RESUMEN
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.
Asunto(s)
Movimiento Celular , Quinasa 1 de Adhesión Focal/metabolismo , Macrófagos Peritoneales/metabolismo , Óxido Nítrico Sintasa de Tipo II/fisiología , Receptores Toll-Like/metabolismo , Familia-src Quinasas/metabolismo , Animales , Western Blotting , Adhesión Celular , Células Cultivadas , Quinasa 1 de Adhesión Focal/genética , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genéticaRESUMEN
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 iNOS-dependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.