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.

ABSTRACTpretreatment with tipifarnib resulted in significant inhibition of TNF-␣, IL-6, MCP-1, IL-1␤, and MIP-1␣ production. Tipifarnib had no effect in vitro or in vivo on LPS-induced IL-8. Studies in THP-1 cells to address potential mechanism(s) showed that tipifarnib partially inhibited LPS-induced p38 phosphorylation. Tipifarnib significantly inhibited inhibitory subunit of nuclear factor-B (NF-B) (IB)-␣ degradation and p65 nuclear translocation induced by LPS, but not by tumor necrosis factor-␣, IL-1␣, or toll-like receptor (TLR)2 ligand, suggesting that the target for inhibition of NF-B activation was exclusive to the LPS/TLR4 signal pathway. The extent of IB-␣ degradation inhibition did not correlate with inhibition of Ras farnesylation, indicating that Ras was not the target for the observed antiinflammatory activity of tipifarnib. Our findings differ from those for other FTIs, which may have relevance for their dissimilar activity in specific tumor repertoires.The links between cancer and inflammation are well established (for review, see Balkwill et al., 2005). Many human and murine cancers are found in a microenvironment rich in cytokines, chemokines, and inflammatory enzymes. It is therefore of interest to investigate the effects of antitumor drugs on inflammation. Tipifarnib [R115777, Zarnestra, (R)-6-amino[(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl2(1H)-quinolinone)] is an orally active inhibitor of protein farnesyltransferase. It has shown significant antitumor activity and is currently in clinical trials for acute myeloid leukemia and myelodysplastic syndrome (Cortes, 2003). Farnesyltransferase is an enzyme that catalyzes the attachment of a farnesyl group, from farnesyl pyrophosphate, to the cysteine-thiol group of protein C-terminal CAAX consensus sequences (Moores et al., 1991;Reiss et al., 1991). A variety of cellular proteins are farnesylated (Schafer and Rine, 1992;Tamanoi et al., 2001), including Ras superfamily G proteins, nuclear lamins A and B, rhodopsin kinase, centromere-binding proteins CENP-E and CENP-F, cochaperone DnaJ/HDJ-2, prostacyclin receptor Kinsella, 2004, 2005), and cytosolic phospholipase A 2 ␥ (Jenkins et al., 2003). Ras farnesylation is critical for oncogenic Ras signaling (Kato et al., 1992), and Ras mutants are associated with ϳ30% of human cancers. Farnesyltransferase inhibitors (FTIs) were developed to Article, publication date, and citation information can be found at

"CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion