Formyl-peptide receptors, NADPH oxidase and replicative senescence

The formyl-peptide receptor family members FPR, FPRL1 and FPRL2, expressed in human cells, belong to pertussis toxin sensitive G-protein coupled seven transmembrane receptor family (GPCR). In polymorphonucleate cells (PMN) binding of small formyl-peptide derivatives, whose prototype is N-formyl-methionyl-leucyl-phenylalanine (N-fMLP), to these receptors triggers a complex program that results in cell migration, reorganization of the actin cytoskeleton and superoxide anion generation through NADPH oxidase activation. FPR and FPRL1 bind N-fMLP with high and low efficiency, respectively. FPRL2 does not respond to formyl-peptides and it was described as a low affinity receptor for several FPRL1 agonists. FPR and FPRL1 were initially detected in phagocytic leukocytes, while FPRL2 was described only in monocytes and in dendritic cells but in the last years many results demonstrate that several other cell types and tissues also express these receptors. In addition to N-fMLP, several nonformylated peptide agonists or other non-proteic ligands that preferentially activate either or both FPR and FPRL1 in nonphagocytic cells have been identified. They include WKYMVm peptide, isolated by screening a random oligonucleotide library, annexin 1, lipoxin A4 (LXA4), urokinase and its receptor, serum amyloid A, humanin and cathelicidin LL-37. Phagocytic NADPH oxidase activation and the subsequent reactive oxygen specie (ROS) generation represents one of the downstream target of the signaling cascade triggered by FPR and/or FPRL1. Proteins homologous to the membrane catalytic subunit gp91phox and to the cytosolic regulatory components p47phox and p67phox of NADPH oxidase also have been identified in nonphagocytic cells. Compared with PMN, much less is understood about the signal transduction pathways involved in the regulation of the activation of nonphagocytic NADPH oxidase, triggered by formyl-peptide receptors/agonist interaction. We demonstrated that human fibroblasts and epithelial cells express FPRL1 and an enzymatic machinery homologous to phagocytic NADPH oxidase. In serum-deprived cells, exposure to growth factors stimulates NADPH oxidase to generate superoxide anion and treatment with NADPH oxidase inhibitors results in impairment of the serum-induced signaling cascade. Furthermore, exposure to N-fMLP or to 10-fold lower concentration of WKYMVm for short times (1’) induces superoxide generation due to serine-phosphorylation and membrane translocation of the regulatory citosolic NADPH oxidase subunit p47phox. These effects are in large part mediated by the rapid activation of ERKs and are inhibited by pertussis toxin, suggesting the involvement of CPCRs. In addition to ERKs, in these cells exposed to N-fMLP or WKYMVm, p47phox phosphorylation and its translocation on membrane also requires Protein kinase C-α and -δ. On the other hand the exposure for longer times (up to 3hrs) induces p21waf1 accumulation, the block of the G1/S cycle, a significative increase of β-gal positive cells and the appearance of a senescent phenotype.