Mol Neurobiol DOI 10.1007/s12035-013-8621-5

NGF Induces the Expression of Group IIA Secretory Phospholipase A2 in PC12 Cells: The Newly Synthesized Enzyme Is Addressed to Growing Neurites Vincenza Nardicchi & Monica Ferrini & Francesca Pilolli & Emanuela Biagioni Angeli & Emanuele Persichetti & Tommaso Beccari & Roberta Mannucci & Cataldo Arcuri & Rosario Donato & Robert V. Dorman & Gianfrancesco Goracci

Received: 26 October 2013 / Accepted: 15 December 2013 # Springer Science+Business Media New York 2014

Abstract We proposed that group IIA secretory phospholipase A2 (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168–2174, 2010). The involvement of other secretory PLA2 isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA2 (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition,

The authors Vincenza Nardicchi and Monica Ferrini contributed equally to this study. V. Nardicchi : M. Ferrini : F. Pilolli : E. B. Angeli : G. Goracci (*) Department of Internal Medicine, Neurochemistry Laboratory, University of Perugia, Via del Giochetto, 06126 Perugia, Italy e-mail: [email protected] E. Persichetti : T. Beccari Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy C. Arcuri : R. Donato Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy R. Mannucci Laboratory of Image Analysis, University of Perugia, Perugia, Italy R. V. Dorman Department of Biological Sciences and School of Biomedical Sciences, Kent State University, Kent, OH, USA

NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation–reacylation reactions necessary for the incorporation of polyunsaturated fatty acids. Keywords Phospholipase A2 . Nerve growth factor . PC12 cells . Differentiation . Protein expression . GIIA sPLA2 Abbreviations ALA cPLA2 CDP-choline DHA ECL ELISA GIIA GV GX IMF Inh I iPLA2 KLH LPLATs lyso-PtdCho lyso-PtdEtn MAPK

α-Linoleic acid Cytosolic PLA2 Cytidine diphosphocholine Docosahexaenoic acid Enhanced chemiluminescence Enzyme-linked immunosorbent assay Group IIA sPLA2 Group V sPLA2 Group X sPLA2 Immunofluorescence microscopy sPLA2 Inhibitor I Ca2+-independent PLA2 Keyhole Limpet hemocyanin Lysophospholipid acyltransferases 1-Acyl-2-lyso-sn-glycero-3phosphocholine 1-Acyl-2-lyso-sn-glycero-3phosphoethanolamine Mitogen-activated protein kinases

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NGF PBS PBS-T PCR PED6

PFA PL PLA2 PtdCho

Nerve growth factor Phosphate buffer saline PBS containing 0.05 % Tween-20 Polymerase chain reaction N-((6-(2,4-Dinitrophenyl) amino)hexanoyl)-2-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-pentanoyl)-sn-glycero3-phosphoethanolamine Paraformaldehyde Phospholipid Phospholipase A2 Phosphatidylcholine

Introduction Mammalian cells express a superfamily of phospholipases, which specifically deacylate the sn-2 position of various glycerophospholipids. These phospholipases A2 (PLA2) include calcium-dependent, calcium-independent, and secretory forms, all of which have been detected in nervous tissues [1]. The secretory isoforms (sPLA2) have low molecular weights (13–18 kDa) and variable numbers of disulfide bridges and may function in intra- or extracellular environments [2]. All mammalian brain regions express an sPLA2 belonging to group IIA (GIIA) [3], and this isoform appears to be responsible for most PLA2 activities detected in rat brain particulate fractions [4]. There is evidence that GIIA is involved in neurodegeneration [5–7], but it may also be involved in normal functions, such as neurotransmission and neuronal differentiation [8–11]. GIIA is localized in cytosol and mitochondria in undifferentiated PC12 cells [12]. Treatment of these cells with NGF causes a time-dependent accumulation of the enzyme in the plasma membrane, neurite extensions, and neuritic tips [9], which suggest the participation of endogenous GIIA in neuritogesis by a mechanism that does not require its release in the extracellular medium. This was demonstrated by confocal immunofluorescence analysis using a monoclonal antirat GIIA antibody and by the addition to the culture medium of a cell-impermeable sPLA2 inhibitor which did not affect neuritogenesis induced by NGF [9]. Although other sPLA2 isoforms are able to stimulate the differentiation of PC12 cells, different mechanisms appear to be involved. Indeed, adding various sPLA2 of different origin to culture medium of PC12 cell causes neurite growth, much like that observed when the cells are treated with a neurotrophin [13–15]. Recombinant group X sPLA2 (GX) is the exogenous sPLA2 most active at inducing neuritogenesis in PC12 cells [16]. In fact, GX enzyme activity is required for the induction of neurite outgrowth, and one of its

products, 1-acyl-2-lyso-sn-glycero-3-phosphocholine (lysoPtdCho), is also active by itself [16]. Thus, GIIA and GX may both participate in the mechanisms involved in neuritogenesis, but it is the former one that appears to be linked to NGF-induced neurite outgrowth through its intracellular migration along growing neurites. Indeed, an extracellular role for GX in neuritogenesis is supported by the observation that GX is secreted by PC12 cells as pro-enzyme, which is converted to mature form only after secretion [17]. NGF-induces the differentiation of PC12 cells and the accumulation of endogenous GIIA in plasma membrane, growth cones, and neurite tips [9]. The current research was designed to clarify the metabolic and positional regulations of this enzyme during NGF-induced neuronal differentiation. To accomplish this, we employed a newly prepared antiGIIA antibody (Ab) for Western blot and immunofluorescence analyses, after it was found the Ab did not cross-react with other sPLA2 isoforms. In addition, real-time polymerase chain reaction (PCR) was used to determine the effects of NGF on the expressions of GIIA and GX, while immunofluorescence microscopy (IMF) was used to localize both the GIIA and GX isoforms. Finally, we analyzed the subcellular localization of the GIIA-myc fusion protein during NGF-induced differentiation of PC12 cells using an anti-myc antibody.

Experimental Procedures Cell Culture Rat pheochromocytoma PC12 cells were cultured in RPMI 1640 (Euro Clone) supplemented with 5 % (v/v) fetal bovine serum, 10 % (v/v) horse serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified atmosphere of 95 % air and 5 % CO2. NGF (100 ng/mL) (Invitrogen, Carlsbad, CA, USA) was added to the cells at the beginning and after 48 h of culturing on poly(L-lysine)-coated cover glasses (24 mm dia) in multi-well plates (six wells, 6×105 cells/well), in order to stimulate differentiation. Real-Time PCR Real-time PCR was used to quantify mRNA for GIIA and GX in control and NGF-treated (72 h) PC12 cells. Eukaryotic 18S rRNA was used as an endogenous control gene. Pre-designed TaqMan® Gene expression assays were purchased from Applied Biosystems (primer probe set IDs: GIIA Rn01424865_g1; GX Rn00668379_g1; 18S Hs99999901_s1), and a 7300 Real-Time PCR System (Applied Biosystems) was used according to the manufacturer’s instructions. Threshold cycle (Ct) data for target and control genes were used to calculate ΔCt values [ΔCt=Ct (target gene)−–Ct (18 s RNA)]. Then, ΔΔCt values were calculated [ΔΔCt=ΔCt (treated cells)−ΔCt (untreated cells)].

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The fold change was calculated as 2− ΔΔCt. All treatments were performed in duplicate, each one analyzed in triplicate. Cloning Recombinant Rat GIIA sPLA2 Total RNA from rat liver was reverse-transcribed using the SuperScript III kit (Invitrogen) and oligodT as primers. The GIIA PLA2, containing the full-length coding sequence including the prope pt ide seq ue nc e w a s PC R - a m p l i f i e d u s i n g t h e DyNAzymeTaq polymerase (Finnzymes, Vantaa, Finland) and primers IIAfor-EcoRI (5′-ATGAATTCGCATGAAGGT CCTCCTGTTGC-3′) and IIArev-XhoI (5′-ATCTCGAGTGCAACTGGGCGTCTTCCCTTTG-3′). The cycling parameters were: 94 °C for 2 min, then 35 cycles of 94 °C for 1 min, 61 °C for 1 min, and 72 °C for 30 s, then 72 °C for 2 min. The fragment obtained from the amplification reaction was cloned into the pcDNA3.1-myc-His vector (Invitrogen, Carlsbad, CA, USA), in order to obtain a C-terminal myctagged GIIA protein. Both strands of the cloned cDNA were sequenced to exclude the presence of PCR-induced mutations. Expression of GIIA-myc by Transient Transfection The pcDNA GIIA-myc recombinant vector was transfected into PC12 cells using LipofectamineTm 2000 in Dulbecco’s modified Eagle’s medium (DMEM) medium as described [18]. Briefly, pcDNA-GIIA myc recombinant vector (3 μg) was mixed with 10 μL LipofectamineTm 2000 (Invitrogen, Carlsbad, CA, USA) in 500 μL of DMEM (Euro Clone) and added into sub-confluent PC12 cells in 800 μL of DMEM in six-well plates. The cells were incubated with the Lipofectamine– plasmid suspension for 5 h in a H2O-saturated 5 % CO2 atmosphere at 37 °C, and then the medium was replaced with fresh RPMI 1640 medium containing 10 % (v/v) horse serum and 5 % (v/v) fetal bovine serum. After 24 or 48 h, the cells were processed for enzymatic assay or Western blotting analysis following 24 or 48 h of incubation. In addition, GIIA localization was evaluated using confocal immunocytochemistry and the anti-myc antibody as described below. Confocal Immunofluorescence Microscopy PC12 cells were plated, cultured, and treated with NGF, as described above. Confluent cells were washed two times with (PBS) and loaded with 500 nM Mito-Tracker Red CM-XRos for 15 min at 37 °C. Cells were washed two times with PBS and fixed with 3.7 % (w/v) paraformaldehyde (PFA) in PBS for 20 min at room temperature. The cells were rinsed in PBS and permeabilized with 0.1 % (w/v) Triton X-100 in PBS for 10 min at room temperature. Non-specific binding of antibodies or antisera was blocked by prior incubation of fixed cells with 3 % (w/v) bovine serum albumin (BSA) and 1 % (w/v) glycine in PBS (blocking buffer) for 1 h at room temperature. This was followed by incubation at room temperature with rabbit polyclonal antiserum against rat-GIIA (1:100 in blocking buffer for 1 h) or with rabbit polyclonal antiserum against

GX (1:100 in blocking buffer for 1 h) or with a monoclonal antibody against mouse anti-myc (1:1,000 in blocking buffer for 1 h). The cells were washed for 10 min five times in PBS prior to incubation for 1 h at room temperature with the appropriate secondary antibodies conjugated with Alexa 488 diluted 1:500 in blocking buffer. After two washes with PBS and one with dH2O, the cells were dried and mounted with ProLong Antifade medium (MolecularProbes®, Invitrogen, Carlsbad, CA, USA). The fluorescence of Mito-Tracker and Alexa 488 were detected using a Bio-Rad MRC 1024 confocal microscope with an Ar/Kr laser. The fluorescence of MitoTracker and Alexa 488 (MolecularProbes®, Invitrogen, Carlsbad, CA, USA) were detected with excitation wavelengths of 568 and 488, respectively. Images were elaborated on a SGI Octane work station (SGI, Mountain View, CA, USA).

Measurements of Phospholipase A2 Activity (a) Assay with Radiolabelled Substrate: PLA2 activity in cell lysates were assayed by measuring the amounts of radiolabeled oleic acid released from [3H]oleate-labeled Escherichia coli membranes, as previously described [9, 19]. Briefly, PC12 cells were washed with PBS two times, harvested, and pelleted by centrifugation for 10 min at 800×g. The resultant pellet was suspended in PBS, and the cells were disrupted by sonication (4 cycles for 45 s, with 15-s intervals in between). Unbroken cells and cellular debris were removed by further centrifugation, and the subsequent supernatants were used for enzyme assays. Protein contents were determined using bovine serum albumin as standard [20]. Aliquots of cell lysates (30 μg protein) were taken for PLA2 assays. They were incubated with [3H]oleate-labeled E. coli (30 nCi per sample) for 1 h at 37 °C in a total volume of 300 μL containing 125 mM Tris–HCl, pH 7.4, 2 mM CaCl2, and 1 mg/mL BSA (fatty acid free). The reactions were stopped by the addition of 150 μL of 2 N HCl and 150 μL of BSA (20 mg/mL). After 20 min at 4 °C, labeled bacteria were pelleted for 5 min at 10,000×g. The radioactivity of the supernatants, which contained the free [3H]oleic acid bound to BSA, was determined by liquid scintillation counting. The PLA2 activity in each sample was calculated by subtracting the radioactivity of an enzyme-free blank. This method has good selectivity, because it uses a natural substrate with the radiolabeled fatty acid mainly in phosphatidylethanolamine which is the preferred substrate for GIIA [21]. This assured detectability of even low levels of GIIA activities when the enzyme is bound to a fusion protein. (b) Assay with Fluorogenic Substrate: PLA2 activity in cell lysates was assayed using the fluorogenic substrate N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-

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2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine (PED6, MolecularProbes®, Invitrogen, Carlsbad, CA, USA). PED6 (1 μM), Tris–HCl (50 mM, pH7.4), NaCl (100 mM), CaCl2 (1 mM), Triton X-100 (0.04 mM), and 15 μg of protein lysates in a total volume of 0.2 mL were incubated at 37 °C in 96-well microplates. Fluorescence intensity was measured for 25 min at Ex/Em=485/510 nm. As previously demonstrated, the increase in fluorescence was linear for at least 20 min [22]. The initial rate of substrate hydrolysis was determined after subtraction of T0 fluorescence observed in the absence of the enzyme. PLA2 specific activity was calculated by comparison of the experimental values with the fluorescence of known concentrations of the PLA2 product Bodipy-FL5 (Molecular Probes®, Invitrogen, Carlsbad, CA, USA) determined under identical conditions. The fluorogenic substrate is less well selected by the enzyme, which translates to lower specific activities of the enzyme. However, this method detects sPLA2 activities in the lysate preparations, and it has higher throughput and is less expensive. When indicated, 10 μM Group IIA sPLA2 inhibitor I (cyclic(2-naphthylAla-Leu-Ser-2-naphthylAla-Arg) trifluoroacetate salt (GIIA-Inh, Calbiochem, Darmstadt, Germany) was added to the incubation system. At this concentration, no inhibition of cPLA2 or iPLA2 was observed or other sPLA2 isoforms were observed [22, 23]. Western Blot Analysis Cell lysates were solubilized by boiling for 5 min in Laemmli sample buffer. Proteins (50 ng protein/ lane in Fig. 1; 40 μg cell lysate/lane in Figs. 2 and 5) were resolved using 15 % (w/v) polyacrylamide sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membrane at 100 V for 1 h and 30 min, blocked with 5 % (w/v) dried milk PBS containing 0.05 % (w/v) Tween-20 and incubated overnight at 4 °C with an anti-rat GIIA antibody (see below) diluted

Fig. 1 The newly prepared antibody specifically recognizes GIIA protein. Newly prepared anti-rat GIIA antibody was probed against sPLA2 protein isoforms GIIA, GV, and GX by Western blotting. Lane 1, 50 ng rat GIIA (source I); Lane 2, empty; Lane 3, 50 ng mouse GIIA; Lane 4, 50 ng mouse GV; Lane 5, 50 ng mouse GX; Lane 6, 25 ng rat GIIA (source II); Lane 7, 50 ng rat GV

Fig. 2 NGF stimulated GIIA expression in PC12 Cells. PC12 cells were cultured and treated with 100 ng/mL of NGF for 72 h, as described above. a Western blot analyses of the GIIA levels (40 μg of protein/lane). Lane 1, Control and Lane 2, NGF-treated PC12 cells (100 ng/mL NGF). b Relative density GIIA in control PC12 cells lysate/β-tubulin (light gray bar) and NGF-treated PC12/βtubulin (gray bar). c Relative expressions of GIIA mRNA contents in PC12 cells are shown in the absence (open bar) or presence of NGF (100 ng/mL) for 72 h (filled bar). The results are expressed as mean±SEM values for two different experiments in triplicate. a = significantly different from untreated control

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1:1,000, or anti-myc antibody diluted 1:5,000 in blocking solution. After five washes with PBS containing 0.05 % (w/v) Tween-20, the membranes were incubated with the secondary goat anti-mouse antibody conjugated with peroxidase diluted 1:5,000 in blocking solution. After five washes with PBS containing 0.05 % (w/v) Tween-20, immune reactions were revealed using ECL kit (Thermo Scientific, Rockford, IL, USA). Images were acquired using the VersaDoc Imaging System, and signals were analyzed using Quantity One software (Bio-Rad, Milan, Italy). Preparation of Anti-rat GIIA Antibody Anti-rat GIIA antibody was prepared by a procedure similar to that previously reported for obtaining antisera against other secretory PLA2 groups [24]. The rabbits used in this study were handled in accordance with all local and state regulations for research with animals. Peptide ARNKKSYSLKYQFYPNKFC, corresponding to amino acids 120-139 of rat GIIA protein, was synthesized by Genscript (Piscataway, NJ, USA.) and conjugated to KLH carrier protein. This peptide did not exhibit any sequence identity with other sPLA2 groups. Rabbits were immunized at day 0 and boosted again at the days 14 and 21 with the conjugated peptide. Immunized serum was tested by ELISA before bleeding the rabbits, and the serum obtained was preserved with 0.1 % (w/v) sodium azide. Affinity chromatography using a Pierce AminoLink Plus Immobilization Kit (Thermo Scientific, Rockford, IL, USA) was used to purify the antibody according to the manufacturer’s instructions. A rabbit polyclonal anti-GX antibody was kindly provided by Dr. Miriam Kolko [24] whereas rat GIIA proteins were provided by (I) Prof. Henk van den Bosch [25] and (II) Prof. Gerard Lambeau. Rat GVand mouse GIIA, GX, and GV proteins were also kindly provided by G. Lambeau [26]. These proteins were probed by Western blot analysis using the newly prepared anti-rat GIIA antibody.

Lambeau (source II). The Ab did not recognize either GV or GX, which assured that the results obtained with that Ab were specific for the GIIA protein. This antibody was then used for Western blot analysis of NGF-treated cells. As shown in Fig. 2a–b, a 143 % increase was observed in GIIA in PC12 cells at 72 h of exposure. Consistent with the increase in GIIA content, there was almost a doubling of GIIA mRNA (Fig. 2c). In contrast, GX mRNA was not detected in control or NGF-treated cells. NGF was shown to increase the activity of PLA2 in PC12 cells [9], but no information was provided on the specific isoenzyme that it stimulated. Here, a selective inhibitor of GIIA activity was used to quantitate the involvement of this isoform in the stimulated PLA2 activity (Fig. 3). PC12 cells were exposed to 100 ng/mL of NGF for 3 or 6 days, and then PLA2 activity was assayed in cell lysates in the absence or presence of Inh 1 (10 μM), a selective inhibitor of GIIA [22, 23]. NGF increased PLA2 activities, with respect to the corresponding controls, by 26 or 49 % at 3 or 6 days, respectively. It appeared that GIIA provided most of the PLA2 activity in control cells, since Inh 1 reduced PLA2 activity in untreated cells by about 70 % at 3 and 6 days. It also appeared that GIIA was responsible for the NGF-induced activation of PLA2, since Inh 1 eliminated the growth factor-stimulated increases in enzyme activities observed at 3 or 6 days. The NGF-induced differentiation of PC12 cells into neuron-like cells involves a number of metabolic alterations, including the transport of membrane proteins to surface sites and the reorganization of selected proteins at growing

Statistical Analyses The results are expressed as means± SEM. The number of samples and replications are indicated in the figure legends. One-way ANOVA was used for statistical analyses, followed by Sheffe’s multiple comparison test. p