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Endocrinology, doi:10.1210/en.2008-0314
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Endocrinology Vol. 149, No. 11 5888-5896
Copyright © 2008 by The Endocrine Society

Lysophosphatidic Acid Mediates Interleukin-8 Expression in Human Endometrial Stromal Cells through Its Receptor and Nuclear Factor-{kappa}B-Dependent Pathway: A Possible Role in Angiogenesis of Endometrium and Placenta

Shee-Uan Chen, Hsinyu Lee, Daw-Yuan Chang, Chia-Hung Chou, Chih-Yuan Chang, Kuang-Han Chao, Chung-Wu Lin and Yu-Shih Yang

Departments of Obstetrics and Gynecology (S.-U.C., D.-Y.C., C.-H.C., C.-Y.C., K.-H.C., Y.-S.Y.), Life Science (H.L.), and Pathology (C.-W.L.), National Taiwan University, 106 Taipei, Taiwan

Address all correspondence and requests for reprints to: Dr. Yu-Shih Yang, Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, 106 Taipei, Taiwan. E-mail: ysyang{at}ha.mc.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lysophosphatidic acid (LPA) is a pleiotropic phospholipid molecule involved in inflammation, angiogenesis, would healing, and cancer invasion. Whereas serum lysophospholipase D activity increases in women with pregnancy, the role of LPA in pregnancy remains unclear. We investigated the expression of LPA receptors and function of LPA in endometrial stromal cells. Histologically normal endometrium was obtained from surgical specimens of women undergoing hysterectomy for leiomyoma. First-trimester decidua was obtained from women receiving elective termination of pregnancy. We examined the expressions of LPA1, LPA2, and LPA3 receptors in endometrial stromal cells. The effects of LPA on the expression of vascular endothelial growth factor, IL-6, and IL-8 were examined. Signal pathways of LPA were delineated. Functions of secretory angiogenic factors were tested using human endometrial microvascular endothelial cells. Immunoreactivity and mRNA of LPA1 receptors were identified in endometrial stromal cells. LPA enhanced IL-8 expression in a dose- and time-dependent manner, whereas vascular endothelial growth factor or IL-6 expression was not affected by LPA treatment. Mechanistic dissection disclosed that LPA functioned via the Gi protein, MAPK/p38 and nuclear factor-{kappa}B pathway. LPA-induced IL-8 enhanced migration, permeability, capillary tube formation, and proliferation of human endometrial microvascular endothelial cells. Endometrial stromal cells express LPA1 receptors. Through the LPA1 receptor, LPA induces IL-8 expression via a nuclear factor-{kappa}B-dependent signal pathway. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROCESSES OF successful pregnancy include embryo implantation, placentation, and local immune suppression for maternal tolerance to fetal foreign antigens. Maternal oxygen and nutrient supply via an extensive vascular network of endometrium to placenta are indispensable for fetal growth. However, the relevant molecular mechanisms of neovascularization of endometrium and placenta are still obscure. A number of growth factors, cytokines, and phospholipids may exert modulatory effects. These include vascular endothelial growth factor (VEGF), IL-8, IL-6, angiopoietin-1 and -2, angiogenin, human chorionic gonadotropin (hCG), prostaglandins, and platelet-activating factor (1, 2, 3, 4, 5, 6, 7, 8, 9).

An increase in serum lysophospholipase D activity had been found during pregnancy in human (10). It was suggested that lysophosphatidic acid (LPA) formed by increased activity of lysophospholipase D might participate in maintenance of pregnancy. LPA, a biologically active phospholipid, plays critical roles in physiological and pathological processes including inflammation, cell proliferation, angiogenesis, wound healing, and cancer invasion (11, 12, 13, 14, 15). LPA could be produced through the hydrolysis of phospholipids by extracellular lysophospholipase D (10) or activated platelets, leukocytes, epithelial cells, and tumor cells (16, 17, 18). However, the physiological roles of increased LPA during pregnancy remain unclear.

LPA exhibits pleiotropic functions via the interaction with specific G protein (Go, Gs, Gi, or G12/13)-coupled endothelial differentiation gene (Edg) receptors including LPA1/Edg2, LPA2/Edg4, or LPA3/Edg7 (19). In humans, whereas the expression of the LPA receptors has been reported in some healthy tissues and cancer cells (13, 20), the expression of the LPA receptor isoforms in the endometrium is unknown. Recently LPA was demonstrated to promote ovarian cancer growth by inducing angiogenic factors including VEGF, IL-6, and IL-8 through various LPA receptors (21, 22, 23). The relation between LPA and increased angiogenic factors in pregnancy merits further investigation.

In the present study, we attempted to determine the possible role of LPA in pregnancy. We first examined the LPA receptors of endometrial stromal cells. The effects of LPA on endometrial stromal cells regarding expressions of angiogenic factors of VEGF, IL-6, or IL-8 were examined, and the signaling pathways were investigated. We then explored whether LPA-induced angiogenic factors modified migration, permeability, capillary tube formation, or proliferation of human endometrial microvascular endothelial cells (hEMVECs).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Endometrial tissues
Endometrial specimens were obtained from 38 premenopausal women (aged 35–44 yr) undergoing hysterectomy for leiomyoma. Decidua or placenta specimens were obtained from 12 women receiving elective termination of pregnancy (7–10 wk). This study was approved by the Ethics Committee of National Taiwan University Hospital, and informed consent was obtained from patients. The phases of endometrium were determined by histological criteria. The dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Endometrial stromal cells in the filtrate were cultured in phenol-red-free DMEM/F-12 containing 10% charcoal-stripped fetal bovine serum. The purity of endometrial stromal cells was more than 99%, as determined by staining with antibodies to vimentin and cytokeratin.

Antibodies and reagents
Pertussis toxin (PTX), Ki16425, AG1478, LY294002, PD98059, SB203580, 1-oleoyl-LPA, and fatty acid-free BSA were purchased from Sigma-Aldrich (St. Louis, MO). LPA was dissolved in vehicle of PBS containing 1% fatty acid-free BSA. Recombinant human IL-8 and IL-8 neutralizing antibodies were obtained from R&D Systems (Minneapolis, MN). Antibodies to human p38 and phospho-p38 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Flow cytometry analysis
Endometrial stromal cells were incubated with anti-LPA1, LPA2, or LPA3 antibodies (1:100 dilutions; BD PharMingen, San Diego, CA) for 60 min. Isotype antibodies were used for background controls. Using fluorescein isothiocyanate-labeled secondary antibody, the cell surface expression of LPA1, LPA2, or LPA3 receptor was analyzed by flow cytometry (BD Biosciences, San Jose, CA).

RT-PCR
The total RNA was isolated from the endometrial stromal cells using the RNAzol B reagent (Biotecx Laboratories, Houston, TX). Then cDNA was prepared from 2 µg of the total RNA with random hexamer primers (ImProm-II RT system; Promega, Southampton, UK). The specific oligonucleotide primer pairs for LPA receptors were as follows: LPA1, 5'-CAA AAT GAG GCC TTA CGA CGC CA-3' and 5'-TCC CAT TCT GAA GTG CTG CGT TC-3'; LPA2, 5'-GCG CGC GGA TCC ACC ATG GTC ATC ATG GGC CAG TGC-3' and 5'-GCG CGG TCG ACT CAG TCC TGT TGG TTG GGT TGA-3'; LPA3, 5'-CTG ATG TTT AAC ACA GGC CC-3' and 5'-GAC GTT GGT TTT CCT CTT GA-3' (12).

Immunohistochemistry (IHC)
The formalin-fixed and paraffin-embedded endometrial or placental tissues were used for IHC. Sections were incubated with anti-LPA1 or anti-IL-8 antibodies (Santa Cruz Biotechnology) for 60 min. Then the sections were incubated with biotinylated secondary antibody. The localization of LPA1 receptors or IL-8 was detected using a streptavidin-biotin immunoperoxidase kit (Dako Ltd., Buckinghamshire, UK).

Enzyme immunoassay (EIA)
Endometrial stromal cells were plated into 6-well culture plates at a density of 2 x 105 cells/well. After cell attachment, cell layers were washed and incubated with serum-free medium for 24 h. The cells were then treated with either vehicle or indicated conditions. After 24 h, the supernatant were collected. Levels of IL-6, IL-8, and VEGF were determined using EIA kits (R&D Systems).

RNA interference
Small interfering RNA (siRNA) duplexes were purchased from Santa Cruz Biotechnology. The targeted siRNA of LPA1 was sc-43746. Endometrial stromal cells were transfected with siRNA at the concentration of 25 nM in serum-free Opti-MEM by using the Oligofectamine method (Invitrogen Corp., Carlsbad, CA).

Real-time quantitative RT-PCR
We quantified IL-8 mRNA expression in various conditions using a fluorescein quantitative real-time PCR detection system (Light Cycler DNA master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). The primer pairs were: 5'-TTT CTG CAG CTC TCT GTG AGG-3' and 5'-CTG CTG TTG TTG TTG CTT CTC-3' for IL-8; 5'-GGG AAG GTG AAG GTC GG-3' and 5'-TGG ACT CCA CGA CGT ACT CAG-3' for glyceraldehyde-3-phosphate dehydrogenase. Amplification was followed by melting curve analysis to verify the correctness of the amplicon. The amount of IL-8 mRNA was normalized by that of glyceraldehyde-3-phosphate dehydrogenase mRNA and is presented in arbitrary units, with 1 U corresponding to the value in cells treated with a vehicle control.

Promoter construction and reporter assays
Transfections of plasmid of human IL8–1.4 Kb or nuclear factor-{kappa}B (NF-{kappa}B) binding site-driven luciferase plasmids (BD Bioscience) into endometrial stromal cells was performed using the Transfast transfection reagent (Promega). At 24 h after transfection, cells were serum starved for 24 h and then treated with the indicated conditions.

Western blotting
The endometrial stromal cell lysates were centrifuged at 12,000 rpm for 25 min at 4 C. The protein concentration then was measured using a Bio-Rad protein assay (Hercules, CA). A 50-µg protein sample was separated using SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and immunoblotted with various antibodies.

NF-{kappa}B and activator protein-1 (AP-1) decoy oligodeoxynucleotides to endometrial stromal cells
The sequences of the phosphorothioate oligodeoxynucleotides of NF-{kappa}B decoy, AP-1 decoy, or scrambled decoys had been described previously (20). The oligodeoxynucleotides was mixed with the Transfast transfection reagent (Promega) for 15 min and then incubated with the endometrial stromal cells in a serum-free medium.

hEMVEC migration assay
hEMVECs were separated from fresh first trimester decidua. The endometrial specimen was homogenized and resuspended in 1 ml of cold M199 medium containing 0.3 mg/ml magnetic tosyl-activated M-450 Dynabeads (Dynal, Oslo, Norway), which had been coated with mouse antihuman CD31. The endothelial cells bound to the magnetic beads were isolated using a MPC-1 magnet (Dynal) and were cultured in endothelial cell culture medium (Sigma-Aldrich).

hEMVEC migration was measured using the in vitro wound healing assay. hEMVECs were grown to confluence on 12-well culture dishes. Wounds were made with an approximate width of 110 µm using a sterile blunt glass bar. The medium was changed to indicate conditions. After 6 h, the hEMVECs were fixed with 1% formaldehyde and stained with 0.05% crystal violet. The distances of wounds were measured and photographed.

hEMVEC monolayer permeability assay
hEMVEC were cultured in trans-well chambers (0.4 µm pore-size polycarbonate filters; Costar Corp., Cambridge, MA). After reaching confluence, the medium was replaced with the indicated conditions (0.3 ml in the upper chamber and 1 ml in the lower chamber). Horseradish peroxidase molecules (type VI-A, 44 kDa; Sigma-Aldrich) at a concentration of 0.126 µM were added to the upper compartment. After incubation for 1 h, the medium in the lower compartment was assayed for enzymatic activity using a photometric guaiacol substrate assay (Sigma-Aldrich).

hEMVEC capillary tube formation assay
hEMVECs (5 x 104) in the medium of indicated conditions were plated on a growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ)-coated 24-well plates. After incubation for 6 h, the wells were examined for tube formation under a phase-contrast microscope.

hEMVEC proliferation assay
hEMVECs were seeded in 96-well plates at densities of 1 x 104 cells/well using 0.2 ml of endothelial cell medium. After 24 h for cell attachment, the medium was changed to the indicated conditions. After incubation for 72 h, the number of hEMVECs was analyzed using the trypan blue exclusion assay.

Statistics
In this study, each experiment was repeated at least three times on different occasions. Data were presented as mean ± SD. The data were examined with one-way ANOVA, followed by Tukey test for multiple comparisons. Significance level was set as P < 0.05 by two-tailed test. SAS software version 8.01 (SAS Institute Inc., Cary, NC) was used for calculation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPA1 receptors are expressed in human endometrial stromal cells
We first detected LPA receptors with specific LPA1, LPA2, and LPA3 antibodies in primary culture of endometrial stromal cells by flow cytometry. The cells incubated with LPA1-specific antibody displayed a significant area shift, whereas those with LPA2 or LPA3 did not exhibit this change (Fig. 1AGo). The quantitative data from nine cases revealed that LPA1 receptors were highly expressed in endometrial stromal cells (Fig. 1BGo). No differences of LPA1 expressions were found among the proliferative phase, secretory phase and first-trimester endometrium.


Figure 1
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  		FIG. 1. Expression of LPA receptors in endometrial stromal cells. A, The expression pattern of LPA receptors by flow cytometry. Diagrams presented here were from three of the nine cases. B, The quantitative results of LPA1, LPA2, and LPA3 receptors in nine cases. C, The mRNA expression of LPA receptors by RT-PCR. The total RNA of the cultured endometrial stromal cells and SK-OV3 cells were extracted under normal culture conditions. LPA1, LPA2, and LPA3 receptors were detected using RT-PCR with specific primers.

 
We further confirmed expression patterns of LPA receptors by RT-PCR. Those were compared with SK-OV3 cell line, which was a well-known ovarian cancer cell line with expressions of LPA1, LPA2, and LPA3 receptors. The results indicated that only LPA1 receptors’ mRNA was expressed in endometrial stromal cells of the proliferative and secretory phases as well as first-trimester decidua (Fig. 1CGo). The IHC results further demonstrated that LPA1 receptors located in the stromal cells and glandular epithelium of human endometrium as well as in the endothelial cells (Fig. 2Go, A–C). In addition, IL-8 was also present in the endometrium (Fig. 2Go, D and E).


Figure 2
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  		FIG. 2. Immunohistochemical localization of LPA1 receptors and IL-8 in the human endometrium. A, Late secretory endometrium. LPA1 receptors are present in the stromal cells (S) and glandular epithelium (G). The endothelial cells show positive stain for LPA1 receptors (arrow). B, LPA1 receptors in the first-trimester decidua. C, First-trimester placenta. LPA1 receptors are present in the endothelial cells (arrow) and trophoblasts (double arrows). D, Late secretory endometrium. IL-8 is present in the stromal cells and glandular epithelium. E, IL-8 in the first-trimester decidua. F, First-trimester decidua. Negative control without treatment of primary antibodies to LPA1 receptors or IL-8. (Magnification, x40).

 
LPA enhances IL-8 expression in endometrial stromal cells at both protein and mRNA levels
Via the primary culture system, LPA was applied to the endometrial stromal cells of the secretory phase and first-trimester decidua. Using EIA, we found that LPA significantly increased IL-8 protein secretion (8.9 ± 1.2- and 8.7 ± 1.1-fold, respectively) at 24 h and also at 48 and 72 h. However, LPA did not enhance IL-6 or VEGF secretions (Fig. 3AGo). The hCG did not obviously increase VEGF secretion at 24 h but significantly increased it at 48 and 72 h. In contrast, hCG did not induce IL-8 or IL-6 secretions (Fig. 3BGo).


Figure 3
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  		FIG. 3. Different induction patterns of IL-8 and VEGF expressions between LPA and hCG in endometrial stromal cells. The cultured cells were treated with LPA (10 µM) (A) or hCG (1 IU/ml) (B). After 24, 48, and 72 h, VEGF, IL-6, and IL-8 proteins in supernatants were determined by EIA. Data are the relative fold of induction, compared with vehicle-treated controls. *, P < 0.05; n = 5 for the secretory-phase endometrium (S) and first-trimester decidua (D), respectively. C, Dosage effect of LPA on IL-8 secretion. The cultured stromal cells from the secretory-phase endometrium were stimulated with indicated doses of LPA. After 24 h, the supernatants were detected for IL-8. Data are concentrations of IL-8 proteins and are compared with vehicle-treated controls. *, P < 0.05; n = 5. D, LPA transcriptionally regulated IL-8 mRNA expression. The cells were stimulated with LPA (10 µM). At indicated times, the IL-8 mRNA was measured by real-time quantitative RT-PCR. Data are compared with the starting time point. *, P < 0.05 (n = 4).

 
We examined the dosage effect of LPA on IL-8 secretion (Fig. 3CGo). The results revealed that upon stimulation of LPA, the secretion of IL-8 was enhanced in a dose-dependent manner from 1 to 20 µM. Using real-time quantitative RT-PCR (Fig. 3DGo), we found that mRNA expression of IL-8 increased in a time-dependent manner when treated with LPA. After the 8 h of treatment, the level of IL-8 (6.7 ± 0.6-fold, compared with controls) reached the maximum.

Signal transduction pathway involved in LPA-induced IL-8 expression
We investigated the role of LPA receptor in LPA-induced IL-8 expression in endometrial stromal cells. Using EIA (Fig. 4AGo), we found that both the Ki16425, an antagonist of LPA1 and LPA3, and PTX specifically abolished the LPA-enhanced IL-8 secretion. However, the epidermal growth factor receptor inhibitor (AG1478) did not diminish the enhancing effect. We further applied specific LPA1 receptor siRNA to affect mRNA expressions of LPA1 receptors (Fig. 4BGo, upper panel). We found that the LPA1 siRNA significantly reduced LPA-enhanced IL-8 secretion (Fig. 4BGo, lower panel). These results indicated an involvement of the PTX-sensitive Gi protein-coupled LPA1 receptor, but not epidermal growth factor receptor, in the LPA-induced IL-8 expression in human endometrial stromal cells.


Figure 4
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  		FIG. 4. Signal transduction pathway of LPA-induced IL-8 expression in endometrial stromal cells. A, Receptors involved in LPA-mediated IL-8 secretion. Endometrial stromal cells were pretreated with Ki16425 (10 µM), PTX (100 ng/ml), or AG1478 (10 µM) for 1 h before LPA (10 µM) treatment. After 24 h, the supernatants were collected for IL-8 protein detection by EIA. Data are compared between the LPA-treated-only group and different inhibitor groups. *, P < 0.05 (n = 5). B, upper panel, Confirmation of the effect of specific LPA1 siRNA on the mRNA expression of LPA1 receptor using RT-PCR. Lower panel, The effect of LPA1 siRNA on the LPA-induced IL-8 protein secretions. The targeted siRNA of LPA2 (sc-39926) was used for a mock control. *, P < 0.05 (n = 5). C, Examination of signal transduction mediators. The cells were pretreated with LY294002 (50 µg/ml), SB203580 (5 µg/ml), or PD98059 (50 µg/ml) for 1 h before LPA treatment. After 24 h, IL-8 protein secretions were detected and compared. *, P < 0.05 (n = 5). D, LPA induction of p38 phosphorylation. The cells were pretreated with indicated inhibitors for 1 h and then treated with LPA. After 1 h, the cell lysates were collected for detection of protein levels of phospho (p)-p38 and p38 using Western blotting. Data were measured as relative density of p-p38/p38. The p-p38/p38 of the vehicle control was defined as 1 (n = 3).

 
We further explored signal transduction mediators involved in LPA-induced IL-8 expression. Using EIA (Fig. 4CGo), we verified that MAPK/p38 inhibitor (SB203580) significantly decreased LPA-enhanced IL-8 secretion but not phosphatidylinositol 3-kinase/Akt inhibitor (LY294002) or MAPK/ERK inhibitor (PD98059). Using Western blotting and densitometry (Fig. 4DGo), we found that LPA strongly induced phosphorylation of p38. In addition, the phosphorylation was significantly diminished by pretreatments with Ki16425, PTX, or SB203580.

Because the upstream promoter sequences of IL-8 gene contain conserved putative binding sites for NF-{kappa}B and AP-1, we used synthetic double-stranded oligodeoxynucleotides as decoy cis elements to examine the role of NF-{kappa}B and AP-1 in LPA-induced IL-8 expression. We found that treatment with NF-{kappa}B decoy, but not the AP-1 decoy, significantly decreased LPA-induced IL-8 promoter activity (Fig. 5AGo). Using real-time quantitative RT-PCR, we also verified that LPA-enhanced IL-8 mRNA expression was significantly diminished by NF-{kappa}B decoy but not AP-1 decoy (Fig. 5BGo). Using Western blotting and densitometry (Fig. 5CGo), we observed that LPA induced NF-{kappa}B nuclear translocation. The LPA-enhanced NF-{kappa}B nuclear translocation was decreased by inhibitors of Gi (PTX), p38 (SB203580), and NF-{kappa}B (BAY117082). We further verified that LPA potently enhanced NF-{kappa}B binding site-driven luciferase activity (12.0 ± 1.7-fold), and the enhanced activity was significantly reduced by inhibitors of Gi (PTX), p38 (SB203580), and NF-{kappa}B (BAY117082) (Fig. 5DGo). These results indicated that NF-{kappa}B played a critical role in LPA-induced IL-8 expression in human endometrial stromal cells.


Figure 5
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  		FIG. 5. NF-{kappa}B is critically involved in LPA-induced IL-8 expression. A, Endometrial stromal cells were transfected with IL-8 promoter plasmid. The cells were then treated with NF-{kappa}B decoy (dy), AP-1 decoy, or scrambled (s) decoy. After treatment with LPA (10 µM) for 8 h, the luciferase activity of the IL-8 promoter was measured by luminometer. Data are compared between the LPA-treated-only group and different decoy groups. *, P < 0.05 (n = 5). B, Endometrial stromal cells were transfected with NF-{kappa}B decoy, AP-1 decoy, or scrambled decoy and then treated with LPA. After 8 h, IL-8 mRNA levels were determined by real-time quantitative RT-PCR and compared. C, Nuclear translocation of NF-{kappa}B. Endometrial stromal cells were pretreated with PTX (100 ng/ml), SB203580 (50 µg/ml), or BAY117082 (5 µM) for 1 h before LPA treatment. Nuclear and cytosolic proteins were collected after 1 h. The levels of NF-{kappa}B p65 subunits were examined by Western blotting. Data were measured as relative density of nuclear p65/cytosol p65. The nuclear p65/cytosol p65 of the vehicle control was defined as 1 (n = 3). D, NF-{kappa}B binding site-driven luciferase activity assay. Endometrial stromal cells were transfected with NF-{kappa}B reporter plasmids. The cells were then pretreated with indicated chemical inhibitors for 1 h before stimulation with LPA. After 4 h, the NF-{kappa}B binding site-driven luciferase activity was measured. Data are compared between the LPA-treated-only group and different inhibitor groups. *, P < 0.05 (n = 5).

 
LPA-induced IL-8 protein secretion from endometrial stromal cells enhances angiogenesis
Using in vitro wound healing assay (Fig. 6AGo, left panel), we observed increased migration of hEMVECs when incubated with LPA-treated conditioned medium (CM) (b), compared with vehicle CM (a). LPA-containing medium with 24 h incubation did not enhance migration (c). In addition, LPA-treated CM preincubated with IL-8 neutralizing antibodies (d) or LPA-treated CM from endometrial stromal cells that had been pretreated with antisense IL-8 oligonucleotides (f) did not increase hEMVEC migration. In contrast, LPA-treated CM preincubated with nonspecific IgG (e) or LPA-treated CM from cells pretreated with sense oligonucleotides (g) significantly enhanced hEMVEC migration. On the other hand, LPA-treated CM from cells pretreated with antisense IL-8 oligonucleotides added with recombinant human (rh) IL-8 (h) or the rhIL-8-containing medium (i) significantly enhanced hEMVEC migration. The quantitative data demonstrated the specificity and direct effect of LPA-induced IL-8 in mediating endothelial cell migration (Fig. 6AGo, right panel).


Figure 6
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  		FIG. 6. Angiogenic functions of LPA-induced IL-8 protein. CM from primary culture of endometrial stromal cells with indicated conditions was examined. A, hEMVEC migration assay. Left panel, a, Vehicle-treated CM; b, LPA (10 µM)-treated CM; c, LPA (10 µM)-containing medium after 24 h incubation; d, LPA-treated CM preincubated with IL-8 neutralizing antibody (IL-8 Ab) (2 µg/ml); e, LPA-treated CM preincubated with nonspecific IgG (2 µg/ml); f, LPA-treated CM from endometrial stromal cells that had been pretreated with antisense (AS) IL-8 oligonucleotides (10 µM) for 24 h before LPA treatment; g, LPA-treated CM from endometrial stromal cells pretreated with sense (S) IL-8 oligonucleotides (10 µM); h, LPA-treated CM from endometrial stromal cells pretreated with antisense IL-8 oligonucleotides added with rhIL-8; i, hEMVECs were treated with rhIL-8 (10 ng/ml). Right panel, Quantitative results of migrated hEMVECs are presented. Comparison is made between the LPA-treated CM group and different treatment groups. *, P < 0.05 (n = 5). B, hEMVEC monolayer permeability assay. Data are the relative permeability percent of indicated conditions in that vehicle-treated CM in lane 1 is defined as 100%. *, P < 0.05 (n = 5). C, hEMVEC capillary tube formation assay. Left panel, The indicated conditions were same as those of the migration assay. Right panel, Quantitative results of the number of hEMVEC tubing are shown. *, P < 0.05 (n = 5). D, hEMVEC proliferation assay. Data are the relative cell number percent of indicated conditions in that vehicle-treated CM in lane 1 is defined as 100%. *, P < 0.05 (n = 5).

 
With hEMVEC permeability assay (Fig. 6BGo), we found increased permeability of hEMVEC monolayer when incubated with LPA-treated CM (lane 2), compared with vehicle-treated CM (lane 1). LPA-containing medium with 24 h incubation did not enhance permeability (lane 3). In addition, LPA-treated CM preincubated with IL-8 antibodies (lane 4) or LPA-treated CM from endometrial stromal cells pretreated with antisense IL-8 oligonucleotides (lane 6) did not increase permeability. In contrast, LPA-treated CM preincubated with nonspecific IgG (lane 5) or LPA-treated CM from cells pretreated with sense oligonucleotides (lane 7) augmented permeability. Alternatively, LPA-treated CM from cells pretreated with antisense IL-8 oligonucleotides added with rhIL-8 (lane 8) or the rhIL-8-containing medium (lane 9) enhanced permeability. These results verified the specificity and direct effect of LPA-induced IL-8 in increasing endothelial cell permeability.

In the test of hEMVEC capillary tube formation (Fig. 6CGo, left panel), we found increased tube formation of hEMVECs when incubated with LPA-treated CM (b), compared with vehicle-treated CM (a). LPA-containing medium with 24 h incubation did not augment tube formation (c). In addition, LPA-treated CM preincubated with IL-8 neutralizing antibodies (d) or LPA-treated CM from endometrial stromal cells pretreated with antisense IL-8 oligonucleotides (f) did not increase hEMVEC tube formation. On the contrary, LPA-treated CM preincubated with nonspecific IgG (e) or LPA-treated CM from cells pretreated with sense oligonucleotides (g) significantly augmented hEMVEC tube formation capability. Conversely, LPA-treated CM from cells pretreated with antisense IL-8 oligonucleotides added with rhIL-8 (h) or the rhIL-8-containing medium (i) significantly enhanced hEMVEC tube formation. The quantitative data indicated the specificity and direct effect of LPA-induced IL-8 in augmenting hEMVEC tube formation (Fig. 6CGo, right panel).

In the examination of hEMVEC proliferation (Fig. 6DGo), we observed increased growth of hEMVECs when incubated with LPA-treated CM (lane 2), compared with vehicle-treated CM (lane 1). LPA-containing medium with 24 h incubation did not enhance hEMVEC growth (lane 3). However, LPA-treated CM preincubated with IL-8 antibodies (lane 4) or LPA-treated CM from endometrial stromal cells pretreated with antisense IL-8 oligonucleotides (lane 6) did not augment hEMVEC growth. In contrast, LPA-treated CM preincubated with nonspecific IgG (lane 5) or LPA-treated CM from cells pretreated with sense oligonucleotides (lane 7) augmented hEMVEC growth. LPA-treated CM from cells pretreated with antisense IL-8 oligonucleotides added with rhIL-8 (lane 8) or the rhIL-8-containing medium (lane 9) increased hEMVEC growth. These results indicated the specificity and direct effect of LPA-induced IL-8 in enhancing endothelial cell growth.

The representation of LPA function through LPA1 receptors and signaling pathways in endometrial stromal cells was schematically summarized in Fig. 7Go.


Figure 7
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  		FIG. 7. Schematic signaling and possible function of LPA-enhanced IL-8 expressions in endometrial stromal cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we first demonstrate that LPA1 receptors are expressed in human endometrial stromal cells in both the proliferative and secretory phases. In primary culture of endometrial stromal cells, LPA up-regulates IL-8 expression in a concentration- and time-dependent manner. We further prove that LPA-induced IL-8 protein functionally enhances multistep processes of angiogenesis including migration, permeability, capillary tube formation, and proliferation of endothelial cells. The process of implantation and pregnancy entails angiogenesis of endometrium and placenta. Increased concentration of LPA in pregnancy may mediate endometrial stromal cells to secrete IL-8 and regulate neovasculogenesis. Therefore, LPA may play a role in angiogenesis for the establishment of pregnancy.

The expression of hCG/LH receptors had been verified in human endometrium throughout the proliferative and secretory phases (24, 25). Via its receptors to induce VEGF secretion, hCG was suggested to enhance angiogenesis of endometrium for promoting blastocyst implantation (7). IL-8 has been implicated in the involvement of normal menstruation, endometrial receptivity for embryonic implantation, and pregnancy (26, 27, 28). The modulation of IL-8 secretion was found to be enhanced by other cytokines such as TNF-{alpha}, IL-1β, and thrombin in human first-trimester decidua cells (29). Recently IL-8 mRNA expression was detected to be significantly induced by prokineticin 1 in human endometrium during early pregnancy (30). Adiponectin decreased IL-1β-induced secretion of IL-8 from endometrial stromal cells (31). In this work, we demonstrated that LPA significantly induced IL-8 secretion but not VEGF in human endometrial stromal cells. On the contrary, hCG induced VEGF secretion but not IL-8. The LPA-induced IL-8 stimulated angiogenesis of endothelial cells in vitro. Our results suggest that LPA may play a role in up-regulation of IL-8 during pregnancy.

We validate that through LPA1 receptors, the Gi protein, MAPK/p38, and NF-{kappa}B pathway is involved in LPA-induced IL-8 expression in endometrial stromal cells. Recently LPA was found to induce IL-8 expression in bronchial epithelial cells (17) and human umbilical vein endothelial cells (32) as well as IL-8 and IL-6 expressions in dendritic cells (33) and granulosa-lutein cells (20). These enhancement effects are also NF-{kappa}B dependent (17, 20, 32). LPA appears to be an important physiological regulator for IL-8 or IL-6 secretions among human tissues.

In developmental studies of mice, LPA has been found to play important roles in embryo development and implantation. LPA enhanced the growth of two- or four-cell mouse embryos to blastocysts (34) and accelerated blastocyst outgrowth and differentiation (35, 36). Mouse uteri expressed LPA3 receptors, and LPA may induce uterine cyclooxygenase-2 to enhance prostaglandins E2 and I2 generation (37). Targeted deletion of the LPA3 receptor resulted in delayed implantation and altered embryo spacing and significantly reduced litter size. In humans, we first indicate that through LPA1 receptors, LPA induces IL-8 in endometrial stromal cells that may enhance angiogenesis of the endometrium and placenta.

In our previous study (20), we found that granulosa-lutein cells expressed LPA1, LPA2, and LPA3 receptors. Through LPA receptors, LPA induced IL-8 and IL-6 secretions that IL-8 enhanced multisteps of angiogenesis of endothelial cells and IL-6 enhanced permeability. Ovarian cancer had been found to express LPA1, LPA2, and LPA3 receptors and to produce excessive LPA. LPA stimulated VEGF, IL-6, and IL-8 secretions in ovarian cancer and augmented tumor growth, angiogenesis, and invasion (14, 21, 22, 23, 38). It appears that in contrast to ovarian cancer inducing angiogenesis by LPA, the effect of LPA on physiological angiogenesis of endometrium and corpus luteum is mainly by IL-8 but not through VEGF.

LPA could be produced by extracellular lysophospholipase D or activated platelets, leukocytes, or epithelial cells. It had been found that serum lysophospholipase D activity increased during pregnancy (10). High lysophospholipase activity was present in the human placental tissues and amnion (39). However, the origin and local concentrations of LPA and its regulation by autocrine/paracrine factors in the human uterine endometrium during the menstrual cycle and pregnancy remain unclear that deserve further investigation.

We demonstrate that LPA1 receptors are equally expressed in human endometrial stromal cells in the proliferative and secretory phases as well as first-trimester decidua. LPA induces IL-8 expression through the LPA1 receptor, Gi protein, MAPK/p38, and NF-{kappa}B signal pathway. LPA-induced IL-8 protein stimulates migration, permeability, capillary tube formation, and proliferation of endothelial cells, which are critical steps involved in the angiogenesis. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.


   Acknowledgments
 
The authors are grateful for Ms. Tzu-Hsin Chen and Ms. Chung-Ru Chen for the technical assistance.


   Footnotes
 
This study was supported in part by Grant 96-2314-B-090-MY2 from the National Science Council and Grant 95A707 from the National Taiwan University Hospital, Taipei, Taiwan.

Disclosure Statement: All authors have nothing to declare.

First Published Online July 10, 2008

Abbreviations: AP-1, Activator protein-1; CM, conditioned medium; Edg, endothelial differentiation gene; EIA, enzyme immunoassay; hCG, human chorionic gonadotropin; hEMVEC, human endometrial microvascular endothelial cell; IHC, immunohistochemistry; LPA, lysophosphatidic acid; NF-{kappa}B, nuclear factor-{kappa}B; PTX, pertussis toxin; rh, recombinant human ; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Received March 6, 2008.

Accepted for publication July 1, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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