BSA (essentially fatty acid-free), BF3/MeOH, CDP-choline, DMSO for cell culture, EDTA, SDS, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and PMSF were purchased from Sigma (St. Louis, MO, USA). Solvents were from Lab-Scan (Ireland). Cell culture media, Fetal Calf Serum (FCS), Trypan Blue, and reagents for cell cultures were from Gibco (Invitrogen Corporation, Carlsbad, USA). Palmitoyl-CoA lithium salt, Silica gel 60 and cellulose TLC plates without fluorescent indicator were from Merck (Darmstadt, Germany) and solvents were from Lab-Scan (Ireland). CDP-[methyl-14C]choline (55 mCi/mmol) was supplied by American Radiolabelled Chemicals Inc.; [Palmitoyl-1-14C]-CoA (60 mCi/mmol, NEC-555) was supplied from Du Pont (Du Pont NEN Products, Boston, MA). C12-NBD- PtdCho, C12-NBD-FA, lyso-PtdCho and standard lipids for MS were purchased from Avanti Polar Lipids (Pelham, AL, USA). Standard methyl esters of free fatty acids for GC–MS were from Sigma (St. Louis, MO, USA). Molecular mass standards for SDS-PAGE were from Fermentas Life Sciences (Germany). Bis-acrylamide, Coomassie Brilliant Blue R-250 and Immuno-Blot PVDF membrane were from Bio-Rad (Life Science Research, Segrate Italy). Monoclonal mouse anti-1-Cys Peroxiredoxin was from Chemicon (Eagle Close, UK). Horseradish peroxidase-conjugated rabbit anti-mouse IgG was from Pierce Biotechnology (Rockford, USA). Enhanced chemiluminescence kit from Amersham Pharmacia Biotech, (UK) and X-ray film was from Kodak (France).
Human lung adenocarcinoma A549 cells (ATCC-CCL 185 Manassas, VA) were routinely grown at 37 °C in a complete medium consisting of Hams F12 K (Gibco) supplemented with 10 % w/v heat-inactivated FCS, antimycotic-antibiotic containing penicillin, streptomycin and amphotericin, 200 mΜ glutamine and 14.2 mM Na2CO3, pH 7.4. The cells were grown at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2, until they reached 85 % confluence, (almost 48 h), on BioFlex Collagen type I plates carrying a flexible- hydrophilic growth surface. Afterwards, the complete medium was replaced with serum-free medium to minimize the effect of residual growth factors . Then, the cells were subjected to a static mechanical stretch for 1 h with a Flexcell 5,000 cell stretching device (Dunn Labotechnik Europe), achieving an even cell deformation of 4 %. Unstretched cells were used as control.
After stretching, the cells were washed twice with ice-cold phosphate buffered saline (PBS), pH 7.4, and were then re-suspended in a buffer solution, pH 7.4, containing 20 mM Tris-HCI, 50 mM NaCl, 1 mM EDTA and 1 mM PMSF, or otherwise indicated. Homogenization was performed by sonication for 3 × 20 s with intervals between, at 30 W and the homogenate was aliquoted and stored at −80 °C until the time of analysis. The cell supernatants were collected and tested for possible release of LDH and or phospholipids. Release of lactate dehydrogenase (LDH) into the culture media, in the case of cell damage, was measured with an Olympus analyzer (Olympus AU400). Protein determination was performed according to the method of Bradford .
Phospholipid Analysis and Quantitation
Aliquots from the cell homogenates or their supernatants were extracted by the method of Bligh-Dyer . The chloroform phase was dried under a stream of N2 and was used for the quantitation of total phospholipids after digestion with 70 % (v/v) HClO4, according to the method of Bartlett . For the determination of individual phospholipid classes, the total lipid extract was analyzed by TLC using chloroform/methanol/water (65:35:7, v/v/v) as solvent system. The plate was visualized under a UV lamp after spraying with 1 mM 6-p-toluidine-2-naphthalene sulfonic acid solution (TNS) in 50 mM Tris–HCl, pH 7.4. The regions corresponding to the R
s of authentic phospholipids were scraped off the TLC plate and quantified according to the method of Bartlett . The recovery of the sum of individual lipids was 97.5 ± 4.5 %, considering the lipid phosphorus determined in the initial lipid extract as 100 %.
The DP-PtdCho content as well as the DP-PtdCho/C16-CerPCho ratio was determined by a targeted lipidomics approach, using an ESI-LTQ-ORBITRAP XL unit (Thermo Fisher Scientific) at the full scan positive mode. The ESI LTQ ORBITRAP XL unit was operated with a spray voltage of 3.4 kV, while the sheath gas flow rate and auxiliary gas flow rate were adjusted to 30 and 8 arbitrary units, respectively. The capillary voltage and the tube lens voltage were set to 40 and 110 V, respectively. The mass resolution was set to 60,000, while the scan ranged from m/z: 150 up to m/z: 1,200. The MS parameters were optimized for each individual phospholipid class. Each sample was analyzed three times and the results are expressed as the mean values ± SD. The MS data were analyzed by the Xcalibur 2.1.0 software. The determination of intact phospholipids was assessed by integrating the area of a selected exact molecular ion, using the extracted ion chromatogram mode. DP-PtdCho was quantified by using 1-heptadecanoyl-2-(9Z-tetradecenoyl)-sn-glycero-3-phosphocholine (C17-PtdCho) (Avanti Polar Lipids, USA) as internal standard, while authentic DP-PtdCho (Avanti Polar Lipids, USA) was used as an external standard. From the molecular species of CerPCho we quantified its C16:0 analog, using N-palmitoyl-d-erythro-sphingosylphosphorylcholine (Avanti Polar Lipids, USA) as external standard. The mass tolerance for all the phospholipids was <5 ppm. Chromatographic separation was performed with a Hypersil Gold column (Thermo Fisher Scientific), 5 μm particle size and 150 × 2.1 mm column, using a linear gradient elution from A–B: (35:65, v/v) to B: 100 %, whereas A: 1 % triethylamine (TEA), 0.1 % formic acid in water and 10 % of mobile phase B (v/v) and B: 1 % TEA, 0.1 % formic acid in methanol and 40 % acetonitrile. Injection volume: 20 μL, Column oven temperature: 27 °C. The flow rate was adjusted at 200 μL/min.
Site-Specific Analysis of PtdCho
In certain experiments the PtdCho fraction was isolated by TLC and subjected to fatty acyl chain analysis and identification of the positional distribution of palmitate: The esterified fatty acyl moieties of the PtdCho class were analyzed in the form of fatty acid methyl esters FAME, by gas chromatography coupled with mass spectrometry, (GC–MS) (Shimadzu Q5000) with electron impact (EI) ionization in the full scan mode, using a DB-5 column, after transesterification with BF3/MeOH . The injection temperature was set to 240 °C and Helium was used as carrier gas. The total running time of analysis was 45 min. Total FAME pertaining to both sn-1 and sn-2 positions of the glyceryl backbone of the PtdCho fraction were obtained after direct transesterification of the PtdCho fraction. For the analysis of the acyl-groups esterified at the sn-2 position alone, PtdCho was pre-treated with pancreatic phospholipase A2, the liberated fatty acids were first purified by TLC and then transesterified as previously described. Palmitate identification was performed by comparison with established mass spectra (for palmitate methyl ester: m/z = 270). All the other fragments reflected rearrangements of methyl esters fragments [22, 23]. The relative percentage of palmitate was calculated from the integrated area of the peak corresponding to palmitate versus the total chromatogram area after subtraction of the solvent peak. The data were expressed as percent difference from the control. Three individual cell cultures were prepared for each condition (stretch and control), while each sample was tested twice.
CPT activity was measured in four individual cultures for every condition, while each sample was tested twice, as previously described . The reaction mixture contained 100 mM Tris–HCl, 0.5 mM EDTA, 10 mM MgCl2, 20 μg/mL BSA, pH 8.5, 200 μM of purified 1,2-dipalmitoylglycerol and 200 μM CDP-[methyl-14C]choline (0.1 μCi). The reaction started with the addition of 100 μg protein in a final volume of 0.5 mL, it was carried out at 37 °C for 15 min and was stopped with 80 μL acetic acid, 10 % (v/v). The lipid products were extracted and separated by TLC using chloroform/methanol/water (65:35:7, v/v/v). The area corresponding to the R
of authentic PtdCho was scraped off the plate for radioactivity measurements.
Prior to CPT assay the substrates were purified as follows: Commercial 1,2-diacylglycerol was analyzed by TLC to examine whether the inactive 1,3-diacylglycerol isomeric form was present. Analysis was run on silica gel G60 (Merck, Darmstadt, Germany) pre-coated TLC plates using chloroform/methanol/acetic acid (95:5:1, v/v/v) as solvent system prior to the experiment . The two isomers were visualized after spraying the plate with FeSO4/H2SO4 and charring. The percentage of each isomer was determined with a Vilber-Lourmat image analyzer with CNIH Bio-1D Ver. 97 software (France, Marne-La Valée). The substrate was used in the assay in its initial composition, but the specific activity of CPT was corrected on the basis of the actual concentration in 1,2-diacylglycerol. CDP-[methyl-14C]choline was analyzed by TLC on cellulose-precoated plates, (Avicell, Alltech), after two consecutive developments to the same direction, up to the top of the plate, with the following solvent systems: a) n-butanol/acetic acid/water (50:20:30, v/v/v) and b) 0.02 N acetic acid in 60 % v/v ethanol, according to the technical data sheet. The spots were visualized under UV lamp after spraying with TNS solution. All the radioactivity was recovered at the R
of authentic CDP-choline (~0.35), indicating that the material was pure. The radioactivity was measured by a beta Liquid Scintillation Counter (Tri-Carb 2100 TR, Packard Instrument Company, Meriden, USA).
The detection of aiPLA2 was performed by western blotting: at the end of each experiment the cells were washed with ice-cold phosphate buffered saline (PBS) and harvested by scrapping directly into 2 × sample buffer (50 mM Tris–HCl, 1 % (w/v) SDS, 10 % (v/v) glycerol, 2 % (v/v) β-mercaptoethanol and 0.01 % (w/v) bromophenol blue, pH 6.8, as described by Kim et al. . The cell extracts were aliquoted, boiled for 4 min and stored at −80 °C until use. The protein extracts were separated by 12.5 % SDS-PAGE (8 μg protein/well)  using Hela cells total protein extract as a positive control. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes by a semi-dry transfer apparatus using Towbin buffer (25 mM Trizma, 192 mM glycine, 1.3 mM SDS), at 75 V for 90 min. Non specific binding sites were blocked with 5 % (w/v) dried milk in TBST solution (20 mM Trizma, 1.4 M NaCl, 0.1 % v/v Tween-20) for 3 h at room temperature. The blot was then incubated overnight with a mouse anti-1-cys peroxiredoxin monoclonal antibody (Chemicon International, Germany), clone: 8H11, diluted 1:500 in TBS (20 mM Trizma, 1.4 M NaCl) buffer, pH 7.4, at 4 °C. After washing thee times with TBST the membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (HP-labelled) in 5 % w/v blocking solution (1:10,000), washed twice with TBST and twice with TBS, each one for 5 min. Immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer’s instructions. Blots were also probed with anti-β-actin antibody (Cell Signalling) to check for equal loading. Densitometric analysis was performed after scanning by using the Image J Launcher Software (Ver. 1.36, USA). For each experimental condition, four individual cultures were tested.
Furthermore, aiPLA2 activity was determined fluorometrically according to a method developed in our laboratory, with modifications in the incubation mixture [28, 29]. In particular, we used as substrate the fluorescent analogue of phosphatidylcholine C12-NBD-PtdCho at concentrations greater than the critical micellar one (CMC). Under these conditions, due to the formation of micelles, the fluorescence of C12-NBD-PtdCho was quenched and kept at low levels (baseline). In the presence of PLA2, the liberated fluorescent fatty acid (C12-NBD-FA) released in the aqueous environment causes a linear fluorescence increase, depending on the quantity of C12-NBD-FA released. PLA2 activity is determined from the slope of the curve representing the increase of fluorescence intensity over time. The incubation buffer contained 40 mM sodium acetate, 5 mM EDTA, pH 4.0, and 5 nM C12-NBD-PtdCho as substrate. Under acidic pH and in the absence of Ca2+ the contribution of cPLA2, and sPLA2 to the measured activity is kept to a minimum level. Excitation and emission wavelengths were adjusted to 475 and 535 nm, respectively. The reaction started with the addition of 5 μg protein from the cell homogenate and took place for 4 h at 37 °C. At indicated time intervals, approximately every 20 min, aliquots were subjected to HPLC analysis to separate the produced fatty acid from the relevant substrate .
In certain experiments, (three individual cell cultures for each condition), the cell homogenates were pre-incubated for 30 min in the dark with 60 μΜ MJ33, an inhibitor of PLA2, for the characterization of the enzymatic activity. Under our experimental conditions (absence of Ca2+ and acidic pH), the contribution of sPLA2 or cPLA2 to the measured activity was minimal.
LPCAT activity was determined radiometrically in four individual cultures for every condition, each measured twice, according to Wykle et al. . First, the optimal conditions for the assay were determined: The activity increased proportionally with time up to 7 min, reaching a plateau afterwards, for both stretched and control cells. Thus, 5 min were selected as incubation time. Maximal activity was obtained at 20 μg protein, which was applied in the assay. The incubation mixture (1 mL) contained 200 μΜ lyso-PtdCho, 25 μΜ palmitoyl-CoA, 10 μΜ (60 mCi/mmol) [1-14C]palmitoyl-CoA and 100 μM Tris-HCI buffer, pH 7.4. The reaction started with the addition of 20 μg protein at 37 °C and was terminated after 5 min by extraction of lipids with 1 mL methanol and 1 mL chloroform. The products were separated by TLC, using chloroform/methanol/acetic acid/water (50:25:8:4, v/v/v/v) as solvent system. The PtdCho fraction was scraped off the plate and the radioactivity was measured by a beta scintillation counter.
Total RNA Isolation and Real-Time PCR
After stretching, the medium was immediately discarded, the A549 stretched and unstretched (control) cells were washed once with ice-cold phosphate buffered saline (PBS), and then cell lysis buffer solution, provided by the NucleoSpin RNA II kit (Macherey–Nagel, GmbH and Co. KG, Germany), was added. Total RNA was isolated according to the manufacturer’s recommended protocol. RNA integrity and purity was checked electrophoretically and verified with the criterion of an OD260/OD280 absorption ratio >1.8.
Real-Time PCR was performed using the iScript One-Step RT-PCR kit with SYBR green (Bio-Rad Laboratories, Hercules, CA), using forward and reverse primers from Qiagen (USA) for CHPT1, an isozyme of CPT expressed in the lung, PRDX6, LPCAT1, and GAPDH human genes, with the last used as reference housekeeping gene. The expected sizes of the amplicons were 86, 139, 103 and 119 bp, respectively.
Total RNA (10 ng) in a 25-μL total volume, was first incubated at 50 °C for 10 min to synthesize cDNA, heated at 95 °C for 5 min to inactivate the reverse transcriptase, and then subjected to 35 thermal cycles (94 °C for 40 s, 60 °C for 40 s, and 72 °C for 1 min) of PCR amplification and 40 cycles from 55 to 95 °C (1 °C increase/cycle) for melting curve analysis using an MJ mini thermal cycler (Bio-Rad, Hercules, CA). Four separate RNA isolations were analyzed in duplicates for each experimental condition.
Occasionally, in order to verify the RT-PCR data, the reaction products were electrophoretically analyzed on a 2 % (w/v) agarose gel with ethidium bromide staining and visualized with a UV transilluminator (model LMS-26E, UVP CA, USA). Relative quantitation of qPCR data was carried out according to the method of Pfaffl , using GAPDH as an internal reference. Quantitative RT-PCR results were calculated as fold-increase in gene mRNA versus fold-increase in GAPDH mRNA.
Each set of experiment was performed using at least three individual cultures. Each sample was measured in duplicate and these two values were averaged. Then, the average values for the three or four different experiments were applied to determine the mean value, the SE and the statistical significance. Results are expressed as means ± SE of the values or the percentage differences between stretched and unstretched (control) cells, unless otherwise stated. The percentage differences were calculated as [(stretched value–unstretched value)/unstretched value] × 100. The control value was set at zero. Statistical analysis was performed by the Mann–Whitney U non-parametric test using the statistical package for social sciences (SPSS ver. 18). The difference was considered statistically significant for a p value <0.05.