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.

Eicosanoids are a family of bioactive compounds derived from AA that play crucial roles in pathophysiology, including inflammatory conditions of multiple organ systems.They include prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), and lipoxins (LX).Once released by the catalytic action of cPLA2, AA can be metabolized by cyclooxygenases (COXs) that catalyze the transformation of AA in PGH2 [6,7].The aberrant AA metabolism observed in cancer cells results in a high concentration of PGs, in particular PGE 2 [8,9].Alternatively, AA can be metabolized by the enzyme 5-lipoxygenase (5-LOX) to generate LTs.5-LOX interacts with 5-lipoxygenase activating protein (FLAP) and converts AA to LTA4 [10].Therefore, LTs biosynthesis is highly dependent on the activities of cPLA2 and 5-LOX.In addition, AA can be metabolized by cytochrome P450, generating epoxides and a wide spectrum of biologically active fatty acid mediators [9].
The biosynthesis of eicosanoids requires several catalytic steps regulated by inflammatory and stress signals via different molecular mechanisms.In fact, phosphorylation at Ser505 residue of cPLA2 by extracellular response kinases (ERKs) and the stress-regulated p38MAPK regulates the activity of this enzyme [11,12], whereas 5-LOX is phosphorylated at Ser271 and Ser663 residues by p38MAPK-regulated MAPKAPK-2/3, ERKs, and CaMKII.Conversely, phosphorylation at Ser523 residue by PKA suppresses 5-LOX activity [13].The G-protein coupled receptors (GPCRs) can sense extracellular metabolites and thus regulate inflammatory responses, including eicosanoid production [10].Signaling triggered by GPCRs includes calcium mobilization and the activation of several downstream kinases, such as ERKs, p38MAPK, and p38MAPK-regulated MAPKAPK, which in turn can regulate cPLA2 and 5-LOX activity.
FPR2 is considered the most versatile FPR isoform, being able to recognize an array of structurally and chemically unrelated ligands [32,33].FPR2 agonists include both endogenous ligands and exogenous ligands [32].Depending on the nature of its ligands and/or FPR2 rearrangement with other FPR isoforms or with the scavenger macrophage receptor with collagenous structure (MARCO) [34], FPR2 can modulate pro-or anti-inflammatory responses [14,35].
We recently demonstrated that the FPR2 agonists WKYMVm and ANXA1 elicit intracellular redox signaling pathways involved in glucose uptake and aerobic metabolism of glucose typical of the Warburg effect in the human lung adenocarcinoma cell line CaLu-6 [44,45].Moreover, we proved that exposure to FPR2 agonists enhances the non-oxidative phase of pentose phosphate pathway (PPP), improves the expression of the glutamine transporter ASCT2, and induces the de novo synthesis of pyrimidine nucleotides [46].
We herein report our metabolomic data showing a significant increase in AA in FPR2stimulated CaLu-6 cells.Therefore, we dissect the molecular mechanisms involved in FPR2-dependent cPLA2 and 5-LOX activation that trigger AA release and LTs synthesis, respectively.We also evaluated the role of redox signaling in the cell metabolism of AA, disclosing that FPR2-mediated NADPH oxidase-dependent ROS generation plays a key role in both cPLA2 and 5-LOX phosphorylation and, in turn, in AA metabolism.
In other experiments, CaLu-6 cells were preincubated with the selective FPR2 antagonist WRWWWW (WRW4) (Primm, Milan, Italy) for 15 min at a final concentration of 10 µM, or with 100 µM apocynin, the selective inhibitor of NADPH oxidase (Sigma Chemical, St. Louis, MO, USA), for 2 h, or with 50 µM PD09805, a selective inhibitor of MEK (Sigma), for 90 min, or with 10 µM SB203580, a selective inhibitor of p38MAPK (Sigma), for 1 h.FPR2-unstimulated CaLu-6 cells only preincubated with the appropriate amount of the above-mentioned selective inhibitor were used as a negative control of pretreatments.

Metabolomic Analysis by LC-MS
Metabolomic analysis by LC-MS was performed in growing and in 24 h serum-starved CaLu-6 cells stimulated or not with WKYMVm in the presence or absence of WRW4 as previously described [46].Briefly, 24 h serum-starved cells were treated as above mentioned and lysed in 400 µL of a 1:1 prechilled MetOH:H 2 O solution.The samples were centrifuged at 10,000× g at 4 • C for 10 min.Supernatants were dried and then reconstituted with 125 µL of methanol/acetonitrile/water (50:25:25).Extracted metabolites were analyzed using an ACQUITY UPLC system online coupled to a Synapt G2-Si QTOF-MS (Waters Corporation, Milford, MA, USA) in positive and negative modes in the following settings: reverse-phase ACQUITY UPLC CSH C18 (1.7 µm, 100 × 2.1 mm 2 ) column (Waters), 0.3 mL/min flow rate, mobile phases composed of acetonitrile/H 2 O (60:40) containing 0.1% formic acid and 10 mM ammonium formate (Phase A), and isopropanol/acetonitrile (90:10) containing 0.1% formic acid and 10 mM ammonium formate (Phase B).Peak detection, metabolite identification, and quantitation were performed as previously described [44], fitting experimental data with internal standard and calibration curves.Data analysis and heatmap were generated with the online software MetaboAnalyst 5.0 (https://www.metaboanalyst.ca,accessed on 1 June 2021).

Statistical Analysis
All data reported are expressed as means ± standard error mean (SEM) and are representative of at least three or more independent experiments.Statistical analyses were performed with unpaired t-test to compare the mean of two independent groups of experiments or by one-way analysis of variance (ANOVA).GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) was used to compare more than two experiments.A p value of less than 0.05 was considered to be statistically significant.

FPR2 Stimulation Induces Arachidonic Acid Release by Activating cPLA2
We previously demonstrated that FPR2 stimulation triggers the metabolic reprogramming of CaLu-6 cells by modulating aerobic glycolysis, PPP, glutamine transport, and the de novo synthesis of pyrimidine nucleotides [44][45][46].Besides glycolysis and glutamine metabolism, an emerging role of lipids in metabolic reprogramming of many types of cancers cells has been observed [48].
By metabolomic analysis, we observed that AA concentration was significantly enhanced in WKYMVm-stimulated CaLu-6 cells (Figure 1B) compared to unstimulated cells (Figure 1A), whereas concentrations of other fatty acids, such as stearate, pentadecanoate, and laurate, were unaffected (Figure 1A,B).AA levels remained unchanged when cells were preincubated with the FPR2 antagonist WRWWWW (WRW4) (Figure 1C), thus indicating that it depends on FPR2 stimulation.
cPLA2 activity is tightly regulated in cells by at least three mechanisms.The increase in the intracellular Ca 2+ concentration represents a key regulator of cPLA2 activity in several cell types [52,53].In addition to Ca 2+ , cPLA2 is also regulated by intracellular lipids, which allosterically activate the enzyme and increase its residence time in membranes [54].cPLA2 is also regulated by phosphorylation at Ser505, Ser515, and Ser727 residues, which controls agonist-induced AA mobilization [55].Ser515 and Ser727 phosphorylation depends on cell type and stimulation conditions, whereas only Ser505 phosphorylation is required for cPLA2 full activation and cell membrane translocation [55].
In WKYMVm-stimulated cells, we observed a time-dependent increase in cPLA2 Ser505 phosphorylation (Figure 1D), which was prevented by preincubation with the FPR2 antagonist (Figure 1E).
FPR2 ability to induce an increase in PLA2 expression was previously observed in SAAstimulated cells [56], as well as in conjunctival goblet cells and in neutrophils incubated with ANXA1 or WKYMVm, respectively [57,58].
Our results demonstrate, for the first time, that FPR2 stimulation triggers cPLA2 phosphorylation at Ser505 residue, thereby suggesting that cPLA2 activation can contribute to the concomitant increase in AA concentration.

FPR2-Mediated cPLA2 Ser505 Phosphorylation Depends on NOX Activation
Ser505 residue of cPLA2 is phosphorylated by different kinases, such as ERKs [11] and p38MAPK [12].Furthermore, there is increasing evidence that NOX-dependent ROS generation can activate cPLA2 [59] and that hydrolytic products of cPLA2, including AA, could enhance NOX activity [60][61][62].The assembled NOX complex serves as a target for anchoring cPLA2 to the plasma membranes [63].Therefore, it is conceivable that these two enzymes share a common mechanism for activation by intracellular kinases.ERKs and Growing cells (ctrl) were serum-starved for 24 h and then stimulated or not with 10 µM WKYMVm for 1 h in presence or absence of 10 µM WRW4.Metabolomic analysis was performed as described in Materials and Methods.cPLA2 Serine 505 (Ser505) phosphorylation is promoted by FPR2 stimulation with WKYMVm (D,E).Serum-starved CaLu-6 cells were exposed to 10 µM WKYMVm for 5, 10, 30, or 60 min (D), or pre-treated for 15 min with WRW4 before the stimulation with WKYMVm (E).Sixty micrograms of whole lysates were resolved on 10% SDS-PAGE and incubated with an anti-phospho-cPLA2 (Ser505) (α-p-cPLA2(Ser505)) antibody.An anti-GAPDH (α-GAPDH) antibody was used as control for protein loading.Data are representative of three independent experiments.* p < 0.05 compared to unstimulated cells.§ p < 0.05 compared to stimulated cells.

FPR2-Mediated cPLA2 Ser505 Phosphorylation Depends on NOX Activation
Ser505 residue of cPLA2 is phosphorylated by different kinases, such as ERKs [11] and p38MAPK [12].Furthermore, there is increasing evidence that NOX-dependent ROS generation can activate cPLA2 [59] and that hydrolytic products of cPLA2, including AA, could enhance NOX activity [60][61][62].The assembled NOX complex serves as a target for anchoring cPLA2 to the plasma membranes [63].Therefore, it is conceivable that these two enzymes share a common mechanism for activation by intracellular kinases.ERKs and p38MAPK trigger both cPLA2 phosphorylation and ROS production, and both events are prevented by NADPH oxidase inhibitors [64].
Several GPCR agonists elicit an increase in NOX-dependent ROS concentration and trigger the activity of several kinases, such as p38MAPK, ERKs, and JNK, which are able to phosphorylate cPLA2 [65,66].These kinases can be also activated by the canonical pathway of TKRs elicited by GPCR-dependent TKRs transactivation [67].
Previously, we demonstrated that intracellular domains of activated FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signaling cascades, including activation of PLC, PKC isoforms, p38MAPK, PI3K/Akt, and MAPK pathway.Phosphorylation of cytosolic tyrosine kinases, TKRs transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, and release of calcium and production of oxidants also belong to the distinct intracellular pathways elicited by FPR2 [32].

FPR2 Stimulation Induces 5-LOX Activation
Eicosanoids, including PGs, TXs, LTs, and lipoxins, are generated during the various phases of AA metabolism along the COX and LOX pathways [70].Release of AA and activation of 5-LOX, in response to several stimuli, initiates the biosynthesis of proinflammatory LTs, a family of lipid mediators with pivotal roles in inflammatory disorders [71].
Notably, ROS play an important role in inflammation processes and in signal transduction.According with our findings, NOX activation increases ROS levels that in turn inactivate the function of phosphothyrosine and serine/threonine phosphatases containing cysteine moieties within their active centers, which are susceptible to oxidation by ROS.Therefore, inactivation of phosphatases results in activation of MAPKs, p38MAPK, and ERKs which, in turn, activate cPLA2 [68].Very interestingly, protein phosphatase inhibition also induces activation of non-receptor tyrosine kinase family Src, which, in turn, leads to EGFR phosphorylation [38] and thus to the transduction of this signal into MAPK cascades [69], further improving cPLA2 activation.

FPR2 Stimulation Induces 5-LOX Activation
Eicosanoids, including PGs, TXs, LTs, and lipoxins, are generated during the various phases of AA metabolism along the COX and LOX pathways [70].Release of AA and activation of 5-LOX, in response to several stimuli, initiates the biosynthesis of proinflammatory LTs, a family of lipid mediators with pivotal roles in inflammatory disorders [71].
In addition to the normal expression in the various leukocyte types, aberrant expression of 5-LOX has been detected in many tumor cells [72][73][74] and, besides inflammatory processes, 5-LOX is involved in cell differentiation, oxidative stress, and in the progression of different diseases [75][76][77].
In the resting cell, 5-LOX is localized in either the cytosol or a soluble compartment inside the nucleus.Activation of cellular 5-LOX involves enzyme translocation to the nuclear envelope, where it colocalizes with cPLA2 and FLAP [78][79][80].
5-LOX is a substrate for several protein kinases, and phosphorylation of different residues has divergent consequences for 5-LOX subcellular localization and activity.Phosphorylation at Ser271 and Ser663 residues increases 5-LOX expression and is strongly promoted by unsaturated fatty acids, including AA. Enhanced 5-LOX activity following dual phosphorylation at Ser271 and Ser663 residues is observed when intracellular Ca 2+ levels are low and, thus, insufficient to activate 5-LOX alone.In addition, Ser271 phosphorylation promotes nuclear localization of 5-LOX [81].An increase in cAMP levels activates PKA, which represses 5-LOX activity through phosphorylation at Ser523 residue [82].This phosphorylation prevents nuclear import of the enzyme and, in turn, its cytoplasmic enrichment [83].
In PMN, AA and fMLP, an FPR1 agonist, activate p38MAPK, leading to 5-LOX activation [84].ERKs have been described as kinases that phosphorylate 5-LOX at Ser663 residue, and dual phosphorylation by ERK2 and p38MAPKAPKs at different sites is necessary for AA-induced 5-LOX activation [86].AA plays a central role in the convergence of MAPK signaling cascades, leading to phosphorylation and activation of 5-LOX.Synergistic actions of MAPK pathways were also observed for the activation of cPLA2 [13,87].
Because catalysis by 5-LOX requires oxidation of Fe 2+ to Fe 3+ in the active site of the enzyme, cellular redox conditions represent a crucial factor that modulates 5-LOX activity.Conditions that promote the formation of ROS upregulate 5-LOX, whereas reducing conditions, as well as the presence of suitable thiols (GSH or DTT), prevent 5-LOX activity [81,88].
In mammalian cells, ROS are generated via a variety of cellular oxidative processes, including the activities of NOX, xanthine oxidases, and the mitochondrial respiratory chain.NOX-generated ROS are the best characterized examples of ROS, although they are also generated by the oxidative metabolism of AA, released from the membrane phospholipids via cPLA2 activity.In fact, LOX-and COX-generated AA metabolites can induce ROS generation by stimulating NOX, highlighting a potential signaling connection between LOX/COX metabolites and NOX [89].
Ser271 is phosphorylated by p38MAPK-regulated MAPKAPKs, and among MAPK families, p38MAPK is also activated when cells are exposed to various cellular stress.We previously proved that in CaLu-6 cells FPR2 stimulation with WKYMVm or ANXA1 induced NOX-dependent activation of p38MAPK [40].Thus, an increase in ROS in these cells may activate p38MAPK and, consequently, 5-LOX by promoting Ser271 phosphorylation [90].
This latter evidence and the results herein presented strongly demonstrate that FPR2 signaling induces 5-LOX phosphorylation at Ser271 and Ser663, which requires p38MAPK and ERKs activation, respectively.Furthermore, since NOX inhibition prevents Ser271 phosphorylation, we can hypothesize that FPR2-dependent NOX activation might contribute to 5-LOX nuclear translocation.

Conclusions
In the last decade, much progress has been made in understanding the effects of ROS on inflammatory processes that are associated with many human diseases.In many cases, the disease, or its progression, is associated with NOX activation, which generates ROS that trigger inflammatory reactions, representing one of the risk factors for cancer.
Although 5-LOX catalyzes the first step of LTs molecules with proinflammatory functions, it is also involved in the biosynthesis of lipid mediators with anti-inflammatory properties, such as lipoxins.In many diseases, an imbalance between proinflammatory LTs and anti-inflammatory lipoxins is observed.However, some questions remain unresolved.How does intranuclear localization of 5-LOX confer a higher activity?Does 5-LOX have other roles inside the nucleus?Since inhibition of LTs biosynthesis shows beneficial effects in various inflammatory diseases, new findings on 5-LOX activation/translocation may lead to future novel therapeutic possibilities.In this context, the elucidation of the molecular mechanisms underlying inflammatory reactions triggered by NOX-dependent ROS generation (Figure 5) can provide new strategies to inhibit 5-LOX activation, whereas the control of FPR2-mediated nuclear import of 5-LOX might represent an important step in resolving the inflammatory process in many human diseases.

Conclusions
In the last decade, much progress has been made in understanding the effects of ROS on inflammatory processes that are associated with many human diseases.In many cases, the disease, or its progression, is associated with NOX activation, which generates ROS that trigger inflammatory reactions, representing one of the risk factors for cancer.
Although 5-LOX catalyzes the first step of LTs molecules with proinflammatory functions, it is also involved in the biosynthesis of lipid mediators with anti-inflammatory properties, such as lipoxins.In many diseases, an imbalance between proinflammatory LTs and anti-inflammatory lipoxins is observed.However, some questions remain unresolved.How does intranuclear localization of 5-LOX confer a higher activity?Does 5-LOX have other roles inside the nucleus?Since inhibition of LTs biosynthesis shows beneficial effects in various inflammatory diseases, new findings on 5-LOX activation/translocation may lead to future novel therapeutic possibilities.In this context, the elucidation of the molecular mechanisms underlying inflammatory reactions triggered by NOX-dependent ROS generation (Figure 5) can provide new strategies to inhibit 5-LOX activation, whereas the control of FPR2-mediated nuclear import of 5-LOX might represent an important step in resolving the inflammatory process in many human diseases.FPR2 stimulation contribute to regulate arachidonic acid metabolism.Upon WKYMVm incubation, FPR2 increases the concentration of arachidonate.FPR2, once stimulated, can contribute to arachidonic acid metabolism by phosphorylating cPLA2 on its Serine 505 (Ser505) in a p38MAPK-, ERKs-, and NADPH oxidase-dependent fashion.FPR2-cPLA2-mediated increase in arachidonic acid can improve 5-LOX activation by phosphorylating its Serine 271 (Ser271) and Serine 663 (Ser663).5-LOX activation can be also promoted by an increase in intracellular Ca 2+ concentration ([Ca 2+ ]).Both 5-LOX phosphorylation required FPR2 stimulation.However, Ser271 required p38MAPK and NADPH oxidase activation, whereas Ser663 ERKs activation.5-LOX, once phosphorylated, can migrate to the nuclear envelope, where it colocalizes with PLA2 and 5-lipoxygenase activating protein (FLAP), and in turn, it can contribute to generating anti-inflammatory mediators.Furthermore, 5-LOX phosphorylation at Ser271 promotes its nuclear localization, where it can favor biosynthesis of pro-inflammatory mediators.