Identification of Synergistic Transcription Factors that Respond to NF-κB and p38 Signaling Pathway in Activated Macrophages
Date Issued
2011
Date
2011
Author(s)
Wu, Shang-Duen
Abstract
Macrophages play a pivotal role in immune system through recognition and elimination of microbial pathogens. Toll-like receptors (TLRs) on macrophages interact with microbial substances and initiate signal transduction through intracellular adapters. TLR4, which is important for response to lipopolysaccharide (LPS), triggers downstream signaling mediators and eventually activates IκB kinase (IKK) and mitogen-activated protein kinases (MAPKs) such as ERKs, JNKs, and p38 MAPKs. Previous reports reveal that, in addition to NF-κB, the induction of some LPS-inducible genes in macrophages requires a second transcription factor whose activity depends on p38. Therefore, the specific aim of this thesis was to identify genes both regulated by NF-κB and p38 pathway, and their transcription factors that interact with NF-κB.
This study showed that 55 candidate genes were identified by microarray analysis using mouse bone marrow derived macrophages (BMDMs) of wild-type, IkkβΔ, and p38-inhibited by SB202190. Among these, promoters of 16 genes involved in immune cell movement were applied for transcription factor binding site prediction. As a result, NF-κB p65 and C/EBPβ binding sites were predicted in the promoters of Tnfaip3 and Zc3h12a. Chromatin immunoprecipitation (ChIP) assay was performed to validate the binding activity of p65 and C/EBPβ on predicted binding sites in mouse macrophages RAW264.7 cells. ChIP assays showed abundant bindings of p65 and C/EBPβ on Tnfaip3 promoter at 4 hours after LPS treatment. However, the binding activities of p65 and C/EBPβ were suppressed in the presence of SB202190, suggesting that C/EBPβ may be the p38-dependent synergistic transcription factor interacting with NF-κB for the regulation of Tnfaip3 expression.
Subjects
macrophages
LPS
Chromatin immunoprecipitation
Type
thesis
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