Protein J DOI 10.1007/s10930-014-9594-6

Molecular Characterisation of Group IVA (Cytosolic) Phospholipase A2 in Murine Osteoblastic MC3T3-E1 Cells Hans Jo¨rg Leis • Werner Windischhofer

Ó Springer Science+Business Media New York 2014

Abstract Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are nontransformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation. Keywords

cPLA2  Bone  Osteoblast  MC3T3-E1

H. J. Leis (&)  W. Windischhofer Research Unit of Analytical Mass Spectrometry, Cell Biology and Biochemistry of Inborn Errors of Metabolism, University Hospital of Youth and Adolescence Medicine, Medical University of Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria e-mail: [email protected]

Abbreviations Prostaglandin E2 PGE2 ET-1 Endothelin-1 IP Immunoprecipitation MS Mass spectrometry PL Phospholipase cPLA2 Group IVA calcium-dependent cytosolic phospholipase A2 (pla2g4a) AA Arachidonic acid PA Phosphatidic acid COX Cyclooxygenase GAPDH Glyceraldehyde-3-phosphate dehydrogenase SDS Sodium dodecylsulphate PAGE Polyacrylamide gel electrophoresis RT-PCR Reverse transcriptase-polymerase chain reaction

1 Introduction Formation of the powerful osteogenic prostaglandin E2 (PGE2) by osteoblasts can be regarded as a key modulatory event in the paracrine and autocrine regulation of bone cell activity [1]. Its biosynthesis, however, is preceded by release of the precursor fatty acid Arachidonic acid (AA) from phospholipid stores. The main routes of AA liberation may involve phospholipase enzymes such as cytosolic phospholipase A2 (cPLA2) which is believed to be the main effector in many cell system due to its preference for AAcontaining lipids [2]. Alternative routes may be the combined activity of PLD and phosphatidate phosphatase, as well as PLC, both leading to diacylglycerol that might release AA through diacylglycerol lipases [3–5].

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Additionally, secretory PLA2s and group VI calciumindependent PLA2s may contribute to free AA levels [6, 7]. MC3T3-E1 cells are non-transformed osteoblasts derived from C57BL/6J mouse strain genetically deficient of IIA PLA2s and are widely used as an in vitro model of osteoblast function [8, 9]. In these cells there is still no clarity about the main release pathway of AA. An essential role of cPLA2 in PGE2-mediated bone resorption was suggested using a cPLA2 knockout mouse model [10] and AA was shown to inhibit MC3T3-E1 cell differentiation through a cPLA2-dependent pathway [11]. cPLA2 was also shown to be responsible for initiating COX-2-dependent delayed PGE2 generation [12]. On the other hand, PLC and PLD pathways lead to sustained elevated DAG production in these cells and are therefore also suggested to play a key role in AA release [13–17]. However, most of the studies concerning involvement of diverse phospholipases in AA release and subsequent PGE2 formation were relying on the use of more or less specific blockers and indirect methods. In spite of the crucial role of osteoblastic prostgalandin formation information on the occurrence and characterisation of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. In an effort to study the contribution of various pathways for AA release it was therefore necessary to characterise the phospholipases and downstream enzymes at the molecular level. It was the aim of this work to provide evidence for the presence of a main effector phospholipase, cPLA2, at the mRNA and protein levels and to demonstrate its role in ET-1-induced PGE2 generation in osteoblastic MC3T3E1 cells.

(Vienna, Austria). Antibodies against cPLA2 (4-4B-3C and H-12) were purchased from Santa Cruz (Santa Cruz, USA). Peroxidase-conjugated anti-mouse IgG was from Rockland (Gilbertsville, PA, USA). NuPAGE Bis–Tris Mini Gels, NuPAGE buffers and SimplyBlue safe stain were obtained through Invitrogen life technologies (Lofer, Austria). ET-1 was purchased from Sigma (Vienna, Austria). Chymotrypsin sequencing grade was from Promega (Vienna, Austria). OligofectamineTM was from Invitrogen (Vienna, Austria). Culture dishes were from Falcon via Szabo (Vienna, Austria). MC3T3-E1 cells were kindly donated by Dr. Klaushofer (Vienna, Austria)). All other chemicals, solvents and reagents were from Merck (Darmstadt, FRG). 2.2 Cell Culture MC3T3-E1 cells (passage number 10–30) were cultured routinely in a-minimum essential medium (a-MEM) containing 5 % fetal calf serum (FCS), 50 lg mL-1 ascorbate and L-glutamine (0.584 g L-1) in a humidified atmosphere of 5 % CO2 in 80 cm2 flasks (initial plating density 2 9 104 cells cm-2) and transferred to 4 cm2 12-well culture dishes or 100 mm round dishes before experiments. Experiments were carried out at confluency (day 6 of culture). For measurement of PGE2 formation medium was replaced by incubation buffer (D-glucose, 5.5 mM; KCl, 5.3 mM; NaCl, 136.8 mM; HEPES, 20.0 mM, ascorbate, 0.28 mM) and cells were stimulated with ET-1 (50 nM) for 30 min. 2.3 RNA Extraction, RT-PCR and Electrophoresis

2 Materials and Methods 2.1 Materials a-MEM and FCS were obtained from Sera-lab (Vienna, Austria). Trypsin/ethylene diamine tetraacetic acid were purchased from Bo¨hringer (Mannheim, FRG). L-glutamine was from Serva (Vienna, Austria). OneStep RT-PCR kit, QIAshredder, RLT Buffer and RNeasy Mini Kit as well as negative control siRNA and Flexitube siRNAs for cPLA2 (set of four siRNAs) were from QUIAGEN (Vienna, Austria). Specific primers for RT-PCR were from TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany). RNeasyAgarose gels were from BioRad (Vienna, Austria). Complete Mini protease inhibitor was from Roche (Vienna, Austria). Pierce classic IP kit, Thermo ScientificTM PierceTM Classic IP Kit, Pierce BCA protein assay, trypsin, pentafluorobenzyl bromide, N-methy-N-(trimethylsilyl) trifluoroacetamide, silylation grade pyridine, acetonitrile, and O-methoxyamine hydrochloride were from Thermo

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For PCR analysis MC3T3-E1 cells were lysed by addition of 600 lL RLT Buffer (QIAGEN) and harvested with a rubber policeman. The lysate was treated with QIAshredder (QIAGEN) and RNA was extracted according to the manufacturer instructions using RNeasy Mini Kit (QIAGEN). To amplify mouse cPLA2 mRNA, the following set of primers was used: (a) forward: 50 -GTGTCTGGGGC AGTGCCTTT-30 ; reverse: 50 -GTTGAAAATGGCG 0 ATTCGGG-3 and (b) forward: 50 -GGCACAGCTACAT TCCCTGT-3; reverse: 50 -TAAAGGTGACAGGCTGG C-30 . The RT-PCR was carried out using OneStep RT-PCR kit, according to the manufacturer instructions. The conditions of PCR-amplification were as follows: 35 cycles; denaturation step 30 s at 94 °C, 30 s at annealing temperature at 59°, extension step 60 s at 72 °C; followed by a final 10 min elongation at 72 °C. The amplified product was resolved on RNeasy Agarose gels supplemented with 0.1 % (v/v) ethidium bromide and visualised using a UV transilluminator (Herolab). To ensure equal RNA loading, RT-PCR for GAPDH was performed for each experiment.

Molecular Characterisation of Group IVA Phospholipase

2.4 Protein Isolation and Immunopurification Confluent cells were washed twice with chilled PBS (pH 7.4). Cell lysis was performed on ice for 10 min with 100 mL IP lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1 % NP-40, 5 % glycerol, pH 7.4, complete Mini protease inhibitor). Insoluble cell debris was removed by centrifugation at 11,000g (4 °C; 10 min). Protein content of cell lysates was determined using the BCA protein assay according to the manufacturer suggestions. Immunoprecipitation was carried out according to the manufacturer instructions using Thermo ScientificTM PierceTM Classic IP Kit. Two antibodies specific to cPLA2 were used for immunoprecipitation and subsequent Western Blotting. 2.5 SDS Gel Electrophoresis and Western Blotting Immunoprecipitated protein was treated on column with 50 lL diluted electrophoresis sample buffer (NuPAGE LDS 49; 1:1, v/v; 100 mM Tris, 1 % LDS, 5 % glycerol, 0.25 mM EDTA, 0.11 mM Serva Blue G250, 0.88 mM phenol red, pH 8.0) and 5 lL DTT (50 mM in water) and heated to 100 °C for 10 min. For mass spectrometry, alkylation of cysteine residues was performed after cooling by adding 5 lL of iodoacetamide (150 mM in water) and reacting in the dark for 1 h. Samples were applied to NuPAGE Bis–Tris Mini Gels and electrophoresis performed using NuPAGEÒ MES SDS Running Buffer according to the manufacturer instructions. After completion, proteins were transferred to nitrocellulose membranes and probed with the antibodies mentioned above. Detection of immunoreactive bands was performed with peroxidase-conjugated anti-mouse IgG followed by incubation with the SuperSignal West Pico chemiluminescent substrate.

1,40,000; MS2 resolution 17,500; isolation with 4.0 amu; data dependent loop count: top 30 with apex triggering (8–15 s) and dynamic exclusion (10 s) with stepped normalised collision energy (28 ± 20 %). Data were analysed with Proteome Discoverer 1.4 software (Thermo Scientific) performing Sequest search in the Swissprot protein database. 2.7 Measurement of PGE2 Production After stimulation the incubation buffer was removed and PGE2 measured by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) [18]. Briefly, PGE2 was converted to its pentafluorobenzyl estertrimethylsilyl ether-O-methyloxime derivative. Quantitation was carried out by use of tetradeuterated PGE2. A ISQ GC–MS system (Thermo) was used. GC was performed on a 15 m TG-SQC fused silica capillary column (Thermo). The temperature of the splitless Grob injector was kept at 290 °C, initial column temperature was 160 °C for 1 min, followed by an increase of 40°/min to 310 °C. NICI was carried out in the single ion recording mode with methane as a moderating gas. 2.8 siRNA Transfection MC3T3-E1 cells were cultured in 6-well plates in a-MEM containing 5 % fetal calf serum FCS without antibiotics. At 30–50 % confluency cells were transfected with four different cPLA2-specific siRNAs (100 nM) using the lipid transfection reagent oligofectamineTM according to the manufacturer’s instructions. A scrambled siRNA was used as a negative control. cPLA2 silencing was monitored by Western Blotting. At confluency, cells were stimulated with ET-1 as indicated and PGE2 formation measured.

2.6 High-Resolution Mass Spectrometry For identification by mass spectrometry the electrophoresis gel was stained with comassie blue (SimplyBlue safe stain) and corresponding bands (as verified by Western Blot) excised. After destaining and tryptic or chymotryptic digestion, peptides were dissolved in 5 % methanol/water, containing 0.1 % formic acid. 20 lL were injected to the HPLC System (Accela 1250, Thermo Scientific) and chromatographed on a Acclaim PepMap 100 column (C18; ˚ ; Thermo Scientific) using 1.0 mm 9 15 cm; 3 lm; 100 A the following gradient from solvent A (0.1 % formic acid) to solvent B (methanol; 0.1 % formic acid) at a flow rate of 50 lL/min: 5–40 % A (30 min); to 100 % B (45 min); hold for 15 min and return to staring conditions. LC was connected to the heated electrospray source of the Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) and data dependent MS2 was performed: full MS resolution

3 Results 3.1 cPLA2 mRNA Expression cPLA2 mRNA expression was investigated using two different sets of primers for RT-PCR. As can be seen in Fig. 1, both primers yield amplicons at the expected size of 867 and 570 bp, respectively. Sequence analysis of the product confirmed the identity with the expected amplification product. Stimulation with ET-1 up to 1 h did not increase mRNA levels. 3.2 cPLA2 Protein Expression Presence of cPLA2 at the protein level was verified with two different antibodies after Western Blotting, as shown

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- ET-1

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H. J. Leis, W. Windischhofer

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Fig. 1 mRNA expression of cPLA2 in MC3T3-E1 cells. Confluent cells were stimulated without (no) or with ET-1 (50 nM) for the indicated time points (15–30–60 min) using primer 1 (expected product size 867 bp) and primer 2 (expected product size 570 bp). GAPDH was used a control to show equal loading

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Fig. 2 Western Blot of cPLA2 protein in MC3T3-E1 cells. Confluent cells were stimulated without (no) or with ET-1 (50 nM) for the indicated time points (15–30–60–120 min) using different antibodies (H-12 and Ab 4-4B-3C) for Western Blotting. b-Actin was used as a control to show equal loading

in Fig. 2. In both Western Blots a band at approximately 85 kDa was observed that corresponded with the positive control sample (NIH-3T3 cell lysate). Upon stimulation with ET-1 no increase in protein levels was observed up to 2 h. Subcellular fractions (membrane/cytosol) obtained after ultracentrifugation at 100,000g revealed location of the enzyme in the cytosol as can be seen in Fig. 3. The minor band seen in the membrane fraction is, at least in part, due to contamination from the cytosol, as evidenced by reprobing the blot with the cytosolic marker GAPDH. Upon stimulation with ET-1, a slight increase of membrane cPLA2 content was visible. 3.3 Immunoprecipitation of cPLA2 Specificity of immunoprecipitation was greatly enhanced by using two different antibodies for binding and Western Blotting. As seen in Fig. 4a, coomassie stained gels revealed a faint band of cPLA2 which expectedly did not overlap with the antibody, demonstrating a highly purified product. Western Blotting of the immunopurified cPLA2

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Fig. 3 Western Blot of protein in MC3T3-E1 cells after subcellular fractionation. Confluent cells were stimulated without or with ET-1 (50 nM) for 15 min and the cell lysate centrifuged at 100,000g to obtain membrane and cytosolic fractions. Blots were probed for cPLA2, stripped and reprobed for GAPDH

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B Fig. 4 a SDS-PAGE (coomassie stained) and b Western Blot of MC3T3-E1 whole cell protein lysate and immunopurified cPLA2, using cPLA2 antibody H-12 for IP and 4-4B-3C for Western Blotting

additionally indicated enhancement of enzyme concentration by the process, as shown in Fig. 4b. 3.4 High-Resolution Mass Spectrometry of cPLA2 High-resolution MS data analysis after tryptic and chymotryptic digestion of the iodoacetamide alkylated protein band by Proteome Discoverer 1.4 software was

Molecular Characterisation of Group IVA Phospholipase

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Fig. 5 a ET-1-induced formation of PGE2 and b Western Blot in MC3T3-E1 cells after cPLA2 silencing using four different cPLA2specific siRNAs. b-Actin was used as a control to show equal loading during Western Blotting

accomplished using Sequest HT search of the Swissprot protein data bank. 3.5 siRNA Tranfection and PGE2 Formation The effect of cPLA2 silencing on osteoblastic PGE2 formation is shown in Fig. 5. cPLA2 protein expression is largely reduced with all siRNAs tested and ET-1 induced PGE2 formation is attenuated to basal levels.

4 Discussion Stimulating osteoblast-like MC3T3-E1 cells with ET-1 revealed that cPLA2 mRNA is constitutively expressed. Similarly, at the protein level constitutive presence of cPLA2 enzyme was observed. Subcellular fractionation

demonstrated localisation of the enzyme to the cytosol with translocation to the membrane fraction after ET-1 treatment as a consequence of intracellular calcium elevation. Occurrence of cPLA2 in the membrane fraction was at least in part due to cytosolic contamination of the pellet, as evidenced by the cytosolic marker GAPDH. However, relative band intensities suggest that a significant proportion of membrane-bound cPLA2 originates from other processes. Thus, sedimentation of high molecular weight compounds may occur at high-speed centrifugation, or some basal membrane association might occur by inducing translocation by manipulations during protein harvesting. As already stated in the introduction there is substantial evidence that cPLA2 may not be the sole effector lipase to liberate AA for subsequent prostaglandin formation. Our results after gene silencing with specific siRNAs, however, show a reduction of ET-1-induced PGE2 formation to basal levels. Although silencing does not completely abolish cPLA2 protein expression, levels seem to be sufficiently low to impair prostaglandin formation to a high degree. This can be reasoned by the fact that free AA levels are under efficient control and rapidly removed by reacylation into phospholipids by the Lands cycle [19]. As an essential role for cPLA2 in agonist-induced AA liberation might be expected, the contribution of other phospholipases remains to be established. Thus, PLD produces PA, which might serve either as a substrate for phosphatidate phosphatases and downstream enzymes, or for phospholipase A2 enzymes to generate free AA. PA potentiates activation of cPLA2 [20] and also serves as its substrate, indicating a tight interplay between PLD and cPLA2 pathways in osteoblastic AA liberation. cPLA2 is important for bone homeostasis. cPLA2 knockout mice showed reduced severity and incidence in collagen-induced arthritis [21]. cPLA2 activity is essential for osteoclast formation and LPS-induced bone resorption [22] an effect most likely mediated by osteoblast-derived PGE2 because the EP4 is required for bone resorption and osteoclast formation [23]. The immunopurification protocol for cPLA2 used herein was highly specific due to the use of different antibodies for binding and Western blotting. It is noteworthy that the band of the alkylated products shifts towards higher apparent mass values, which can be attributed in part to the increase of protein mass (8 cysteine residues: ?513 Da) and also to the altered charge state and tertiary structure of cPLA2. As a final identification step high-resolution mass spectrometry on an orbitrap mass spectrometer was performed after tryptic and chymotrypric digestion of the immunopurified enzyme. Data analysis revealed mouse cPLA2 as the only high-scored hit with an overall sequence coverage of 31 %. A total of 35 peptides were identified, 25 of them being unique to mouse cPLA2.

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In summary, our findings evidence the presence of cPLA2 in osteoblast-like MC3T3-E1 cells. Constitutive expression at mRNA and protein level was demonstrated. Receptor-related signal transduction evoked by stimulation with ET-1 did not induce cPLA2 expression within 2 h. Osteoblastic cPLA2 is crucially involved in ET-1-induced prostaglandin formation. Immunopurification in combination with high-resolution mass spectrometry was demonstrated to be an effective tool in targeted characterisation of phospholipases and other proteins of interest. Ongoing studies will focus on the characterisation of other target phospholipases (PLD, PLC) and their distinct and/or concerted activity leading to AA release in these cells. As a wider scope this protocol shall be extended to a targeted proteomic approach for the rapid mass spectral characterisation of these proteins and their possible modifications.

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Molecular characterisation of group IVA (cytosolic) phospholipase A2 in murine osteoblastic MC3T3-E1 cells.

Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell ac...
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