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G protein-coupled receptors (GPCRs) are the most important regulators of cardiac function and are commonly targeted for medical therapeutics. Formyl-Peptide Receptors (FPRs) are members of the GPCR superfamily and play an emerging role in cardiovascular pathologies. FPRs can modulate oxidative stress through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) production whose dysregulation has been observed in different cardiovascular diseases. Therefore, many studies are focused on identifying molecular mechanisms of the regulation of ROS production. FPR1, FPR2 and FPR3 belong to the FPRs family and their stimulation triggers phosphorylation of intracellular signaling molecules and nonsignaling proteins that are required for NADPH oxidase activation. Some FPR agonists trigger inflammatory processes, while other ligands activate proresolving or anti-inflammatory pathways, depending on the nature of the ligands. In general, bacterial and mitochondrial formylated peptides activate a proinflammatory cell response through FPR1, while Annexin A1 and Lipoxin A4 are anti-inflammatory FPR2 ligands. FPR2 can also trigger a proinflammatory pathway and the switch between FPR2-mediated pro- and anti-inflammatory cell responses depends on conformational changes of the receptor upon ligand binding. Here we describe the detrimental or beneficial effects of the main FPR agonists and their potential role as new therapeutic and diagnostic targets in the progression of cardiovascular diseases.

The chromosomal translocation t(4, 11) marks an infant acute lymphoblastic leukemia associated with dismal prognosis. This rearrangement leads to the synthesis of the MLL-AF4 chimera, which exerts its oncogenic activity by upregulating transcription of genes involved in hematopoietic differentiation. Crucial for chimera’s aberrant activity is the recruitment of the AF4/ENL/P-TEFb protein complex. Interestingly, a molecular interactor of AF4 is fibroblast growth factor receptor 2 (FGFR2). We herein analyze the role of FGFR2 in the context of leukemia using t(4, 11) leukemia cell lines. We revealed the interaction between MLL-AF4 and FGFR2 by immunoprecipitation, western blot, and immunofluorescence experiments, we also tested the effects of FGFR2 knockdown, FGFR2 inhibition, and FGFR2 stimulation on the expression of the main MLL-AF4 target genes, i.e., HOXA9 and MEIS1. Our results show that FGFR2 and MLL-AF4 interact in the nucleus of leukemia cells and that FGFR2 knockdown, which is associated with decreased expression of HOXA9 and MEIS1, impairs the binding of MLL-AF4 to the HOXA9 promoter. We also show that stimulation of leukemia cells with FGF2 increases nuclear level of FGFR2 in its phosphorylated form, as well as HOXA9 and MEIS1 expression. In contrast, preincubation with the ATP-mimetic inhibitor PD173074, before FGF2 stimulation, reduced FGFR2 nuclear amount and HOXA9 and MEIS1 transcript level, thereby indicating that MLL-AF4 aberrant activity depends on the nuclear availability of FGFR2. Overall, our study identifies FGFR2 as a new and promising therapeutic target in t(4, 11) leukemia.

Testo di Biochimica Generale utile per lo studio dei principi di Biochimica per studenti universitari.

The human formyl-peptide receptors FPR1, FPR2 and FPR3 belong to the G-protein-coupled receptor (GPCR) family and were first detected in phagocytic leukocytes and in monocytes. Their expression has also been demonstrated in several tissues and cell types at the protein or mRNA level and their relevant biological functions have emerged through the identification of high affinity host-derived agonists. Lung cancer remains the leading cause of cancer death worldwide. Tobacco smoking and air pollution exposure are mainly implicated in lung cancer development. The current use of molecular profiling technologies to assess DNA, RNA, protein and metabolites have provided substantial advances in our understanding of the molecular basis of cancer, leading to the potential development of more effective targeted therapies. The identification of markers involved in cell growth may further allow the molecular profiling of lung cancer. We investigated the possible role of FPRs as molecular markers in several types of lung carcinoma. We collected tumour tissue samples from six groups of patients (48 total) affected by lung cancer. Surgical biopsies were analyzed for expression of FPR isoforms both in tumoral and peritumoral tissue by real-time Polymerase Chain Reaction (PCR), western blot and immunofluorescence. Real-time PCR, western blot and immunofluorescence analyses showed that FPR expression is lower in types of human lung cancer tissues when compared to the surrounding peritumoral tissues. Since FPRs are involved in EGFR transactivation, these results suggest a potential role of the cross-talk mechanisms in tyrosine kinase inhibitors resistance observed in lung cancer treatment. The study of the mechanistic basis for the control of FPR expression in normal peritumoral versus tumoral tissues could provide the basis for new diagnostic and therapeutic interventions., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Molecular mechanisms underlying the generation of reactive oxygen species in LL-37-stimulated cells are poorly understood. Previously we demonstrated that in human fibroblasts the exposure to WKYMVm induced p47phox phosphorylation and translocation and, in turn, NADPH oxidase activation. These effects were mediated by the activation of the Formyl-peptide receptor-like 1 (FPRL1) and the downstream signaling involved ERKs phosphorylation and PKCα- and PKCδ- activation. Since LL-37 uses FPRL1 as a receptor to mediate its action on several cell types, we investigated in LL-37-stimulated IMR90 cells molecular mechanisms involved in NADPH-dependent superoxide generation. The exposure to LL-37, which is expressed in fibroblasts, induced ERKs activation, p47phox phosphorylation and translocation as well as NADPH oxidase activation. These effects were prevented by pertussis toxin, PD098059 and WRWWWW, a FPRL1-selective antagonist. Furthermore, the stimulation with LL-37 of HEK293 cells, transfected to stably express FPRL1, induced a rapid activation of ERKs and p47phox phosphorylation.

Formyl-peptide receptors (FPR) are expressed in several cell types including phagocytic leukocytes, and a wide variety of agonists of FPR and of its FPRL1 variant have been identified. These ligands interact with their specific receptors on the cellular membrane, and activate specific biological functions through a G-protein- coupled pathway. In nonphagocytic cells, agonist/FPR binding also induces transactivation of the constitutive membrane receptors PDGF-R, EGF-R and uPAR that in turn trigger specific, characteristic intracellular signal transduction pathways. The second messengers resulting from the interaction between ligands and formyl- peptide receptors act on various intracellular kinases (mitogen-activated protein kinases, protein kinases C and B, Jun kinase and some tyrosine kinases). Activation of NADPH oxidase expressed in nonphagocytic cells, and phosphorylation and nuclear translocation of regulatory transcriptional factors may be the downstream targets of this signaling cascade. The activated signal transduction pathways also lead to various biochemical cellular responses that can contribute to cell proliferation, and can protect against cell death and the malignant behavior of several human cancer cell lines. Dissection of the signaling cascade triggered by different agonists will shed light on the role of FPRs in nonphagocytic cells in both human physiology and diseases.

The regulation of the activation of non phagocytic NADPH oxidase is poorly understood. Previously we demonstrated that in fibroblasts the exposure to WKYMVm induced p47phox phosphorylation and translocation and that these effects were mediated by ERKs activation. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in polymorphonucleate cells stimulated via FPRL1 receptor, but its involvement in fibroblasts was not demonstrated. Therefore, we investigated in IMR90 cells exposed to WKYMVm the role of PKC isoenzymes in the activation of NADPH oxidase-like enzyme. Preincubation with general pharmacological inhibitors of PKC, before stimulation with WKYMVm, prevented the ERKs activation, p47phox phosphorylation and translocation. The analysis of cellular partitioning of PKC isoenzymes demonstrated that PKCa and PKCd translocated from the cytosolic to the membrane fraction upon stimulation with WKYMVm. Preincubation with Go6976 or with rottlerin prevented the phosphorylation and translocation of NADPH oxidase regulatory subunit.

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.