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Dipalmitoyl-Phosphatidylcholine Biosynthesis is Induced by Non-Injurious Mechanical Stretch in a Model of Alveolar Type II Cells

Abstract

Dipalmitoylphosphatidylcholine, (DP-PtdCho), the major phospholipid component of lung surfactant is biosynthesized via a de novo pathway, the last step of which is catalyzed by CDP-choline:cholinephosphotransferase (CPT) and two remodeling steps: a deacylation and a reacylation one, catalyzed by an acidic, Ca2+-independent phospholipase A2 (aiPLA2) and a lyso-phosphatidylcholine acyltransferase (LPCAT), respectively. The aim of our study was to investigate whether a low magnitude, non-injurious static mode of mechanical stretch can induce phosphatidylcholine (PtdCho) biosynthesis and its remodeling to DP-PtdCho in the A549 cell-line, a model of alveolar type II cells. The deformation of A549 cells did not cause any release of lactate dehydrogenase, or phospholipids into the cell culture supernatants. An increase in PtdCho levels was observed after 1 h of static stretching, especially among the DP-PtdCho molecular species, as indicated by targeted lipidomics approach and site-directed fatty acyl-chain analysis. Moreover, although sphingomyelin (CerPCho) levels were unaffected, the DP-PtdCho/CerPCho ratio increased. Induction was observed in CPT, LPCAT and aiPLA2 enzymatic activities and gene expression. Finally, incubation of the cells with MJ33 suppressed aiPLA2 activity and DP-PtdCho production. Our data suggest that mild static mechanical stretch can promote the biosynthesis of PtdCho and its remodeling to DP-PtdCho in lung epithelial cells. Thus, low magnitude stretch could contribute to protective mechanisms rather than to injurious ones.

Introduction

Lung structure and functionality is influenced by a wide range of mechanical forces generated during tidal breathing, blood flow etc. [1]. Substantial evidence indicates that in normal breathing, the mechanical signal produced from the inflation and deflation of the alveoli triggers the secretion of lung surfactant from epithelial alveolar type II (ATII) cells ([24], for review, see Ref. [5]).

Lung surfactant is a protein-lipid complex that lines the alveoli and maintains lung integrity by equilibrating the pressure in all the alveoli, through its tensioactive properties. It is composed mainly of lipids, (90 %), among which the major constituent is phosphatidylcholine (PtdCho), in the form of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DP-PtdCho), and specific surfactant proteins, SP-A, SP-B, SP-C, SP-D, among which SP-A and SP-D act as collectins. These components provide host-defense and anti-inflammatory properties to the respiratory system [6, 7]. Lung surfactant deficiency is associated with serious lung disorders, such as acute and infant respiratory distress syndromes (ARDS and IRDS, respectively).

Biosynthesis of pulmonary surfactant constituents takes place in the endoplasmic reticulum of AT-II cells. It is excreted in the form of lamellar bodies into the alveolar space, where it unravels to form a monolayer and spreads over the aqueous hypophase that covers the lung epithelium. PtdCho is formed via the de novo pathway, the last step of which involves the addition of a phosphocholine group to diacylglycerol by a CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity, (CPT), (EC 2.7.8.2) (Fig. 1a). Enrichment of the sn-2 position of PtdCho with palmitic acid occurs though two consecutive remodeling reactions (Fig. 1b): (i) A lysosomal-type acidic and Ca2+-independent phospholipase A2 (aiPLA2), (EC 1.11.1.7) [8], catalyzes the cleavage of the sn-2-acyl moiety from PtdCho and the formation of lyso-PtdCho. This isoform has been detected in both the lysosomal fraction and in the lamellar bodies of AT-II cells and exhibits additional Se-independent peroxidase 6 (Prdx-6) activity [9, 10] and (ii) The final step of the remodeling pathway comprises the reacylation of 1-palmitoyl-2-lysophosphatidylcholine (lyso-PtdCho) by palmitoyl-CoA:lyso-phosphatidylcholine acyltransferase activity (LPCAT), (EC 2.3.1.23), which has been detected and characterized in the endoplasmic reticulum of AT-II cells [11]. Thus, co-operativity between the lysosomes and the endoplasmic reticulum for the synthesis of DP-PtdCho by the reacylation pathway has been proposed [10, 12].

Fig. 1
figure 1

Final steps of 1,2-dipalmitoyl-phosphatidylcholine (DP-PtdCho) biosynthesis in AT-II cells. a De novo pathway: The transfer of a phosphocholine group to diacylglycerol is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity, (CPT), (EC 2.7.8.2). b Remodeling pathway: (i) A lysosomal-type acidic and Ca2+-independent phospholipase A2 (aiPLA2), (EC 1.11.1.7) catalyzes the cleavage of the sn-2-acyl-moiety. (ii) The reacylation of 1-palmitoyl-2-lysophosphatidylcholine (lyso-PtdCho) to DP-PtdCho involves palmitoyl-CoA:lyso-phosphatidylcholine acyltransferase activity (LPCAT), (EC 2.3.1.23). R palmitate, R’ unsaturated fatty acyl chain (UFA); DAG 1,2-diacylglycerol

Glucocorticoids, fatty acids, calcium and phorbol esters, among other chemicals, can induce the in vitro production of pulmonary surfactant. In addition to these stimuli, cellular stressors including tidal breathing, osmotic shock and cyclic stretch during mechanical ventilation induce its release [13]. Wirtz and Dobbs [14] were the first to demonstrate that a transient increase in intracellular calcium induced surfactant secretion under stretch. Moreover, the induction of PtdCho metabolism through PLA2 and phospholipase C (PLC) has been studied in a model of traumatic cell injury, induced by stretch using rat astrocytes [15]. Nevertheless, our knowledge on the effect of low magnitude stretching on PtdCho and DP-PtdCho biosynthesis is limited. Furthermore, the enzymatic activities involved in surfactant production under stretch and their expression, have not been studied yet.

The aim of the present work was to investigate the effect of mild, non-injurious static mechanical stretch on PtdCho and DP-PtdCho biosynthesis within alveolar type II cells through the enzymes involved in the last steps of the de novo (Kennedy pathway) and the remodeling (Lands cycle) biosynthetic pathways. In this respect, the human lung adenocarcinoma cell line (A549) was used as a model for type-II cells [16], although they may have altered responses in some circumstances. Alterations in PtdCho levels, activity levels and expression of CPT, aiPLA2 and LPCAT were also investigated under mild stretching conditions. Furthermore, different LC–MS and GC–MS approaches were used to investigate alterations in the profile of PtdCho molecular species induced by stretch.

Materials and Methods

Materials

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).

Cell Cultures

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 [17]. 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 [18].

Phospholipid Analysis and Quantitation

Aliquots from the cell homogenates or their supernatants were extracted by the method of Bligh-Dyer [19]. 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 [20]. 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 f s of authentic phospholipids were scraped off the TLC plate and quantified according to the method of Bartlett [20]. 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 [21]. 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.

Enzymatic Assays

CPT activity was measured in four individual cultures for every condition, while each sample was tested twice, as previously described [24]. 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 f 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 [25]. 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 f 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. [26]. 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) [27] 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 [30].

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. [31]. 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 [32], using GAPDH as an internal reference. Quantitative RT-PCR results were calculated as fold-increase in gene mRNA versus fold-increase in GAPDH mRNA.

Statistical Analysis

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.

Results

Cell Viability

The percentage of the cell viability was not significantly altered after stretching, as indicated from the trypan blue exclusion dye (unstretched-control 95.4 ± 2.8 %, vs stretched cells 94.1 ± 3.0 %), or the release of LDH activity in the cell supernatants (27 ± 15 IU/dL, in the unstretched—control samples, vs 24 ± 12 IU/dL, in the stretched cells). Finally, no release of other proteins or phospholipids was detected in the cell supernatants, confirming that our model is non-injurious.

Lipids

Total phospholipids in the control cell homogenates were found to be 6.84 ± 0.35 μg P/mg protein. After 1 h of stretching, the levels increased to 7.52 ± 0.3 μg P/mg protein, (p = 0.048). The major phospholipid classes were: PtdCho, phosphatidylglycerol (PtdGro), phosphatidylethanolamine (PtdEtn), total CerPCho and lyso-PtdCho (data not shown). Detection of intact DP-PtdCho and C16-CerPCho was performed by LC–MS from the characteristic m/z: 734.5658 and 703.5714, corresponding to the molecular ions, with retention times 25.14 and 22.70 min, respectively. Quantitative determination was assessed with the internal standard C17-PtdCho, with m/z: 718.5350 and retention time 20.96 min (Fig. 2). The CerPCho levels did not change under stretch. However, lipid-phosphate determination showed that the total PtdCho/total CerPCho ratio increased after stretch from 3.94 ± 0.05 in the unstretched-control cells to 7.70 ± 0.50, while LC–MS analysis revealed that the molecular species DP-PtdCho/C16-CerPCho ratio increased from 1.90 ± 0.02 to 5.20 ± 0.50, (Table 1). These indicated that the increase in total phosphorus was mainly due to the increase in DP-PtdCho levels.

Fig. 2
figure 2

Representative LC–MS analysis of total lipid extract from A549 control (2A) and stretched (2B) cells. A549 cells were subjected to static mechanical stretch for 1 h. Unstretched cell homogenate was used as control. Total lipids were extracted from the cells and subjected to LC–MS analysis. I Total ion chromatogram, II Extracted ion chromatogram for m/z 734.5658, corresponding to the molecular ion of DP-PtdCho, III Extracted ion chromatogram for m/z 703.5714, corresponding to the molecular ion of CerPCho, IV Extracted ion chromatogram for m/z 718.5350, corresponding to the molecular ion of C17-PtdCho, used as internal standard for the quantitative determination of DP-PtdCho and CerPCho. The magnitude of each peak (absolute areas in arbitrary units) corresponding to the abundance of DP-PtdCho, CerPCho and C17-PtdCho are also provided. DP-PtdCho dipalmitoyl-phosphatidylcholine, CerPCho sphingomyelin, C17-PtdCho 1-heptadecanoyl-2-(9Z-tetradecenoyl)-sn-glycero-3-phosphocholine

Table 1 Effect of stretch on PtdCho/CerPCho and DP-PtdCho/C16-CerPCho ratios in A549 cells

Additional analysis of the PtdCho fraction after stretching with site-specific hydrolysis of the esterified fatty acids gave a characteristic m/z fragment at 270, corresponding to palmitate. It was shown that although after stretching the amount of palmitate groups esterified on both sn-1 and sn-2 positions of PtdCho presented a non-significant tendency to increase, those of the sn-2 position alone significantly increased, by approximately twofold (p < 0.05), (Fig. 3a, b). This signified that the enrichment in palmitate groups occurred mainly at the sn-2 position.

Fig. 3
figure 3

a Effect of static stretch on the positional distribution of palmitate in the PtdCho fraction. A549 cells were subjected to static mechanical stretch for 1 h. Unstretched cell homogenate was used as control. Total lipid extract from A549 cells was analyzed by TLC. The PtdCho fraction was scrapped off the plate and was directly transesterified with BF3-MeOH for the analysis of fatty acids profile esterified on both sn-1 and sn-2 positions. For the analysis of fatty acids esterified at the sn-2 position alone, PtdCho was pre-treated with pancreatic phospholipase A2; the liberated fatty acids were purified by TLC and then transesterified with BF3-MeOH. The produced FAME were analyzed by GC–MS. 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 express the percent difference ±SE from the control and correspond to three individual cultures for each condition, while each sample was tested twice. Asterisk statistical significance (p < 0.05) for the difference between stretched and unstretched/control cells. sn-1, sn-2: positions of the PtdCho glycerol backbone. b Representative mass spectrum of free fatty acids methyl esters derived from the hydrolysis of PtdCho by PLA2. The produced FAME were analyzed by GC–MS, while palmitate methyl ester identification was based on its characteristic m/z fragment at 270

Enzymatic Activities

CPT, which catalyzes the last step of the de novo pathway of PtdCho formation, exhibited an activity of (29.30 ± 3.03) pmol PtdCho/min/mg protein in the control A549 cell homogenate. After stretching, the CPT activity increased significantly, by (47.33 ± 8.89) % over the control, (p = 0.037) (Fig. 4).

Fig. 4
figure 4

Effect of static stretch on CPT and LPCAT:A549 Cells were subjected to static mechanical stretch for 1 h. The enzymatic activities were measured in whole cell homogenates. Unstretched cell homogenates were used as control. The data represent the % mean increases ±SE over control cells corresponding to four individual cell cultures for each condition, whereas each sample was tested twice. Asterisk statistical significance for p < 0.05 and hash for p < 0.01

The aiPLA2 activity was found to be (0.50 ± 0.20) nmol C12-NBD-FA/h/mg protein in the control A549 cell homogenate. Stretching increased the activity to 1.98 ± 0.50 nmol C12-NBD-FA/h/mg protein, (p < 0.01). In the presence of MJ33, an inhibitor of aiPLA2, an approximate 60 % reduction in the specific activity of both stretched and unstretched-control cells was observed (Fig. 5). After stretch, the expression of aiPLA2 at the protein level was elevated by 20.7 % in comparison with the unstretched cells, from 0.29 ± 0.05 to 0.35 ± 0.06 arbitrary units, (p < 0.05), as shown by the representative western blotting of the aiPLA2 isoform band at 26 kDa (Fig. 6a, b).

Fig. 5
figure 5

Effect of MJ33 on aiPLA2 activity: A549 cells were subjected to static mechanical stretch for 1 h in the presence or absence of the inhibitor MJ33. The aiPLA2 activity was measured fluorometrically in whole cell homogenates (for details see the methods section). Unstretched cell homogenates were used as control. The data represent the mean activity ±SE corresponding to three individual cell cultures for each condition, whereas each sample was tested twice. Asterisk statistical significance p < 0.05

Fig. 6
figure 6

Immunoblot analysis of aiPLA2 from A549 cells subjected to static stretch: Whole cell homogenates from control/unstretched and stretched for 1 h A549 cells were analyzed by SDS-PAGE. a Immunoblotting was performed by using anti-Prdx6 antibody. Hela cell homogenates was used as a positive control, while β-actin was used to confirm equal loading. The picture is representative of four different experiments. b The band densities were measured by image analyzer and the aiPLA2 bands were normalized to the band intensity of β-actin

To investigate whether DP-PtdCho formation could be accomplished with the re-acylation of lyso-PtdCho, LPCAT activity was determined in A549 cells. In the control cell homogenates it was found to be (40.00 ± 5.47) pmol DP-PtdCho/min/mg protein. After mechanical stretching, LPCAT activity increased significantly by a percentage of (22.07 ± 9.32) % over control, (p = 0.002) (Fig. 4).

Gene Expression

The qRT-PCR for CHPT1, PRDX6 and LPCAT1 revealed that all three enzymes CPT, aiPLA2/PRDX6 and LPCAT are expressed in control A549 cells. The expression levels of CHPT1 mRNA presented a 2.2 ± 1.0-fold increase after mechanical stretching. Upon 1 h of stretch, A549 epithelial cells responded by a PRDX6 mRNA increase of 1.6 ± 0.7-fold. Moreover, an LPCAT1 mRNA expression increase by 1.5 ± 0.3-fold was observed in stretched A549 cells as compared with the unstretched-control cells (Fig. 7). The genes’ expression was normalized to the housekeeping gene of GAPDH.

Fig. 7
figure 7

Expression of CHPT1, PRDX6 and LPCAT1 transcripts in A549 cells subjected to static stretch. The expression levels of CHPT1, PRDX6 and LPCAT1 mRNA in A549 cells were analyzed by quantitative RT-PCR. The levels of each mRNA were normalized to those of GAPDH housekeeping gene mRNA. The results represent the fold increase compared to control ±SD and are the means of four separate RNA isolations for each condition analyzed by RT-PCR. Asterisk statistical significance, p < 0.05 for the difference between stretched and unstretched/control cells

Discussion

In this work we demonstrated that mild stretch can promote DP-PtdCho biosynthesis in a model of alveolar type II cells. First, we observed a rapid increase in the levels of PtdCho after non-injurious static stretching, accompanied by increase in DP-PtdCho levels, while CerPCho remained unaffected. This finding was confirmed by different experimental approaches. Stretch induced the increase in CPT, aiPLA2 and LPCAT activities as well as in the mRNA expression levels of the above enzymes (CHPT1, PRDX6 and LPCAT1, respectively). These enzymes catalyze the last steps of the de novo and the remodeling biosynthetic pathways of DP-PtdCho formation.

Mechanical stretch models for lung cell studies are usually designed to investigate mechanisms of ventilation-associated lung injury. Thus, a significant mean deformation of the cells is exerted, leading to increased percentage of cell death [3335]. In contrast, the stretch intensity in our model caused a mild cell deformation (4 %), simulating that of lung epithelium during spontaneous inspiration or during mechanical ventilation with low tidal volume (5–6 mL/kg of ideal weight) [36]. Under these conditions, neither membrane permeability, nor cell death were increased, as indicated by the fact that proteins, in particular LDH, and phospholipids were not released into the cell culture supernatant after stretching. This supports the concept that the cells were not injured, although biochemical alterations and signal transduction pathways are induced: In particular, in a previous work we found activation of cytosolic PLA2 (cPLA2), an enzyme that contributes to lung surfactant secretion from AT-II cells, through phosphorylation involving the MEK/ERK and PI3K pathways [37].

Firstly, we demonstrated that the production of PtdCho and in particular DP-PtdCho, which is a major surfactant component, was promoted by mild stretch. This finding is in agreement with the hypothesis that spontaneous breathing or low tidal volume mechanical ventilation trigger surfactant production. The increase in intact PtdCho was assessed by phosphorus determination, while DP-PtdCho levels and the ratio DP-PtdCho/CerPCho were determined by targeted lipidomics approach. Additional information on the enrichment of the sn-2 position of PtdCho with palmitoyl acyl-groups, forming dipalmitoyl-phosphatidylcholine (DP-PtdCho) was provided through site-specific analysis of the esterified fatty acids of PtdCho. With all these different methodological approaches similar results were obtained. Torday and Rehan, utilizing a radioactive choline-incorporation assay, reported that cyclic stretch on rat alveolar type II cells and fibroblast co-cultures resulted in an increase of PtdCho production and proposed a paracrine network that activates DP-PtdCho formation under stretch [38]. Although our results are not contradictory, we found that mild static stretching conditions are adequate for inducing a significant increase in PtdCho and DP-PtdCho biosynthesis, even without the presence of fibroblasts. These alterations took place shortly after the initiation of mechanical stimulation and this is compatible with changes in the fatty acyl- chain profile, which are known to participate into rapid cell responses.

The increase in the PtdCho levels after low magnitude static mechanical stretch for 1 h can be justified by the induction of CPT activity that catalyzes the last step in the de novo biosynthetic pathway. According to our best knowledge, this has not been reported before. The basal levels of the enzymatic activity agree with previous reports regarding A549 cell homogenates [39]. Furthermore, the up-regulation in CHPT1 mRNA, a CPT isoform, identified in the lung, provides strong evidence that CPT is mobilized under mild stretch.

Subsequently, we investigated whether our stretch model could stimulate the remodeling of PtdCho to dipalmitoyl-phosphatidylcholine (DP-PtdCho). Two enzymatic activities are involved in this pathway: (a) aiPLA2, which preferentially cleaves unsaturated fatty acids from the sn-2 position of pre-formed PtdCho and (b) LPCAT that catalyzes the transfer of saturated fatty acyl-groups, in particular palmitate, to lyso-PtdCho [10, 11]. Although cytosolic and secreted forms of PLA2 have been detected in A549 cells [40], the present work demonstrates for the first time the expression of aiPLA2 protein in these cells by immunoblotting experiments using the specific antibody that recognizes the bifunctional enzyme aiPLA2/peroxiredoxin-6 [41]. In consistence with the above, a significant increase in the PRDX6/aiPLA2 mRNA levels was observed. Further evidence for this remodeling step leading to DP-PtdCho formation was provided by inhibition experiments using MJ33, an inhibitor of the enzyme [42], which caused a significant uniform reduction in the activity of both stretched and unstretched-control cells.

The final step of the PtdCho remodeling pathway in AT-II cells, catalyzed by LPCAT, contributes to the formation of DP-PtdCho by 55–75 % by transferring saturated fatty acyl-chains, especially palmitoyl-groups, to the sn-2 position of lyso-PtdCho. LPCAT in AT-II cells has been cloned and characterized. It shows preference for palmitoyl-CoA over oleoyl-CoA and functions predominantly as a lyso-PtdCho acyltransferase [11, 43, 44]. Under our experimental conditions, with 1-palmitoyl-lyso-PtdCho as substrate and palmitoyl-CoA as the acyl-donor, LPCAT activity was increased after stretching. The upregulation of LPCAT was in consistence with the elevated incorporation of palmitate at the sn-2 position of lyso-PtdCho and consequently, to DP-PtdCho formation. Although the induction of this enzymatic activity upon stretch has not been reported before, the specific activity of LPCAT in mice microsomal adenoma type II cells [31] and in control A549 cells [45] are compatible with those found in our control cells. It is known that LPCAT expression increases upon type II cell-differentiation and dramatically decreases when type II cell cultures de-differentiate to type I-like cell phenotypes [11], but this does not seem to be the case in A549 cancer cells.

In conclusion, our results indicate that a mild, non-injurious mechanical signal can cause a rapid increase in total PtdCho levels within A549 cells, parallel to the enrichment of PtdCho fraction with DP-PtdCho molecular species. This is consistent with the increase in CPT, aiPLA2 and LPCAT activities which appears to reflect the de novo expression of the corresponding mRNAs. The physiological significance of these findings is that stretch could also induce the de novo production of surfactant and not only the secretion of pre-formed surfactant.

Ventilator-associated lung injury is an essential cause of poor clinical outcome in mechanically-ventilated patients with and without acute respiratory distress syndrome. The adverse effect of ventilation on lung surfactant resulting in an increase in surface tension and consequently atelectasis has a key role in developing VALI. The ventilator induced surfactant deficiency may be potentially preventable [46, 47]. Therefore, strategies of mechanical ventilation that reduce the incidence and severity of VALI are being sought. In this context, mechanical ventilation with low tidal volume, resulting in low lung deformation, has been proved lung protective. Based on the results of the present study we could speculate that the surfactant production induced by non injurious stretch could contribute to such a protective mechanism.

Abbreviations

CDP-:

Cytidine diphospho-

Prdx6:

1-Cys-peroxiredoxin

FCS:

Fetal calf serum

PtdCho:

Phosphatidylcholine

DP-PtdCho:

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

CerPCho:

Sphingomyelin

LDH:

Lactate dehydrogenase

LPCAT:

Lyso-PtdCho acyltransferase

PBS:

Phosphate-buffered saline

PtdCho:

Phosphatidylcholine

PLA2 :

Phospholipase A2

ai :

Acidic Ca2+-independent

PMSF:

Phenylmethylsulfonyl fluoride

PCR:

Polymerase chain reaction

TLC:

Thin layer chromatography

TNS:

2-(p-toluidinyl)-naphthalene-6-sulfonic acid

C12-NBD-PtdCho:

1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine

C12-NBD-FA:

6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] dodecanoic acid

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Acknowledgments

This research article was funded is part by the “Reinforcement Programme of Human Research Manpower” (PENED) research project, which was co-financed by National and Community Funds (25 % from the Greek Ministry of Development-General Secretariat of Research and Technology and 75 % from the EU). The authors would like to thank the ORBITRAP-LC–MS Unit of the University of Ioannina-Greece for providing access to the facilities. Acknowledgments are due to M. Karagiannopoulos, M.Sc., for technical assistance.

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Pantazi, D., Kitsiouli, E., Karkabounas, A. et al. Dipalmitoyl-Phosphatidylcholine Biosynthesis is Induced by Non-Injurious Mechanical Stretch in a Model of Alveolar Type II Cells. Lipids 48, 827–838 (2013). https://doi.org/10.1007/s11745-013-3800-8

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Keywords

  • Lung surfactant
  • Phosphatidylcholine biosynthesis
  • Mechanical stretch
  • CDP-choline:cholinephosphotransferase
  • Phospholipase A2
  • Lyso-phosphatidylcholine acyltransferase