NOX Dependent ROS Generation and Cell Metabolism.

Reactive oxygen species (ROS) represent a group of high reactive molecules with dualistic natures since they can induce cytotoxicity or regulate cellular physiology. Among the ROS, the superoxide anion radical (O2·-) is a key redox signaling molecule prominently generated by the NADPH oxidase (NOX) enzyme family and by the mitochondrial electron transport chain. Notably, altered redox balance and deregulated redox signaling are recognized hallmarks of cancer and are involved in malignant progression and resistance to drugs treatment. Since oxidative stress and metabolism of cancer cells are strictly intertwined, in this review, we focus on the emerging roles of NOX enzymes as important modulators of metabolic reprogramming in cancer. The NOX family includes seven isoforms with different activation mechanisms, widely expressed in several tissues. In particular, we dissect the contribute of NOX1, NOX2, and NOX4 enzymes in the modulation of cellular metabolism and highlight their potential role as a new therapeutic target for tumor metabolism rewiring.

MiR-27a downregulates 14-3-3θ, RUNX1, AF4, and MLL-AF4, crucial drivers of blast transformation in t(4;11) leukemia cells.

The chromosomal translocation t(4;11)(q21;q23), a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL), encodes mixed-lineage leukemia (MLL)-AF4 oncogenic chimera that triggers aberrant transcription of genes involved in lymphocyte differentiation, including HOXA9 and MEIS1. The scaffold protein 14-3-3θ, which promotes the binding of MLL-AF4 to the HOXA9 promoter, is a target of MiR-27a, a tumor suppressor in different human leukemia cell types. We herein study the role of MiR-27a in the pathogenesis of t(4;11) ALL. Reverse transcription quantitative PCR (qPCR) reveals that MiR-27a and 14-3-3θ expression is inversely correlated in t(4;11) ALL cell lines; interestingly, MiR-27a relative expression is significantly lower in patients affected by t(4;11) ALL than in patients affected by the less severe t(12;21) leukemia. In t(4;11) leukemia cells, ectopic expression of MiR-27a decreases protein level of 14-3-3θ and of the key transcription factor RUNX1. We show for the first time that MiR-27a also targets AF4 and MLL-AF4; in agreement, MiR-27a overexpression strongly reduces AF4 and MLL-AF4 protein levels in RS4;11 cells. Consequent to AF4 and MLL-AF4 downregulation, MiR-27a overexpression negatively affects transcription of HOXA9 and MEIS1 in different t(4;11) leukemia cell lines. In agreement, we show through chromatin immunoprecipitation experiments that MiR-27a overexpression impairs the binding of MLL-AF4 to the HOXA9 promoter. Lastly, we found that MiR-27a overexpression decreases viability, proliferation, and clonogenicity of t(4;11) cells, whereas it enhances their apoptotic rate. Overall, our study identifies the first microRNAthat strikes in one hit four crucial drivers of blast transformation in t(4;11) leukemia. Therefore, MiR-27a emerges as a new promising therapeutic target for this aggressive and poorly curable form of leukemia.

Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates HOXA9 Gene Expression in t(4;11) Leukemia Cells.

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.

Phosphorylation Sites in Protein Kinases and Phosphatases Regulated by Formyl Peptide Receptor 2 Signaling.

FPR1, FPR2, and FPR3 are members of Formyl Peptides Receptors (FPRs) family belonging to the GPCR superfamily. FPR2 is a low affinity receptor for formyl peptides and it is considered the most promiscuous member of this family. Intracellular signaling cascades triggered by FPRs include the activation of different protein kinases and phosphatase, as well as tyrosine kinase receptors transactivation. Protein kinases and phosphatases act coordinately and any impairment of their activation or regulation represents one of the most common causes of several human diseases. Several phospho-sites has been identified in protein kinases and phosphatases, whose role may be to expand the repertoire of molecular mechanisms of regulation or may be necessary for fine-tuning of switch properties. We previously performed a phospho-proteomic analysis in FPR2-stimulated cells that revealed, among other things, not yet identified phospho-sites on six protein kinases and one protein phosphatase. Herein, we discuss on the selective phosphorylation of Serine/Threonine-protein kinase N2, Serine/Threonine-protein kinase PRP4 homolog, Serine/Threonine-protein kinase MARK2, Serine/Threonine-protein kinase PAK4, Serine/Threonine-protein kinase 10, Dual specificity mitogen-activated protein kinase kinase 2, and Protein phosphatase 1 regulatory subunit 14A, triggered by FPR2 stimulation. We also describe the putative FPR2-dependent signaling cascades upstream to these specific phospho-sites.

Wound healing activity and phytochemical screening of purified fractions of Sempervivum tectorum L. leaves on HCT 116.

INTRODUCTION: Sempervivum tectorum L. (Crassulaceae), is a succulent perennial plant widespread in Mediterranean countries and commonly used in traditional medicine for ear inflammation, ulcers and skin rashes as a refrigerant and astringent. OBJECTIVE: To demonstrate the therapeutic effects of the plant, various fractions were purified and characterised. The potential wound healing activity, proliferation rate and intracellular signalling cascades were investigated by using human epithelial colorectal carcinoma (HCT 116) cells. METHODOLOGY: An extraction method without organic solvents was applied for the first time. The purification was carried out by droplet counter current chromatography (DCCC) coupled with high-performance liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ESI-MS) data. By nuclear magnetic resonance (NMR) [1 H, 13 C and two-dimensional (2D) experiments] pure components were identified. Wound healing and cell proliferation assays were utilised to determine the role of the isolated S. tectorum (SVT) fraction on cellular migration and proliferation. The signalling pathways elicited from the SVT fractions, were analysed by Western blot analysis. RESULTS: In this study two rare natural components were identified, namely monosaccharide sedoheptulose and polyalcohol 2-C-methyl-D-erythritol, along with known organic acids and flavonoids. The fractions with high level of sedoheptulose enhance the proliferation and the cellular migration of epithelial HCT 116 cells. The intracellular signalling cascades elicited from the purified fractions induce the c-Src-mediated transactivation of EGFR and the activation of the STAT3 pathway which, in turn, are crucially involved in the cellular proliferation and migration. CONCLUSIONS: Our study demonstrates the efficacy of purified fractions of S. tectorum L. in enhancing cellular proliferation and migration, suggesting their potential role as topical therapeutic treatments for wound healing.

Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells.

Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H(229) and K(231) residues within the NLS.

Cell-surface receptors transactivation mediated by g protein-coupled receptors.

G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we discuss the main mechanisms of GPCR-mediated cell-surface receptors transactivation and the pathways involved in intracellular responses induced by GPCR agonists. These studies may suggest the design of novel strategies for therapeutic interventions.

Formyl Peptide Receptor 2-Dependent cPLA2 and 5-LOX Activation Requires a Functional NADPH Oxidase.

Phospholipases (PL) A2 catalyzes the hydrolysis of membrane phospholipids and mostly generates arachidonic acid (AA). The enzyme 5-lipoxygenase (5-LOX) can metabolize AA to obtain inflammatory leukotrienes, whose biosynthesis highly depends on cPLA2 and 5-LOX activities. Formyl Peptide Receptor 2 (FPR2) belongs to a subfamily of class A GPCRs and is considered the most versatile FPRs isoform. Signaling triggered by FPR2 includes the activation of several downstream kinases and NADPH oxidase (NOX)-dependent ROS generation. In a metabolomic analysis we observed a significant increase in AA concentration in FPR2-stimulated lung cancer cell line CaLu-6. We analyzed cPLA2 phosphorylation and observed a time-dependent increase in cPLA2 Ser505 phosphorylation in FPR2-stimulated cells, which was prevented by the MEK inhibitor (PD098059) and the p38MAPK inhibitor (SB203580) and by blocking NOX function. Similarly, we demonstrated that phosphorylation of 5-LOX at Ser271 and Ser663 residues requires FPR2-dependent p38MAPK and ERKs activation. Moreover, we showed that 5-LOX Ser271 phosphorylation depends on a functional NOX expression. Our overall data demonstrate for the first time that FPR2-induced ERK- and p38MAPK-dependent phosphorylation/activation of cPLA2 and 5-LOX requires a functional NADPH oxidase. These findings represent an important step towards future novel therapeutic possibilities aimed at resolving the inflammatory processes underlying many human diseases.

Formyl-Peptide Receptor 2 Signaling Modulates SLC7A11/xCT Expression and Activity in Tumor Cells.

Cancer cells exhibit high levels of oxidative stress and consequently require a high amount of cysteine for glutathione synthesis. Solute Carrier Family 7 Member 11 (SLC7A11), or xCT, mediates the cellular uptake of cystine in exchange for intracellular glutamate; imported extracellular cystine is reduced to cysteine in the cytosol through a NADPH-consuming reduction reaction. SLC7A11/xCT expression is under the control of stress-inducing conditions and of several transcription factors, such as NRF2 and ATF4. Formyl-peptide receptor 2 (FPR2) belongs to the FPR family, which transduces chemotactic signals mediating either inflammatory or anti-inflammatory responses according to the nature of its ligands and/or FPR2 binding with other FPR isoforms. The repertoire of FPR2 agonists with anti-inflammatory activities comprises WKYMVm peptide and Annexin A1 (ANXA1), and the downstream effects of the intracellular signaling cascades triggered by FPR2 include NADPH oxidase (NOX)-dependent generation of reactive oxygen species. Herein, we demonstrate that stimulation of CaLu-6 cells with either WKYMVm or ANXA1: (i) induces the redox-regulated activation of SLC7A11/xCT; (ii) promotes the synthesis of glutathione; (iii) prevents lipid peroxidation; and (iv) favors NRF2 nuclear translocation and activation. In conclusion, our overall results demonstrate that FPR2 agonists and NOX modulate SLC7A11/xCT expression and activity, thereby identifying a novel regulative pathway of the cystine/glutamate antiport that represents a new potential therapeutical target for the treatment of human cancers.

Comprehensive Molecular Analysis of Disease-Related Genes as First-Tier Test for Early Diagnosis, Classification, and Management of Patients Affected by Nonsyndromic Ichthyosis.

Inherited ichthyoses are a group of clinically and genetically heterogeneous rare disorders of skin keratinization with overlapping phenotypes. The clinical picture and family history are crucial to formulating the diagnostic hypothesis, but only the identification of the genetic defect allows the correct classification. In the attempt to molecularly classify 17 unrelated Italian patients referred with congenital nonsyndromic ichthyosis, we performed massively parallel sequencing of over 50 ichthyosis-related genes. Genetic data of 300 Italian unaffected subjects were also analyzed to evaluate frequencies of putative disease-causing alleles in our population. For all patients, we identified the molecular cause of the disease. Eight patients were affected by autosomal recessive congenital ichthyosis associated with ALOX12B, NIPAL4, and TGM1 mutations. Three patients had biallelic loss-of-function variants in FLG, whereas 6/11 males were affected by X-linked ichthyosis. Among the 24 different disease-causing alleles we identified, 8 carried novel variants, including a synonymous TGM1 variant that resulted in a splicing defect. Moreover, we generated a priority list of the ichthyosis-related genes that showed a significant number of rare and novel variants in our population. In conclusion, our comprehensive molecular analysis resulted in an effective first-tier test for the early classification of ichthyosis patients. It also expands the genetic, mutational, and phenotypic spectra of inherited ichthyosis and provides new insight into the current understanding of etiologies and epidemiology of this group of rare disorders.

Distinct signaling cascades elicited by different formyl peptide receptor 2 (FPR2) agonists.

The formyl peptide receptor 2 (FPR2) is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR) family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aß)-42 and prion protein (Prp)106-126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP)-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC), protein kinase C (PKC) isoforms, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, the mitogen-activated protein kinase (MAPK) pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2 agonists.

Formyl-peptide receptor 2 signalling triggers aerobic metabolism of glucose through Nox2-dependent modulation of pyruvate dehydrogenase activity.

The human formyl-peptide receptor 2 (FPR2) is activated by an array of ligands. By phospho-proteomic analysis we proved that FPR2 stimulation induces redox-regulated phosphorylation of many proteins involved in cellular metabolic processes. In this study, we investigated metabolic pathways activated in FPR2-stimulated CaLu-6 cells. The results showed an increased concentration of metabolites involved in glucose metabolism, and an enhanced uptake of glucose mediated by GLUT4, the insulin-regulated member of GLUT family. Accordingly, we observed that FPR2 transactivated IGF-IRß/IRß through a molecular mechanism that requires Nox2 activity. Since cancer cells support their metabolism via glycolysis, we analysed glucose oxidation and proved that FPR2 signalling promoted kinase activity of the bifunctional enzyme PFKFB2 through FGFR1/FRS2- and Akt-dependent phosphorylation. Furthermore, FPR2 stimulation induced IGF-IRß/IRß-, PI3K/Akt- and Nox-dependent inhibition of pyruvate dehydrogenase activity, thus preventing the entry of pyruvate in the tricarboxylic acid cycle. Consequently, we observed an enhanced FGFR-dependent lactate dehydrogenase (LDH) activity and lactate production in FPR2-stimulated cells. As LDH expression is transcriptionally regulated by c-Myc and HIF-1, we demonstrated that FPR2 signalling promoted c-Myc phosphorylation and Nox-dependent HIF-1α stabilization. These results strongly indicate that FPR2-dependent signalling can be explored as a new therapeutic target in treatment of human cancers.

De Novo Large Deletions in the PHEX Gene Caused X-Linked Hypophosphataemic Rickets in Two Italian Female Infants Successfully Treated with Burosumab.

Pathogenic variants in the PHEX gene cause rare and severe X-linked dominant hypophosphataemia (XLH), a form of heritable hypophosphatemic rickets (HR) characterized by renal phosphate wasting and elevated fibroblast growth factor 23 (FGF23) levels. Burosumab, the approved human monoclonal anti-FGF23 antibody, is the treatment of choice for XLH. The genetic and phenotypic heterogeneity of HR often delays XLH diagnoses, with critical effects on disease course and therapy. We herein report the clinical and genetic features of two Italian female infants with sporadic HR who successfully responded to burosumab. Their diagnoses were based on clinical and laboratory findings and physical examinations. Next-generation sequencing (NGS) of the genes associated with inherited HR and multiple ligation probe amplification (MLPA) analysis of the PHEX and FGF23 genes were performed. While a conventional analysis of the NGS data did not reveal pathogenic or likely pathogenic small nucleotide variants (SNVs) in the known HR-related genes, a quantitative analysis identified two different heterozygous de novo large intragenic deletions in PHEX, and this was confirmed by MLPA. Our molecular data indicated that deletions in the PHEX gene can be the cause of a significant fraction of XLH; hence, their presence should be evaluated in SNV-negative female patients. Our patients successfully responded to burosumab, demonstrating the efficacy of this drug in the treatment of XLH. In conclusion, the execution of a phenotype-oriented genetic test, guided by known types of variants, including the rarest ones, was crucial to reach the definitive diagnoses and ensure our patients of long-term therapy administration.

Formyl-Peptide Receptor 2 Signaling Redirects Glucose and Glutamine into Anabolic Pathways in Metabolic Reprogramming of Lung Cancer Cells.

Glucose and glutamine play a crucial role in the metabolic reprogramming of cancer cells. Proliferating cells metabolize glucose in the aerobic glycolysis for energy supply, and glucose and glutamine represent the primary sources of carbon atoms for the biosynthesis of nucleotides, amino acids, and lipids. Glutamine is also an important nitrogen donor for the production of nucleotides, amino acids, and nicotinamide. Several membrane receptors strictly control metabolic reprogramming in cancer cells and are considered new potential therapeutic targets. Formyl-peptide receptor 2 (FPR2) belongs to a small family of GPCRs and is implicated in many physiopathological processes. Its stimulation induces, among other things, NADPH oxidase-dependent ROS generation that, in turn, contributes to intracellular signaling. Previously, by phosphoproteomic analysis, we observed that numerous proteins involved in energetic metabolism are uniquely phosphorylated upon FPR2 stimulation. Herein, we investigated the role of FPR2 in cell metabolism, and we observed that the concentrations of several metabolites associated with the pentose phosphate pathway (PPP), tricarboxylic acid cycle, nucleotide synthesis, and glutamine metabolism, were significantly enhanced in FPR2-stimulated cells. In particular, we found that the binding of specific FPR2 agonists: (i) promotes NADPH production; (ii) activates the non-oxidative phase of PPP; (iii) induces the expression of the ASCT2 glutamine transporter; (iv) regulates oxidative phosphorylation; and (v) induces the de novo synthesis of pyrimidine nucleotides, which requires FPR2-dependent ROS generation.

Imbricatolic acid from Juniperus communis L. prevents cell cycle progression in CaLu-6 cells.

Imbricatolic acid was isolated from the methanolic extract of the fresh ripe berries of Juniperus communis (Cupressaceae) together with sixteen known compounds and a new dihydrobenzofuran lignan glycoside named juniperoside A. Their structures were determined by spectroscopic methods and by comparison with the spectral data reported in literature. Imbricatolic acid was evaluated for its ability to prevent cell cycle progression in p53-null CaLu-6 cells. This compound induces the upregulation of cyclin-dependent kinase inhibitors and their accumulation in the G1 phase of the cell cycle, as well as the degradation of cyclins A, D1, and E1. Furthermore, no significant imbricatolic acid-induced apoptosis was observed. Therefore, this plant-derived compound may play a role in the control of cell cycle.

Pro-Resolving FPR2 Agonists Regulate NADPH Oxidase-Dependent Phosphorylation of HSP27, OSR1, and MARCKS and Activation of the Respective Upstream Kinases.

BACKGROUND: Formyl peptide receptor 2 (FPR2) is involved in the pathogenesis of chronic inflammatory diseases, being activated either by pro-resolving or proinflammatory ligands. FPR2-associated signal transduction pathways result in phosphorylation of several proteins and in NADPH oxidase activation. We, herein, investigated molecular mechanisms underlying phosphorylation of heat shock protein 27 (HSP27), oxidative stress responsive kinase 1 (OSR1), and myristolated alanine-rich C-kinase substrate (MARCKS) elicited by the pro-resolving FPR2 agonists WKYMVm and annexin A1 (ANXA1). METHODS: CaLu-6 cells or p22phoxCrispr/Cas9 double nickase CaLu-6 cells were incubated for 5 min with WKYMVm or ANXA1, in the presence or absence of NADPH oxidase inhibitors. Phosphorylation at specific serine residues of HSP27, OSR1, and MARCKS, as well as the respective upstream kinases activated by FPR2 stimulation was analysed. RESULTS: Blockade of NADPH oxidase functions prevents WKYMVm- and ANXA1-induced HSP-27(Ser82), OSR1(Ser339) and MARCKS(Ser170) phosphorylation. Moreover, NADPH oxidase inhibitors prevent WKYMVm- and ANXA1-dependent activation of p38MAPK, PI3K and PKCδ, the kinases upstream to HSP-27, OSR1 and MARCKS, respectively. The same results were obtained in p22phoxCrispr/Cas9 cells. CONCLUSIONS: FPR2 shows an immunomodulatory role by regulating proinflammatory and anti-inflammatory activities and NADPH oxidase is a key regulator of inflammatory pathways. The activation of NADPH oxidase-dependent pro-resolving downstream signals suggests that FPR2 signalling and NADPH oxidase could represent novel targets for inflammation therapeutic intervention.

Regulation of Inflammation and Oxidative Stress by Formyl Peptide Receptors in Cardiovascular Disease Progression.

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.

Expression and signaling of formyl-peptide receptors in the brain.

The human formyl-peptide receptor (FPR) and its variants FPRL1 and FPRL2 belong to the G-protein coupled seven transmembrane receptor (GPCR) family sensitive to pertussis toxin. FPR and FPRL1 were first detected in phagocytic leukocytes, and FPRL2 was found in monocytes and in dendritic cells. The three receptors were subsequently identified in other cell types or tissues, including neuronal cells and brain, where FPR and FPRL1 play a key role in angiogenesis, cell proliferation, protection against and cell death, as well as in neuroendocrine functions. Binding of different agonists to FPRs triggers several signaling pathways, activates NFkB and STAT3 transcriptional factors and induces the accumulation of the CDK inhibitors p21(waf1/cip1), p16(INK4) and p27(kip1). Signaling molecules, such as ERKs, JNK, PKC, p38MAPK, PLC and PLD are involved in these intracellular cascades. In this article we briefly review FPRs expression and signaling in neuronal cells.

Multi-Gene Next-Generation Sequencing for Molecular Diagnosis of Autosomal Recessive Congenital Ichthyosis: A Genotype-Phenotype Study of Four Italian Patients.

Autosomal recessive congenital ichthyoses (ARCI) are rare genodermatosis disorders characterized by phenotypic and genetic heterogeneity. At least fourteen genes so far have been related to ARCI; however, despite genetic heterogeneity, phenotypes associated with mutation of different ARCI genes may overlap, thereby making difficult their clinical and molecular classification. In addition, molecular tests for diagnosis of such an extremely rare heterogeneous inherited disease are not easily available in clinical settings. In the attempt of identifying the genetic cause of the disease in four Italian patients with ARCI, we performed next-generation sequencing (NGS) analysis targeting 4811 genes that have been previously linked to human genetic diseases; we focused our analysis on the 13 known ARCI genes comprised in the panel. Nine different variants including three novel small nucleotide changes and two novel large deletions have been identified and validated in the ABCA12, ALOX12B, CYP4F22, and SULT2B1 genes. Notably, two patients had variants in more than one gene. The identification and validation of new pathogenic ABCA12, ALOX12B, CYP4F22, and SULT2B1 variants through multi-gene NGS in four cases of ARCI further highlight the importance of these genes in proper skin function and development.

FPRL1-mediated induction of superoxide in LL-37-stimulated IMR90 human fibroblast.

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 p47(phox) 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 PKCalpha- and PKCdelta-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, p47(phox) 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 p47(phox) phosphorylation.