Activatory Properties of Lysophosphatidic Acid on HumanTHP-1 Cells

F. D_Aquilio,1 M. Procaccini,1 V. Izzi,1 V. Chiurchiu_,1 V. Giambra,1 F. Carotenuto,2

P. Di Nardo,2 and P. M. Baldini1,3

Abstract—Excessive leukocyte proliferation and proinflammatory mediators release represent com-

mon phenomena in several chronic inflammatory diseases. Multiple evidences identify lysophospha-

tidic acid (LPA), a small lipid endowed with pleiotropic activities, as an important modulator of both

proliferation and activation of different cell types involved in several inflammation-associated patho-

logies. However, its possible role on monocyte proinflammatory activation is not fully understood yet.

Aim of the present study was to investigate LPA effects on THP-1 cells in terms of proliferation,

reactive oxygen intermediates (ROI) production and release of arachidonic acid-derived inflammatory

mediators. Actually, LPA significantly increased both DNA synthesis and ROI production as well as

prostaglandin E2 release and the upregulation of LPA3 receptor expression. These findings identified

LPA as both a growth factor and a triggering mediator of proinflammatory response in THP-1 cells.

KEY WORDS: arachidonic acid metabolites; LPA receptors; lysophosphatidic acid; proliferation; reactiveoxygen intermediates.

INTRODUCTION

Lysophosphatidic acid (LPA), a multifunctional bioactive

phospholipid, influences a broad spectrum of biological

responses which are species, tissue and cell specific [1].

LPA exerts many of its activities binding to a family of

G protein-coupled receptors (GPCRs), nowadays termed

LPA1, LPA2 and LPA3, which are able to activate mul-

tiple intracellular signalling pathways [2].

In the immune system, dendritic cells, mast cells and

macrophages are the principal LPA sources [3]. Recent

evidences have shown that LPA is involved in the de-

velopment of normal immunological reactions modulating

immune cell activities and functions [4]. Moreover, LPA

plays an important mitogenic role which couples with its

stimulatory activity on diverse cell types implicated in the

pathogenesis of several human diseases characterized by

chronic inflammation [5, 6].

Excessive cell proliferation and activation are

important processes involved in vascular damage [7].

These processes were initially believed to exclusively

occur in smooth muscle cells; at present, it is known

that they can also occur in inflammatory cells including

monocytes/macrophages and T-lymphocytes [8]. In this

context, the presence of locally released soluble

mediators, to whom cells respond synthesizing and

0360-3997/06/0400-0129/0 # 2006 Springer Science+Business Media, Inc.

Inflammation, Vol. 29, Nos. 4Y6, December 2005 (# 2006)

DOI: 10.1007/s10753-006-9008-9

129

1 Department of Biology, University of Rome BTor Vergata’’, Via

della Ricerca Scientifica, 00133 Rome, Italy.2 Department of Internal Medicine, Laboratory of Cellular and Molec-

ular Cardiology, University of Rome BTor Vergata’’, Rome, Italy.3 To whom correspondence should be addressed at Department of

Biology, University of Rome BTor Vergata’’, Via della Ricerca

Scientifica, 00133 Rome, Italy. E-mail: baldini@uniroma2.it

Abbreviations: AA, Arachidonic Acid; DCF-DA, Dichlorofluorescein

Diacetate; DPI, Diphenylene Iodinium; EIA, Enzyme Immunoassay;

FBS, Foetal Bovine Serum; GAPDH, Glyceraldehyde Phosphate

Dehydrogenase; GPCRs, G-Protein Coupled Receptors; LPA,

Lysophosphatidic Acid; LTB4, Leukotriene B4; NADPHox, NADPH

oxidase; PBMC, Peripheral Blood Mononuclear Cells; PGE2,

Prostaglandin E2; ROI, Reactive Oxygen Intermediates; RT-PCR,

Reverse Transcriptase-Polymerase Chain Reaction; SDS, Sodium

Docecyl Sulphate; TCA, TriChloroAcetate.

releasing several biologically active molecules, such as

arachidonic acid metabolites and reactive oxygen

intermediates (ROI), may contribute to lesion initiation

and progression [9, 10]. In particular, ROI produced by

NADPH oxidase (NADPHox), the principal ROI source

in phagocytic cells [11], have been demonstrated to play

an important role in determining the oxidative stress at

inflamed sites, thus worsening the effects of arachidonic

acid metabolites release [12, 13]. Among the latter,

prostaglandins and leukotrienes generated by cycloxy-

genases and 5-lypoxygenase, respectively, are the most

powerful inflammatory lipid mediators [14].

In order to shed light on its capability to activate

THP-1 cells, LPA effects on key events of inflammatory

response, such as proliferation, ROI production and

release of arachidonic acid metabolites, were investigat-

ed. Our results demonstrated that LPA is able to promote

THP-1 cell activation leading to enhanced cell growth

and simultaneous ROI and prostaglandin E2 (PGE2)

release probably by means of a mechanism involving

the stimulation of LPA3 receptor.

MATERIALS AND METHODS

Cell Culture

The human monocytic cell line THP-1, used to

avoid inter- and intraspecific variability of peripheral

blood mononuclear cells (PBMC), was obtained from

American Type Culture Collection (Manassas, VA), cul-

tured in Falcon flasks with RPMI-1640 medium supple-

mented with 10% foetal bovine serum (FBS), L-glutamine

(2 mM), streptomycin (100 mg/ml), penicillin (100 U/ml),

sodium pyruvate (100 mg/ml), and maintained at 37-C in a

humidified atmosphere containing 5% CO2. Cells were

serum starved before each experiment to rule out

possible interferences with cell growth due to serum

components. In all experiments, to determine cell via-

bility, monocyte cultures were stained with Trypan blue

at specific timepoints after treatment with different

reagents. Stained vs living cells were counted under a

microscope using a Neubauer modified chamber.

DNA Synthesis Assay

THP-1 cells were cultured in Falcon flasks 75 cm2

and seeded in 30�15 mm dishes with serum-free

medium (1�106 cells/well). Cells were challenged with

different concentrations of LPA (0.1Y20 mM) for 3, 6 and

9 h. In a series of experiments, cells were pretreated with

Ki16425 (10 mM) for 30 min before LPA addition. [3H]-

thymidine incorporation into DNA was used to measure

the mitogenic response of THP-1 cells. Cells were pre-

treated with Ki16425 for 30 min, pulsed with [3H]-

thymidine (1 mCi/ml) and then challenged with LPA for

the requested times. Cells were then harvested by cen-

trifugation and treated with 5% TCA at 4-C for 15 min.

The TCA-insoluble fraction was resuspended in 0.1%

SDS in NaOH (200 mM) and samples were counted for

radioactivity, after addition of 3.5 ml Optifluor, by a

liquid scintillation counter (Tricarb 2180/TR, Packard

Instruments, Downers Grove, IL).

Reverse Transcriptase-Polymerase Chain Reaction

(RT-PCR) and LightCycler-PCR Analysis

Total RNA isolation and purification were carried

out by SV Total RNA Isolation System (Promega)

according to the manufacturer_s instructions. Briefly, total

RNA was extracted from 3�106 THP-1 cells treated or

not with LPA (1 mM) for 6 h. Total RNA quantity and

quality were assessed by UV absorbance at 260 nm and

electrophoresis on agarose/formaldehyde gel, respective-

ly. For RT-PCR analysis, cDNA was retro-transcribed

from 2 mg of total mRNA for 1 h at 37-C in an incubation

buffer containing deoxynucleotide triphosphate (dNTP,

250 mM each), random primers p(dN)5 (Roche), RNase

inhibitor and RNA murine Moloney leukaemia virus

reverse transcriptase (200 U, Promega). For classic PCR

analysis, an aliquot of each RT product (2 ml) was

amplified in a final volume of 50 ml using PCR buffer

containing dNTP (160 mM), primers designed to amplify

the mRNA of human LPA receptors (15 pmol each,

Invitrogen) and Taq polymerase (1 U Amersham Pharma-

cia Biotech.). All samples were subjected to the following

reaction conditions: 30 cycles at 94-C for 2 min, 94-C for

30 s, (60-C for 30 s for LPA2 and 58-C for 30 s for LPA1,

LPA3 and GAPDH), 72-C for 30 s, followed by a final

extension at 72-C for 3 min. Expression levels of recep-

tor mRNAs in THP-1 cells were quantified by normal-

ising their respective mRNAs levels to the housekeeping

human GAPDH mRNA levels. The amplified fragments

were then separated by electrophoresis on agarose gel

1Y2% in TAE buffer and visualized by ultraviolet

transillumination.

For LightCycler-PCR, an aliquot of each RT

reaction product (2 ml) was amplified using the Light-

Cycler FastStart DNA master SYBR Green I (Roche

130 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini

Diagnostic) and primers (10 pmol each). The reactions

for LPA receptors were undergoing to 95-C for 10 min

followed by 45 cycles 94-C for 10 s, (60-C for 5 s for

LPA2, 58-C for 10 s for LPA3, 60-C for 20 s for LPA1),

72-C for 15 s, and one melting curve analysis from

95-C, 65-C for 15 s and to 95-C for 10 min, using the

LightCycler instrument (Roche). The GAPDH reaction

was undergoing to 95-C for 10 min followed by 45

cycles 94-C for 10 s, 58-C for 20 s, 72-C for 15 s, and

one melting curve analysis from 95-C, 65-C for 15 s

and to 95-C, using the LightCycler instrument (Roche).

The expression levels of receptor mRNAs were

quantified by normalizing the mRNA levels to the

housekeeping human GAPDH mRNA by an external

standard curve (LightCycler operator_s Manual). The

external standard curve was created with dilutions of

genomic DNA, amplified with LPA3 primers.

Evaluation of ROI Production by 20,70-Dichlorofluorescein (DCF) Fluorescence

The samples (2�106 cells) were loaded with the

fluorescent indicator DCF-DA (10 mM) for 30 min at

37-C in the dark. DCF-DA diffuses through the cell

membrane and is hydrolyzed by intracellular esterase to

nonfluorescent DCF deacetylated, which is then rapidly

oxidized to highly fluorescent DCF in the presence of

ROI. The DCF fluorescence intensity is proportional to

the amount of intracellular ROI formed [15]. After the

incubation with the fluorescent dye, cells were collected,

centrifuged for 5 min at 1,200 rpm at room temperature,

resuspended in serum-free medium and challenged with

different LPA concentrations (0.1Y20 mM) for 3, 6 and 9 h.

When requested, THP-1 monocytes were pretreated with

diphenylene iodinium (DPI, 10j8 M), a specific inhibitor

of NADPH oxidase, and Ki16425 (10 mM), a LPA1 and

LPA3 receptor antagonist, for 1 h and 30 min, respectively,

before LPA addition. Fluorescence was measured under

continuous magnetic stirring and controlled temperature

(37-C) in a Perkin-Elmer luminescence spectrometer

(Model LS-5) equipped with a chart recorder (Model R

100A), with excitation wavelength at 485 nm and emission

wavelength at 530 nm using 5 and 10 nm slits respectively,

for each light path. Results were expressed as Fluorescence

Intensity, reported as Fluorescence Units (F.U.), respect to

cells loaded with only DCF-DA (C).

Primer Sequence

LPA1 sense 50-CGGCGGGTAGTGGTGGTC-30

LPA1 antisense 50-GTCGCGGTAGGAGTAAATGATG-30

LPA2 sense 50-GTCGAGCCTGCTTGTCTTC-30

LPA2 antisense 50-CCAGGAGCAGTACCACCTG-30

LPA3 sense 50-TCGCGGCAGTGATCAAAAACAGA-30

LPA3 antisense 50-ATGGCCCAGACAAGCAAAATGAGC-30

GAPDH sense 50-CATGGGTGTGAACCATGAGAAG-30

GAPDH antisense 50-GTGGCTGTTGAAGTCAGAGGAG-30

Fig. 1. LPA effects on THP-1 cell growth. THP-1 cells were grown in RPMI-1640 plus 10% FBS and serum-

starved before each experiment to rule out possible interferences with cell growth due to serum components. Cells

were challenged with different LPA concentrations (1Y5 2M) for 3, 6 and 9 h. [3H]-thymidine incorporation into

DNA was assessed as reported in the Materials and Methods section. Data are reported as mean T SD of 4

different experiments. *P < 0.05, as reported from Student_s t test in respect to untreated cells (C).

131Properties of Lysophosphatidic Acid on Human THP-1 Cells

132 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini

Roi Generation as Assessed by Microscopy Analysis

ROI generation was carried out on glass chamber

slides. 3�106 cells were labelled with the peroxide-

sensitive fluorescent dye 20,70dichlorofluorescein diace-

tate (DCF-DA, 10 mM) and incubated for 6 h in the

presence or absence of LPA plus or minus DPI (10j8 M)

or Ki16425 (10 mM), as described above. Intracellular

fluorescence was monitored using a fluorescence

microscope (Leica DMRB; objective: �200). Signal-

based averaging was used to quantitate the fluorescence

signal from five randomly selected fields (Delta System

Software).

[3H]-Arachidonic Acid Release Assays

THP-1 cells were seeded in 30�15 mm dishes with

serum-free medium (1�106 cells/well) and labelled for

3 h with 1 mCi [3H]-arachidonic acid (AA) (spec. act.

202.4 Ci/mmol) at 37-C as previously described [16].

To remove non-specific binding of [3H]-AA to cell

surface prior to agonist stimulation, culture medium

was eliminated, cells resuspended in serum-free

medium and challenged with different LPA concen-

trations (1Y20 mM) for 6 h. When requested, cells were

pretreated with Ki16425 (10 mM) for 30 min before LPA

addition. After treatment, supernatants were collected,

centrifuged at 1,200 rpm for 5 min to remove suspended

cells and 100 ml aliquots were added to 3 ml Optifluor

and analyzed by a liquid scintillator counter. Results

were expressed as [3H]-AA cpm/1�106 cells.

Measurement of Leukotriene B4 and Prostaglandin

E2 Release

3�106 cells/well were pre-treated or not with

Ki16425 (10 mM) for 30 min, challenged or not with

LPA (1 mM) for 6 h and, at the end of experimental

time, supernatants were collected and Leukotriene B4

(LTB4) and Prostaglandin E2 (PGE2) levels in the

medium were determined by enzyme immunoassay

(EIA) (Cayman Chemical), following manufacturer_sinstructions. Briefly, 50 ml LTB4 standard or samples

were added to the pre-coated mouse monoclonal anti-

rabbit IgG macrotitre plates. Subsequently, 50 ml LTB4

tracer and 50 ml monoclonal antiserum of LTB4 were

added into each well and the mixture was incubated

overnight at 4-C. After incubation, the content in each

well was removed and the wells were washed 5 times

with PBS buffer containing 0.05% Tween-20. An

aliquot of 200 ml Ellman_s reagent was added into

each well, and the mixture was incubated for 2 h at

room temperature with occasional shaking. The solution

optical density was determined by a multi-well spectro-

photometer (ELISA reader) at 405 nm. A similar proce-

dure was performed for PGE2 release determination,

using pre-coated goat polyclonal anti-mouse IgG macro-

titre plates. LTB4 and PGE2 concentration in each sample

was calculated as the concentration corresponding to

sample_s optic density (O.D.) values plotted on respec-

tive standard built-in curves. Values were reported as

percentile increase in respect to control (C).

Reagents

RPMI 1640, glutamine, penicillin (100 U/ml), and

streptomycin (100 mg/ml) were from Eurobio Labora-

toires. Foetal Bovine Serum (FBS) was from GIBCO

(Grand Island NY, USA). LPA (C18:1, 1-oleoyl-sn-

glycerol-3-phosphate), trypan blue, DPI, Ki16425 and

20,70-dichlorofluorescein diacetate (DCF-DA) were from

Sigma Chemicals. [3H]-thymidine (20 Ci/mmol), [3H]-

arachidonic acid (202.4 Ci/mmol) from Amersham Bio-

sciences. Random primers, murine Molooney leukaemia

virus reverse transcriptase, LPA receptors and GAPDH

primers were purchased from Invitrogen. Taq polymerase

was from Amersham Pharmacia Biotech. SV Total RNA

Isolation System was from Promega. LightCycler Fast-

Start DNA master SYBR Green I kit and all other reagent

for LightCycler-PCR were from Roche Diagnostic.

Leukotriene B4 and Prostaglandin E2 assay kits were

purchased from Cayman Chemical.

Statistical Analysis

Data distribution was preliminarily verified by the

KolmogorovYSmirnov test. Each experiment set was

independently performed and compared with the same

control by Student_s t-test. Quantitative data were

expressed as the meanTSD of at least four replicate

determinations, except where otherwise indicated. Dif-

Fig. 2. LPA effects on ROI production in THP-1 cells. THP-1 cells were

labelled with DCF-DA (10 2M) as reported in the Materials and Methods

section and ROI production was assessed after cell exposure to different

LPA concentrations (1Y5 2M) for 3, 6 and 9 h (a) or after cell exposure

to LPA (1 2M) for 6 h (b). In the experiments with DPI (10j8 M), cells

were pre-treated with the inhibitor for 1 h before LPA addition. Results

are expressed as Fluorescence Intensity, reported as Fluorescence Units

(F.U.), in respect to cells loaded with DCF-DA only (C). Micrographs

were acquired as reported in the Materials and Methods section.

Original magnification: �200. Data are reported as mean T SD of 4

different experiments. *P < 0.05, as reported from Student_s t test in

respect to untreated cells (C).

133Properties of Lysophosphatidic Acid on Human THP-1 Cells

ferences were regarded as significant when P value was

less than 0.05.

RESULTS

Effects of LPA on THP-1 Cell Growth

To evaluate LPA ability to stimulate THP-1 cell

growth, cells were challenged with different lipid concen-

trations (1Y5 mM) and assayed for [3H]-thymidine

incorporation into DNA at 3, 6 and 9 h. As shown in

Fig. 1, only 1 mM LPA induced an increased DNA syn-

thesis that was statistically significant at 3 h and reached

a maximum (+50% in respect to untreated cells) at 6 h.

Longer experimental times and lower (0.1 and 0.5 mM)

or higher (10 and 20 mM) LPA concentrations (data not

shown), did not exert any significant effect.

LPA Effects on ROI Production and NADPH

Oxidase Activation

To evaluate the ability to stimulate ROI production,

a fundamental marker of cell activation [17], THP-1 cells

were challenged with LPA (1Y5 mM) for different

experimental times (3, 6 and 9 h) after labelling with

Fig. 3. LPA effects on Arachidonic Acid (AA), Leukotriene B4 (LTB4) and

Prostaglandin E2 (PGE2) release in THP-1 cells. (a) THP-1 cells were inc-

ubated for 3 h with [3H]-AA (1 2Ci/ml), centrifuged, resuspended in serum-

free medium and challenged with different LPA concentrations (1Y5 2M)

for 6 h. The [3H]-AA released was determined as reported in the Materials

and Methods section. Data are reported as mean T SD of 4 different

experiments. *P < 0.05, as reported from Student_s t test in respect to

untreated cells (C). (b) THP-1 cells were challenged with LPA (1 mM) for 6

h. LTB4 and PGE2 released into the medium was measured by EIA assay, as

described in the Materials and Methods section. A representative experiment

is shown, which was repeated four additional times with similar results.

Values are reported as percentile increase compared to control (C).

134 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini

Fig. 4. LPA effects on [3H]-thymidine incorporation and ROI production in

the presence of Ki16425 in THP-1 cells. (a) THP-1 cells were pre-treated

with Ki16425 (10 2M) for 30 min and then challenged with LPA (1 2M) for

6 h. [3H]-thymidine incorporation into DNA was assessed as reported in the

Materials and Methods section. Data are reported as mean T SD of 4 different

experiments. *P < 0.05, as reported from Student_s t test in respect to

untreated cells (C). (b) THP-1 cells were labelled with DCF-DA (10 mM),

pre-treated with Ki16425 (10 mM) for 30 min and then challenged with LPA

(1 mM) for 6 h. ROI production was assessed as reported in the Materials and

Methods section and expressed as Fluorescence Intensity, reported as

Fluorescence Units (F.U.), in respect to cells loaded with DCF-DA only (C).

Micrographs were acquired as reported in the Materials and Methods section.

Original magnification: �200. Data are reported as mean T SD of 4 different

experiments. *P < 0.05, as reported from Student_s t test in respect to untreated

cells (C).

135Properties of Lysophosphatidic Acid on Human THP-1 Cells

DCF-DA (10 mM). The time-course and dose-response of

ROI production are shown in Fig. 2a. Similarly to cell

growth, LPA induced a significant increase in ROI

production only at 1 mM after 3 h with a maximal effect

after 6 h. Longer experimental times and lower (0.1 and

0.5 mM) or higher (10 and 20 mM) LPA concentrations

(data not shown), did not exert any significant effect.

Subsequently, the possible involvement of NADPH

oxidase in LPA-induced ROI production was investigated.

Since the maximal production of ROI occurred after 6 h of

stimulation with 1 mM LPA, such a concentration and time

were used in experiments performed in the presence or

absence of DPI, a specific inhibitor of NADPH oxidase. As

shown in Fig. 2b, cells pre-treatment with DPI (10j8 M)

significantly reduced the production of LPA-induced

ROI in respect to untreated cells as further on

confirmed by fluorescence microscopy analysis.

LPA Effects on Arachidonic Acid, Leukotriene B4

and Prostaglandin E2 Release

Since LPA can promote arachidonic acid (AA)

mobilization [18, 19], doseYresponse experiments were

performed to assess [3H]-AA release into the medium. In

the same way of cell growth and ROI production, assays

were carried out after cell incubation for 6 h with

Fig. 5. LPA effects on Arachidonic Acid (AA) and Prostaglandin E2 (PGE2)

release in the presence of Ki16425 in THP-1 cells. (a) THP-1 cells were

treated as reported in Fig. 3a and challenged with LPA (1 2M) for 6 h after

30 min pre-treatment with Ki16425 (10 2M). The [3H]-AA released was

determined as reported in the Materials and Methods section. Data are

reported as mean T SD of 4 different experiments. *P < 0.05, as reported from

Student_s t test in respect to untreated cells (C). (b) THP-1 cells were treated

as reported in Fig. 3b after 30 min pre-treatment with Ki16425 (10 mM).

PGE2 released into the medium was measured by EIA assay, as described in

the Materials and Methods section. A representative experiment is shown,

which was repeated four additional times with similar results. Values are

reported as percentile increase compared to control (C).

136 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini

different LPA concentrations (1Y5 mM). Data shown in

Fig. 3a demonstrate that all LPA concentrations exerted

a significant effect on AA release, the maximal response

occurring at 1 mM and progressively decreasing at higher

lipid concentrations. These results indicated to as

sess LTB4 and PGE2 release in the presence of 1 mM

LPA for 6 h. As shown in Fig. 3b, LPA was able to in-

duce an approximately 15% increase in LTB4 release

(left panel), while PGE2 production increase was

approximately 70% (right panel), suggesting a more

efficient activation of the prostaglandin synthesizing

enzymatic machinery.

LPA Effects on Cell Growth, ROI Production, AA

and PGE2 Release in the Presence of Ki16425

Since LPA1 and LPA3 receptors have been related

to cellular activation and malignant tumour proliferation,

respectively [20, 21], experiments were carried out in the

presence of Ki16425, a selective antagonist of these

receptors [22]. As shown in Fig. 4a, the enhanced LPA-

induced [3H]-thymidine incorporation was significantly

inhibited by Ki16425 (10 mM) pre-treatment. Similarly,

Ki16425 cell pre-treatment totally abolished LPA-stimulated

ROI production, as also confirmed by fluorescence

microscopy analysis (Fig. 4b).

In addition, the antagonist pre-treatment totally

abrogated LPA effects on AA mobilization (Fig. 5a)

and PGE2 release (Fig. 5b). These results suggested that

LPA1 and LPA3 receptors play a fundamental role in

mediating LPA effects on THP-1 cell growth, ROI

production and AA and PGE2 release.

LPA Effects on Receptor Expression Profile

Since cells exposure to LPA can alter the receptor

expression pattern [23], PCR analysis was performed to

further discriminate the relative contribution of each

receptor subtype in LPA effects. RT-PCR analysis

(Fig. 6a) showed that all three LPA receptors are ex-

pressed in THP-1 cells; particularly, un-stimulated cells

expressed predominantly LPA1 and LPA2 mRNAs,

while LPA3 mRNA was poorly expressed. After LPA

(1 mM for 6 h) treatment, a significant increase in

mRNA levels of LPA3 receptor occurred, while no

significant variations in mRNA levels of LPA1 and

LPA2 were detectable. To finely quantify such an

increase, Real Time RT-PCR analysis (Fig. 6b) were

performed demonstrating that LPA addition induced 50-

fold up-regulation in the expression of LPA3 receptor in

respect to control, suggesting a possible role for this

receptor in all the observed effects.

DISCUSSION

The inflammatory response to endothelial injuries is

represented by excessive cell proliferation and activation

[7] that, in turn, can be influenced by several locally

released bioactive factors. Among these substances,

lysophosphatidic acid (LPA), one of the most intriguing

phospholipidic mediators, might act as a powerful

mitogenic and activating factor even for immune cells,

such as T- and B-cells [24, 25].

The present study represents the first attempt to

characterize LPA proinflammatory properties in THP-1

cells. In fact, micromolar concentrations of LPA are able

to stimulate THP-1 cell growth, ROI production and

arachidonic acid metabolites release. Interestingly, a

positive factor in prostaglandins (PGs) and leukotrienes

(LTs) synthesis is considered to be the increased cytosolic

oxidant power [13]. This is generally due to an

enhancement in ROI production, whose primary source

differs depending on cell type [26]. In phagocytes, the

principal ROI source is represented by the NADPH

oxidase enzyme complex [27]. Moreover, ROI release at

inflamed sites causes the activation of different cell types

determining the pathological course of reactions [13]. In

this context, the observed LPA ability to increase both

PGs and LTs production, exerting a stronger effect on

PGE2 synthesis rather than LTB4, and to determine

NADPH oxidase-dependent ROI production accounts for

a possible role of this lipid in the pathogenesis of

different diseases characterized by the development of

local inflammatory reactions. These data are substantiat-

ed by the demonstration that all these effects occurred at

the same concentration and timepoint determining a

remarkable increase in cell proliferation.

To refine our analysis, we tried to determine which

receptor subtype could be mostly involved in mediating

LPA effects. LPA, in fact, regulates a broad variety of

biological processes binding to specific receptors, princi-

pally LPA1, LPA2 and LPA3, which, in turn, activate

pleiotropic signalling pathways [28]. Since previous

studies demonstrated that LPA1 receptor stimulation

was critical for monocyte activation [20] and LPA3

receptor was responsible for cell growth and proliferation

of malignant tumours [21], experiments were carried out

in the presence of Ki16425, a selective LPA1 and LPA3

receptor antagonist [22]. Our results demonstrated that

137Properties of Lysophosphatidic Acid on Human THP-1 Cells

138 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini

both receptors were implicated in LPA effects since they

were totally abolished by the addition of a receptor-specific

inhibitor. In fact, preincubating cells with Ki16425, a

significant inhibition of LPA-induced THP-1 proliferation

and ROI production as well as AA release and PGE2

production occurred. Since LPA proinflammatory activity

depends on the individual expression of LPA receptors

[29] and the exposure to mitogens can alter such an

expression profile [23], it is possible that, in THP-1 cells,

the abundance of each LPA receptor may significantly

differ in resting vs activated cells. Therefore, to further

unravel the relative contribute of each receptor subtype in

LPA effects, PCR analysis was performed demonstrating

that the lipid addition caused a 50-fold increase in LPA3

receptor mRNA expression, while the other receptors did

not undergo any significant change. These findings

suggest a possible role for LPA3 receptor in mediating

LPA signalling in THP-1 cells, even though the relative

contribute of LPA1 receptor cannot be excluded.

Taken together, the results obtained demonstrate that

LPA can modulate THP-1 cells proinflammatory activa-

tion implying that this lipid, acting as an autocrine/

paracrine factor, could sustain an ongoing complex

cascade of inflammatory reactions ultimately leading to

a pathological chronic condition. In conclusion, our study

furnishes new evidences on LPA proinflammatory prop-

erties, whose understanding might indicate a new prom-

ising therapeutic strategy in monocytes-related human

diseases management.

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Fig. 6. LPA effects on the expression of LPA1, LPA2 and LPA3 re-

ceptors in THP-1 cells. Total RNA was extracted from THP-1 cells

treated or not with LPA (1 2M) for 6 h. RT-PCR (a) and Real-Time

RT-PCR (b) analysis were performed as reported in the Materials and

Methods section. The expression levels of LPA receptors in THP-1

cells were quantified by normalising their respective mRNA levels

with the housekeeping human GAPDH mRNA levels. A representa-

tive experiment is shown, which was repeated three additional times

with similar results.

139Properties of Lysophosphatidic Acid on Human THP-1 Cells

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140 D_Aquilio, Procaccini, Izzi, Chiurchiu_, Giambra, Carotenuto, Di Nardo, and Baldini