Previously, we have demonstrated the induction of Src in lipopolysaccharide (LPS)-stimulated macrophages. In this study, we observed that pharmacological blockade or knockout of inducible nitric-oxide synthase (iNOS) reduced LPS-mediated Src induction and macrophage migration. Either SNAP (a NO donor) or 8-Br-cGMP (a cGMP analogue) could rescue these defects in iNOS-null macrophages, which indicated the participation of NO/cGMP in LPS-elicited Src expression and mobilization. In addition, Src family kinase (SFK)-specific inhibitor, PP2, inhibited SNAP-and 8-Br-cGMP-evoked motility implicating the involvement of SFKs downstream of NO/cGMP. Analysis of the expression of SFKs indicated LPS dramatically induced Src, which could be attributable to the increased level of the src transcript. Attenuation of Src by src-specific siRNA reduced LPS-and SNAP-evoked mobilization in Raw264.7 macrophages, and reintroduction of avian Src could rescue their motility. Furthermore, LPS-mediated Src induction led to increased FAK Pi-Tyr-397 and Pi-Tyr-861, which was also iNOSdependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.Macrophages are important participants in innate immunity. Due to their ability to eliminate opsonized pathogens through a spectrum of surface receptors and antigen presentation to cells of the adaptive immunity, macrophage recruitment to sites of infection is an important physiological process in host defenses. Disturbed regulation of this event results in a variety of diseases such as sepsis, atherosclerosis, and autoimmune disorders.Nitric oxide (NO), 3 a unique diffusible messenger molecule, is produced via the oxidation of L-arginine by enzymes known as nitric-oxide synthases (NOSs) (1). Three distinct isoforms of the enzyme have been identified and characterized. Whereas Ca ϩ2 /calmodulin can regulate the activity of neuronal (n) and endothelial (e) NOS that are constitutively expressed, the activity of inducible (i) NOS is independent of Ca ϩ2 /calmodulin and only induced by bacterial products as well as inflammatory cytokines. A low level of constitutively produced NO is a crucial mediator for a spectrum of physiological functions such as regulation of neurotransmission, vasodilation, smooth muscle relaxation, and inhibition of platelet aggregation. In contrast, a high level of NO generated by macrophages and other effector cells under inducible conditions mediates host defense, including antibacterial and antitumor functions (2, 3). However, it is also well documented that some pathological processes such as inflammation and tumor can be induced by sustained, chronically produced NO (4, 5). As a main target of NO, sGC is a cytosolic, heme-containing heterodimer of ␣ and ␤ subunits (6). When NO binds to the sGC heme prosthetic group, it activates the enzyme to convert guanosine 5Ј-triphosphate to cGMP, an intracellular second messenger (7). Accumulation of cGMP results in transmission of NO signals to t...

The Toll-like receptors (TLRs) play a pivotal role in innate immunity for the detection of highly conserved, pathogen-expressed molecules. Previously, we demonstrated that lipopolysaccharide (LPS, TLR4 ligand)-increased macrophage motility required the participation of Src and FAK, which was inducible nitric oxide synthase (iNOS)-dependent. To investigate whether this iNOS/Src/FAK pathway is a general mechanism for macrophages to mobilize in response to engagement of TLRs other than TLR4, peptidoglycan (PGN, TLR2 ligand), polyinosinic-polycytidylic acid (polyI:C, TLR3 ligand) and CpG-oligodeoxynucleotides (CpG, TLR9 ligand) were used to treat macrophages in this study. Like LPS stimulation, simultaneous increase of cell motility and Src (but not Fgr, Hck, and Lyn) was detected in RAW264.7, peritoneal macrophages, and bone marrow-derived macrophages exposed to PGN, polyI:C and CpG. Attenuation of Src suppressed PGN-, polyI:C-, and CpG-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Besides, knockdown of FAK reduced the mobility of macrophages stimulated with anyone of these TLR ligands. Remarkably, PGN-, polyI:C-, and CpG-induced Src expression, FAK Pi-Tyr861, and cell mobility were inhibited in macrophages devoid of iNOS, indicating the importance of iNOS. These findings corroborate that iNOS/Src/FAK axis occupies a central role in macrophage locomotion in response to engagement of TLRs.