Abstract
Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation-mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9-MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.
MeSH terms
- Actin Cytoskeleton / drug effects
- Actin Cytoskeleton / metabolism
- Adaptor Proteins, Signal Transducing / genetics
- Androstadienes / pharmacology
- Animals
- Antigens, CD / metabolism
- Cell Adhesion / drug effects
- Cell Line, Tumor
- Cell Membrane / drug effects
- Cell Membrane / metabolism
- Chloroquine / pharmacology
- CpG Islands*
- Cytokines / metabolism*
- Cytoskeleton / drug effects
- Cytoskeleton / metabolism
- Dendritic Cells / drug effects
- Dendritic Cells / metabolism
- Humans
- I-kappa B Proteins / metabolism
- Interferon-alpha / metabolism
- Interleukin-6 / metabolism
- Intracellular Signaling Peptides and Proteins / metabolism
- Macrophages / drug effects
- Macrophages / metabolism
- Mice
- Mice, Knockout
- Models, Biological
- Monocytes / drug effects
- Monocytes / metabolism
- Myeloid Differentiation Factor 88
- NF-KappaB Inhibitor alpha
- Oligodeoxyribonucleotides / pharmacology*
- Phosphorylation / drug effects
- Protein Kinase Inhibitors / pharmacology
- Protein-Tyrosine Kinases / metabolism
- Proto-Oncogene Proteins c-akt / metabolism
- Proto-Oncogene Proteins c-hck / genetics
- Proto-Oncogene Proteins c-hck / metabolism
- Pyrazoles / pharmacology
- Pyrimidines / pharmacology
- Quinacrine / pharmacology
- RNA, Small Interfering / genetics
- Syk Kinase
- Toll-Like Receptor 9 / genetics
- Toll-Like Receptor 9 / metabolism
- Toll-Like Receptor 9 / physiology*
- Tyrosine / metabolism
- Wortmannin
- src-Family Kinases / antagonists & inhibitors
- src-Family Kinases / genetics
- src-Family Kinases / metabolism*
Substances
- 4-amino-7-phenylpyrazol(3,4-d)pyrimidine
- AG 1879
- Adaptor Proteins, Signal Transducing
- Androstadienes
- Antigens, CD
- Cytokines
- I-kappa B Proteins
- Interferon-alpha
- Interleukin-6
- Intracellular Signaling Peptides and Proteins
- MYD88 protein, human
- Myd88 protein, mouse
- Myeloid Differentiation Factor 88
- NFKBIA protein, human
- Nfkbia protein, mouse
- Oligodeoxyribonucleotides
- Protein Kinase Inhibitors
- Pyrazoles
- Pyrimidines
- RNA, Small Interfering
- Toll-Like Receptor 9
- NF-KappaB Inhibitor alpha
- Tyrosine
- Chloroquine
- Protein-Tyrosine Kinases
- HCK protein, human
- Proto-Oncogene Proteins c-hck
- SYK protein, human
- Syk Kinase
- Syk protein, mouse
- lyn protein-tyrosine kinase
- src-Family Kinases
- Proto-Oncogene Proteins c-akt
- Quinacrine
- Wortmannin