induced by LPS. Lipopolysaccharide (LPS) is the major constitute of the outer membrane of Gram-negative bacteria. In blood, LPS forms a complex with serum protein LBP (LPS-binding protein). LPS binds cell the surface receptor CD14 to enhance Toll-like receptor 4 (TLR4)-dependent LPS recognition. After TLR4 forms a complex with protein MD-2, the complex is transported to the cell surface. TLR4, part of the Toll-like receptor family, is a membrane protein with an extracellular leucine-rich repeat domain and a cytoplasmic TIR domain. The activation of TLR4 together with the MyD88 adaptor family members, (including MyD88, TIRAP, TRIF, and TRAM) initiates the intracellular response. MyD88 adaptor protein appears to link TLR4 to IRAK4 kinases, also known as the MyD88-dependent pathway. Upon activation of the cells with LPS, the MyD88-dependent pathway leads to IRAK phosphorylation and TRAF6 ubiquitination. Ubiquitinated TRAF6 activates TAK1, resulting in the activation of the NF-kB, p38, and JNK pathways. TIRAP seems to be responsible for the MyD88-independent response but the precise role is unknown. Both TRIF and TRAM link TLR4 to pathways that activate IRF3 and TBK1. Engagement of TLR4 recruits proximal adaptors TRIF and TRAM, leading to phosphorylation and activation of IRF3 and delayed NF-kB activation. 1. Number of Regulated Genes (>3 fold) in RAW by LPS + LBP Results Early responses (1h) by LPS stimulation. Early growth response 1 and 2 (EGR1 and 2), lipopolysaccharide-induced molecular possessing ankyrin-repeats (MAIL-pending), and small inducible cytokines such as cxcl2, cxcl10, ccl4 were significantly induced at early time points. Immunoresponsive gene 1 (IRG1), cxcl10 (IP10), and IFNb are early products of IRF3 pathway. IL1b, TNF, Myc, CSF3, and IL10 were also highly upregulated. Intermediate responses (4h). Many genes in this group are interferon-activated, interferon- induced, and interferon-stimulated genes. Interestingly, some G-protein-related genes, such as GBP3 (guanylate nucleotide binding protein 3), GBP1, GBP2, and RGS16 were induced. IL6, STAT1, and STAT2 were also upregulated. Late responses (16h). Late responses include glutathione S-transferase theta 1, Fc receptor IgG low affinity IIb, caspase 1, histone H1, histone gene complex 2, and inducible nitric oxide synthase 2 (iNOS2). Throughout the time points tested, there were few downregulated genes and the magnitude of downregulation was relatively small. The copy number of the transcript was roughly measured by comparing the hybridization intensity. Bio-Plex (Dallas lab) results revealed that cytokines such as IL1a, IL1b, IL6, IL10, IL12, G-CSF, MIP-1a, RANTES, and TNFa were induced. The array data were consistent with LPS-responses of Bio-Plex experiments except IL12, where Bio-Plex tested IL12 p70 dimer was positive whereas array tested p35 and 40 subunits were negative. The NFkB pathway map with mRNA expression levels shown provides a basis for the hypothesis that the cellular responses in macrophages, through the recognition of LPS, transcriptionally regulate not only the actual production of the cytokine but also the downstream elements resulting in the release of many cytokines and their related products. 1.Transcriptional regulation of pathways was activated by Toll-like receptor 4 (3 to more than 100 fold). 2.Apparent waves of transcription: Immediate early (1hr), Intermediate (4hr), Late (16hr). 3.Families of genes include specific subsets upregulated by LPS: TNF, Interferon, Interleukin, and chemokine. 4.Possible autocrine loops, and other feedback mechanisms to readjust the patterns of signal transduction.