Elsevier

Cellular Signalling

Volume 18, Issue 12, December 2006, Pages 2252-2261
Cellular Signalling

CpG DNA enhances macrophage cell spreading by promoting the Src-family kinase-mediated phosphorylation of paxillin

https://doi.org/10.1016/j.cellsig.2006.05.007Get rights and content

Abstract

Macrophages are an important component of the innate immune response to infection by microbial pathogens. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). Bacterial DNA, which contains unmethylated CpG dinucleotide motifs, is specifically recognised by TLR9 and triggers the activation of a complex network of intracellular signalling pathways that orchestrates the ensuing inflammatory responses of macrophages to the pathogen. Here, we have established that CpG DNA promotes reorganisation of the actin cytoskeleton and enhances cell spreading by primary mouse bone marrow macrophages. CpG DNA stimulation resulted in an approximately 70% increase in cell size. Notably, CpG DNA-induced cell spreading was dependent on the activity of Src-family kinases. Tyrosine phosphorylation of several proteins was increased in a Src-family kinase-dependent manner following CpG DNA stimulation of bone marrow macrophages, including the cytoskeletal protein paxillin. Paxillin was phosphorylated both in vitro and in vivo by the Src-family kinase Hck. Significantly, paxillin from CpG DNA-stimulated bone marrow macrophages had a greater capacity to bind the SH2 domain of the adapter protein Crk than did paxillin from unstimulated bone marrow macrophages. Furthermore, phosphorylation of paxillin by Hck created a binding site for Crk. We propose that the formation of paxillin-Crk complexes may mediate the cytoskeletal changes that underlie the increased cell spreading of macrophages following their activation by CpG DNA.

Introduction

The innate immune system is the first line of defence against host infection by microbial pathogens [1]. Macrophages are a key component of the innate immune system as they have the capacity to secrete inflammatory cytokines (e.g., TNFα and IL-1), chemokines, proteases and reactive metabolites of oxygen and nitrogen. They also have the capacity to phagocytose and degrade pathogens, with the subsequent presentation of pathogen-derived peptides to T-cells being important for adaptive immunity [1].

The activation of macrophages by microbial pathogens is largely mediated by Toll-like receptors (TLRs) [2]. The different members of the TLR family are activated by specific components of pathogens, referred to as pathogen-associated molecular patterns. For example, TLR4 is activated by bacterial lipopolysaccharide (LPS), whereas TLR9 is activated by bacterial DNA [3]. The recognition of pathogen-associated molecular patterns by TLRs triggers the activation of a complex network of intracellular signalling pathways that coordinates the ensuing inflammatory response of the macrophage to the pathogen. Recruitment of MyD88 and the protein kinases IRAK-1 and IRAK-4 to the activated TLR is the initial event in TLR signalling. Formation of this complex leads to the activation of IRAK-4, which in turn phosphorylates and thereby activates IRAK-1. Subsequent binding of TRAF6 to IRAK-1 ultimately results in the activation of various MAP kinases (e.g., p38 MAP kinase, JNK and Erk1/2) and the transcription factors NF-κB and AP-1. Expression of NF-κB and AP-1 target genes (e.g., TNFα, IL-1, IL-6, and IL-12) then mediates the ensuing innate and adaptive immune responses [3].

Cell adhesion and spreading is necessary for the efficient migration of macrophages to sites of infection. LPS is a potent stimulator of macrophage adhesion and cell spreading [4], [5]. Significantly, the ability of LPS to promote macrophage adhesion and spreading is dependent on the activity of Src-family kinases [6], [7]. In particular, Williams and Ridley reported that the Src-family kinase-dependent phosphorylation of paxillin was likely to directly contribute to the triggering of macrophage adhesion and spreading by LPS [7]. Paxillin is an approximately 68 kDa cytoskeletal adaptor protein that functions as a scaffold to recruit specific proteins (e.g., Crk, c-Src, PKL, FAK and Pyk2) to sites of cell adhesion [8]. The binding of proteins to paxillin is mediated by a large array of protein–protein interaction motifs on paxillin, including those for SH2 and SH3 domains [8].

In the present study, we have investigated the ability of the TLR9 ligand CpG DNA to induce macrophage cell spreading and the molecular mechanism controlling this process. Notably, CpG DNA-induced macrophage spreading was dependent on Src-family kinase activity and correlated with tyrosine phosphorylation of paxillin. Paxillin was phosphorylated in vitro and in vivo by the Src-family kinase Hck, with the resulting phosphorylation mediating the binding of the adaptor protein Crk to paxillin. Based on these findings, we propose that the formation of paxillin-Crk complexes may govern the cytoskeletal changes required for CpG DNA to promote cell spreading by macrophages.

Section snippets

Reagents

Cell culture medium and supplements, fetal calf serum (FCS), and pre-cast SDS-PAGE gels were from Invitrogen. The CpG oligonucleotide (ODN 1826), non-CpG oligonucleotide (ODN 1826 control), and lipopolysaccharide (Escherichia coli 0111:B4) were from InvivoGen. Pfu DNA polymerase and restriction enzymes were supplied by Promega and New England Biolabs, respectively. The HRP-conjugated anti-FLAG and agarose-coupled anti-FLAG monoclonal (M2) antibodies were obtained from Sigma. The

CpG DNA induces the Src-family kinase-dependent cell spreading of macrophages

LPS, a ligand for TLR4, promotes the reorganisation of the actin cytoskeleton in macrophages, leading to increased cell spreading [7]. To establish if the TLR9 ligand CpG DNA also triggers the reorganisation of the actin cytoskeleton and increased cell spreading of macrophages, primary mouse bone marrow macrophages were stimulated with either a synthetic oligonucleotide containing a CpG motif (i.e., CpG DNA) or an inactive form of the oligonucleotide with an inverted CpG motif (i.e., non-CpG

Discussion

The coordinated spreading and retraction of their cell membranes is crucial for the efficient migration of macrophages to sites of infection. Thus, the molecular mechanisms that control cell spreading are important for the successful resolution of infections. Here, we have established that CpG DNA, which activates macrophages via TLR9, markedly enhanced cell spreading by bone marrow macrophages. Significantly, the increased cell spreading correlated with increased tyrosine phosphorylation of

Acknowledgements

The authors thank Dr. Heung-Chin Cheng (Department of Biochemistry and Molecular Biology, The University of Melbourne) for the generous gift of recombinant Hck, and Dr. Hisataka Sabe (Osaka Bioscience Institute) and Dr. Michiyuki Matsuda (Osaka University) for Paxillin and Crk plasmids, respectively. This work was supported in part by the Cooperative Research Centre (CRC) for Chronic Inflammatory Diseases and grants from the National Health and Medical Research Council to JAH and GS.

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