NFκB p50-CCAAT/Enhancer-binding Protein β (C/EBPβ)-mediated Transcriptional Repression of MicroRNA let-7i following Microbial Infection

Abstract

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(؊) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFB p50-C/EBP␤ silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.Intrahepatic bile ducts constitute a complex 3-dimensional tubular network, the biliary tract, through which bile is transported to the duodenum. Human bile is sterile under normal physiological conditions (1); however, the biliary tract is periodically exposed to pathogens, including Escherichia coli and the protozoan parasite Cryptosporidium parvum, or pathogenderived molecules, including Gram-negative bacteria-derived lipopolysaccharide (LPS).2 Upon pathogen recognition, a phenotypic transition occurs through which biliary epithelial cells (cholangiocytes) promote the innate and adaptive immune responses (1-4). Indeed, cholangiocytes express a variety of pathogen recognition receptors and actively participate in the innate immune response through the secretion of cytokines/ chemokines (5, 6), expression of adhesion molecules (7-9), and antimicrobial peptides (1, 10). Expression of these immuneassociated genes is a highly regulated process to assure that the epithelium recognizes and responds to invading pathogens, but does not induce injury through an inappropriate immune response. Recent reports suggest the microRNA machinery contributes to the regulation of the immune-associated gene expression. MicroRNAs are small (21-23 nt) RNA molecules that target and regulate the stability or translational efficiency of mRNAs (11). These regulatory RNAs are transcribed as mono-or polycistronic primary microRNAs (pri-microRNAs), which are sequentially processed to precursor and the functionally active mature microRNA. The molecular mechanisms regulating the expression of most microRNAs remain largely unknown.Using a human cholangiocyte cell culture model of biliary cryptosporidiosis, we previously reported that let-7i, a member of the let-7 family of microRNAs, targets Toll-like receptor 4 mRNA and limits the expression of...

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Coordinated Regulation of miR-155 and miR-146a Genes during Induction of Endotoxin Tolerance in Macrophages

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