CpG DNA enhances macrophage cell spreading by promoting the Src-family kinase-mediated phosphorylation of paxillin
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.
References (51)
- et al.
J. Biol. Chem.
(2000) - et al.
J. Biol. Chem.
(2004) - et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(2001) - et al.
J. Biol. Chem.
(1997) - et al.
J. Biol. Chem.
(1997) - et al.
Gene
(1988) - et al.
J. Biol. Chem.
(1996) - et al.
J. Biol. Chem.
(2004) - et al.
J. Biol. Chem.
(2004)
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Cell
Annu. Rev. Immunol.
Nat. Rev. Immunol.
J. Exp. Med.
J. Immunol.
J. Immunol.
Oncogene
Nucleic Acids Res.
Mol. Cell. Biol.
Cited by (16)
Mechano-regulatory cellular behaviors of NIH/3T3 in response to the storage modulus of liquid crystalline substrates
2016, Journal of the Mechanical Behavior of Biomedical MaterialsCitation Excerpt :Furthermore, cells grown on both 12,312 Pa and 7228 Pa substrates exhibited high phosphorylation levels of paxillin and ERK1/2. Combined with the above cell morphology observations, we hypothesized that paxillin activation was significantly correlated with the increased cell spreading (Achuthan et al., 2006; Choi et al., 2007) and the high ERK activity in NIH/3T3 might lead to elongated morphology and well-organized cytoskeleton (Chang et al., 2014; Pan et al., 2013). The results in this study revealed that cells could respond to the mechanical signals from OPC substrate elasticity leading to the variations in activation of FAK, paxillin and ERK followed by the control of cell adhesion, spreading area and morphology (Mruthyunjaya et al., 2010).
Regulation of tyrosine phosphorylation in macrophage phagocytosis and chemotaxis
2011, Archives of Biochemistry and BiophysicsDasatinib inhibits the secretion of TNF-α following TLR stimulation in vitro and in vivo
2009, Experimental HematologyHematopoietic cell kinase (Hck) isoforms and phagocyte duties - From signaling and actin reorganization to migration and phagocytosis
2008, European Journal of Cell BiologySelf-DNA Sensing by cGAS-STING and TLR9 in Autoimmunity: Is the Cytoskeleton in Control?
2021, Frontiers in Immunology15-Epi-LXA<inf>4</inf> and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation
2020, Proceedings of the National Academy of Sciences of the United States of America