HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhu, X. Articles by Choi, S. Search for Related Content PubMed PubMed Citation Articles by Zhu, X. Articles by Choi, S.

Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression^1,2,3

Xiaocui Zhu^*, Mi Sook Chang^*, Robert C. Hsueh, Ron Taussig, Kelly D. Smith, Melvin I. Simon^* and Sangdun Choi^4,*,

^* Molecular Biology Laboratory, Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125; Cell Preparation and Analysis Laboratory, Alliance for Cellular Signaling, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; Department of Pathology, University of Washington, Seattle, WA 98195; and Department of Molecular Science and Technology, Ajou University, Suwon, Korea

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-, 2-methyl-thio-ATP (2MA), PGE₂, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE₂ and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN- and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN--induced changes in gene expression at 1 h, IFN- signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-- response genes by augmenting STAT1 activity and by activating NF-B, which synergizes with IFN--induced transcriptional factors; 2) IFN- attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN- and inhibiting IL-10 gene expression; 4) PGE₂ and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by contributions from public and private sources, including the National Institute of General Medical Sciences Glue Grant Initiative (U54 GM062114).

² A complete listing of the Alliance for Cellular Signaling sponsors can be found at www.signaling-gateway.org/aboutus/sponsors.html.

³ The microarray data used in this study were deposited into the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/, Gene Expression Omnibus) under the accession numbers of GSM18191-18226, GSM111939-112028, and GSM112042-112129.

⁴ Address correspondence and reprint requests to Dr. Sangdun Choi, Division of Biology, 147-75, California Institute of Technology, Pasadena, CA 91125; E-mail address: schoi{at}caltech.edu or Department of Molecular Science and Technology, Ajou University, Suwon, 443-749, Korea; E-mail address: sangdunchoi{at}ajou.ac.kr

⁵ Abbreviations used in this paper: ISO, isoproterenol; 2MA, 2-methyl-thio-ATP; iNOS, inducible NO synthase; QRT-PCR, quantitative RT-PCR; RGS, regulator of G protein signaling; GAS, IFN--activated sequence; Socs1, suppressor of cytokine signaling 1; Cish, cytokine-inducible SH2-containing protein; Mlp, MARCKS-like protein.

⁶ The online version of this article contains supplemental material.

This article has been cited by other articles:

				J. E. Wilson, B. Katkere, and J. R. Drake Francisella tularensis Induces Ubiquitin-Dependent Major Histocompatibility Complex Class II Degradation in Activated Macrophages Infect. Immun., November 1, 2009; 77(11): 4953 - 4965. [Abstract] [Full Text] [PDF]