Transcriptional Regulation of the Novel Toll-like Receptor Tlr13

Zhongcheng Shi,Zhenyu Cai,Shu Wen,Caoyi Chen,Christi Gendron,Amir Sanchez,Kevin Patterson,Songbin Fu,Jianhua Yang,Derek Wildman,Richard H Finnell,Dekai Zhang

doi:10.1074/jbc.m109.022541

Abstract

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-kappaB appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-kappaB p65 dramatically inhibited it. Additionally, interferon-beta is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.

Highlights

Recent studies on the recognition of microbial pathogenassociated molecular patterns have highlighted the vital role of one group of pattern recognition receptors, the Toll-like receptors (TLRs)2 [9, 10]
Which type of cells expresses Tlr13? Which transcription factors control Tlr13 expression? How do different pathogen-associated molecular patterns from different pathogens regulate Tlr13 expression? This information will perhaps help us understand how this novel TLR responds to different infections and which pathogens might be recognized by Tlr13 to activate the innate immune response
Characterization of Tlr13 Gene Expression in Macrophage RAW 264.7 Cells—Tlr13 is a novel member of the mammalian Toll-like receptor family, and little is known about its expression and function [3, 13]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Reagents—RAW 264.7, NIH 3T3, and HEK 293 cells were purchased from ATCC. An approximately 1.9-kb fragment that contains the immediate 5Ј-flanking Tlr sequence of the putative murine Tlr promoter (GenBankTM number EU588988) was obtained This 1.9-kb KpnI/BglII fragment was subcloned into the pGL3 basic vector (Promega). 2.5 ␮g of nuclear extract was incubated with 10 ng of each labeled probe in binding buffer containing 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, 1 mM MgCl2, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.05 mg/ml poly(dI-dC)-poly(dI-dC) for 20 min at room temperature. The reverse transcription reaction was diluted 1:10, and 2 ␮l of the diluted sample was added to an 18-␮l PCR assay mixture containing a 0.5 ␮M concentration of each primer and 1ϫ SYBR Green JumpStart Taq ReadyMix (Sigma). All assays were performed at least three times from independent RNA preparations

RESULTS

DISCUSSION

Dekai Zhang