Chemotaxis of chinese hamster ovary cells expressing the human neutrophil formyl peptide receptor: role of signal transduction molecules and alpha5beta1 integrin

Activation of the N-formyl peptide receptor (FPR) of human neutrophils by ligands such as N-formyl-methionine-leucine-phenylalanine (fMLP) induces mobilization of intracellular calcium, cell adhesion, chemotaxis, superoxide production and degranulation. Chinese hamster ovary (CHO) cells are normally devoid of FPR and unresponsive to fMLP, but when stably transfected with a human FPR cDNA, exhibited some of these same responses. Specifically, stimulation with fMLP resulted in release of intracellular calcium and chemotactic migration toward a gradient of fMLP. As in neutrophils, both processes were inhibited through receptor desensitization by prior exposure to a higher or equal concentration of ligand or by treatment with pertussis toxin. Soluble and membrane-bound fibronectin greatly increased fMLP-induced chemotaxis of CHO cells expressing FPR, but not of wild-type CHO cells, suggesting a role for FPR in activation of integrin function. Evidence for this hypothesis was obtained by demonstrating that CHO cells expressing FPR rapidly increased their adhesion to a fibronectin-coated surface after stimulation with fMLP. Both chemotaxis and adhesion were largely inhibited by RGDS peptide and a function-blocking antibody against alpha5 integrin. FPR-mediated chemotaxis of the CHO transfectants was partly inhibited by a tyrosine kinase inhibitor, herbimycin A, and blocked by a phosphoinositide 3-kinase inhibitor, wortmannin. These data suggest that stimulation of CHO FPR transfectants with a gradient of fMLP results in phosphoinositide 3-kinase-dependent chemotactic migration, which is enhanced by binding of activated alpha5beta1 to fibronectin. This non-myeloid, non-lymphoid fibroblastic cell line will thus serve as a useful model to investigate additional requirements of signal transduction molecules, adhesion molecules and cytoskeletal elements in FPR-mediated chemotaxis.