help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1512
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, S.-U.
Right arrow Articles by Yang, Y.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, S.-U.
Right arrow Articles by Yang, Y.-S.
Related Collections
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 3 935-943
Copyright © 2008 by The Endocrine Society

Lysophosphatidic Acid Up-Regulates Expression of Interleukin-8 and -6 in Granulosa-Lutein Cells through Its Receptors and Nuclear Factor-{kappa}B Dependent Pathways: Implications for Angiogenesis of Corpus Luteum and Ovarian Hyperstimulation Syndrome

Shee-Uan Chen, Chia-Hung Chou, Hsinyu Lee, Chi-Hong Ho, Chung-Wu Lin and Yu-Shih Yang

Departments of Obstetrics and Gynecology (S.-U.C., C.-H.C., C.-H.H., Y.-S.Y.), of Life Science (H.L.), and of Pathology (C.-W.L.), National Taiwan University, Taipei, 100 Taiwan

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


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Lysophosphatidic acid (LPA) was found at significant amounts in follicular fluid of preovulatory follicle. The lysophospholipase D activity of serum from women receiving ovarian stimulation was higher than women with natural cycles. Angiogenic cytokines, including IL-6, IL-8, and vascular endothelial growth factor, increased in plasma and ascites of patients with ovarian hyperstimulation syndrome. The role of LPA in ovarian follicles is unclear.

Objective: Our objective was to investigate the expression of LPA receptors and function of LPA in granulosa-lutein cells.

Design: Granulosa-lutein cells were obtained from women undergoing in vitro fertilization. We examined the expression of LPA receptors using RT-PCR. The effects of LPA on the expression of IL-6, IL-8, and vascular endothelial growth factor were examined. Signal pathways of LPA were delineated. The functions of secretory angiogenic factors were tested using human umbilical vein endothelial cells.

Results: The LPA1, LPA2, and LPA3 receptors’ mRNA was identified in granulosa-lutein cells. LPA enhanced IL-8 and IL-6 expressions in a dose- and time-dependent manner. LPA functioned via LPA receptors, Gi protein, MAPK/ERK, p38, phosphatidylinositol 3-kinase/Akt, and nuclear factor-{kappa}B, and transactivation of epidermal growth factor receptor. LPA induced IL-8 and IL-6 through different pathways. LPA-induced IL-8 and IL-6 increased permeability of human umbilical vein endothelial cell monolayer.

Conclusions: LPA induces IL-8 and IL-6 expressions through LPA receptors and nuclear factor-{kappa}B dependent pathways in granulosa-lutein cells. The LPA in preovulatory follicles may play a role in the angiogenesis of corpus luteum. Large amounts of LPA-induced IL-8 and IL-6 from multiple corpora luteae of stimulated ovaries may be one of the pathophysiological causes of ovarian hyperstimulation syndrome.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
In the natural menstrual cycle of women, out of a cohort of ovarian follicles, a dominant follicle is selected to ovulate. The development of the preovulatory follicle is associated with increased density of blood vessels within the theca cell layers. These vessels do not penetrate into the granulosa cell layers. After the LH surge and ovulation, the luteinized granulosa cells and theca cells form a corpus luteum with high levels of angiogenesis (1). Neovascularization may facilitate steroid hormone biosynthesis and transportation. However, the relevant molecular mechanisms of follicle rupture and neovascularization are still obscure. A number of growth factors, cytokines, and phospholipids may exert modulatory effects (2, 3). These include IL-6, vascular endothelial growth factor (VEGF), secreted protein acidic and rich in cysteine, IL-8, thrombospondin, angiogenin, basic fibroblast growth factor, angiopoietins 1 and 2, and sphingosine 1-phosphate (3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

Lysophosphatidic acid (LPA), a biologically active phospholipid, plays critical roles in physiological and pathological processes, including inflammation, cell proliferation, angiogenesis, wound healing, and cancer invasion (13, 14). It could be produced through the hydrolysis of phospholipids by extracellular lysophospholipase D (15) or by activated platelets, leukocytes, epithelial cells, and tumor cells (16, 17, 18). The serum concentration of LPA in healthy subjects ranges from 0.1–6.3 µM (19). LPA had been detected to exist at considerable amounts in follicular fluid of preovulatory follicles at levels up to 25 µM (20). However, the physiological role of LPA in human ovarian follicles remains elusive.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of ovarian stimulation. In severe cases a critical condition develops with massive ascites, pleural effusion, hemoconcentration, and oliguria. The underlying mechanism is due to an increase in the capillary permeability with acute fluid shift out of the intravascular space (21, 22). Several angiogenic cytokines, including IL-6, IL-8, and VEGF, have been abundant in the plasma and ascites of OHSS patients, and been attributed to pathogenetic factors (23, 24). It is thought that these factors are mainly secreted by multiple corpora luteae (25, 26). The lysophospholipase D activity of the serum of women receiving ovarian stimulation was significantly higher than that of women with natural cycles (20). The relation of LPA and increased angiogenic cytokines in patients undergoing ovarian stimulation merits further investigation.

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 (27). In humans, whereas the expression of the LPA receptors has been reported in some healthy tissues and cancer cells (14), the expression of the LPA receptor isoforms in the granulosa-lutein cells is unknown. In the present study, we attempt to determine the possible role of LPA in ovulation, angiogenesis of corpus luteum, and OHSS. We first investigated the LPA receptors of granulosa-lutein cells. The effects of LPA on granulosa-lutein cells regarding expressions of angiogenic factors of IL-6, IL-8, and VEGF, and the signaling pathways were explored. We then examined whether LPA-induced angiogenic factors modified migration, permeability, capillary tube formation, or proliferation of endothelial cells.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Granulosa-lutein cell culture

This study was approved by the ethics committee of the National Taiwan University Hospital. Granulosa-lutein cells were obtained from women undergoing oocyte retrieval for in vitro fertilization treatment. Informed consent was obtained from each patient. Follicular fluid from all follicles was collected and then centrifuged at 350 x g for 5 min. The cells were resuspended in 10 ml HEPES-buffered human tubal fluid medium. The cell suspensions were added on 10 ml Ficoll (Sigma-Aldrich, St. Louis, MO). After centrifugation at 450 x g for 15 min, the interphase cells were collected. The cells were treated with 80 IU/ml hyaluronidase in 1 ml human tubal fluid for 10 min. The cells were washed and suspended in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. The granulosa-lutein cells were seeded in a flask. On the second day since collection, the cells were washed to remove remaining red blood cells or leukocytes because these did not adhere to the plastic surface. The cells were then incubated at 37 C in a humidified atmosphere with 5% CO2 in air.

Antibodies and reagents

Pertussis toxin (PTX), Ki16425, AG1478, LY294002, PD98059, SB203580, 1-Oleoyl-LPA, and fatty acid-free BSA were purchased from Sigma-Aldrich. LPA was dissolved in vehicle of PBS containing 1% fatty acid-free BSA. Recombinant human IL-8 and IL-6 neutralizing antibodies were obtained from R&D Systems, Inc. (Minneapolis, MN). Antibodies to human phospho-p38, ERK, and Akt were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

RT-PCR

The total RNA was isolated from the granulosa-lutein cells using the RNAzol B reagent (Biotecx Laboratories, Houston, TX). cDNA was then prepared from 2 µg of the total RNA with random hexamer primers (ImProm-II RT system; Promega, Southampton, UK). The specific oligonucleotide primer pairs were as follows: LPA1, LPA2, and LPA3 described by Fang et al. (28); IL-8, 5'-ACT TCC AAG CTG GCC GTG GCT CTC TTG GCA-3' and 5'-TGA ATT CTC AGC CCT CTT CAA AAA CTT CTC-3' (295 bp); IL-6, 5'-CTT CGG TCC AGT TGC CTT CT-3' and 5'-AGG AAC TCC TTA AAG CTG CG-3' (609 bp); and β-actin, 5'-CTT CTA CAA TGA GCT GCG TG-3' and 5'-TCA TGA GGT AGT CAG TCA GG-3' (305 bp).

Real-time quantitative RT-PCR

We further quantified IL-6 and IL-8 mRNA expression in various conditions. The IL-6 and IL-8 cDNA was analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). The primer pairs were: for IL6, 5'-GCC TTC GGT CCA GTT GCC TT-3' and 5'-GCA GAA TGA GAT GAG TTG TC-3'; for IL-8, 5'-TTT CTG CAG CTC TCT GTG AGG-3' and 5'-CTG CTG TTG TTG TTG CTT CTC-3'; and for glyceraldehyde-3-phosphate dehydrogenase, 5'-GGG AAG GTG AAG GTC GG-3' and 5'-TGG ACT CCA CGA CGT ACT CAG-3'. Amplification was followed by melting curve analysis to verify the correctness of the amplicon. A negative control without cDNA was run with every PCR to assess the specificity of the reaction. Analysis of data was performed using LightCycler software (Roche Diagnostics Ltd., Burgess Hill, UK). PCR efficiency was determined by analyzing a dilution series of cDNA (external standard curve). The amount of IL-6 or 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.

Enzyme immunoassay (EIA)

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

Promoter construction and reporter assays

The human IL (hIL)-6 and hIL-8 promoters in a luciferase activity reporter system were constructed. Transfections of phIL6–1.2Kb, phIL8–1.4Kb, or nuclear factor (NF)-{kappa}B binding site-driven luciferase plasmids (BD Bioscience, Palo Alto, CA) into granulosa-lutein cells were performed in six-well plates 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. To control the transfection efficiency, cells were cotransfected with pSV-β-galactosidase, and data normalizations after all transient transfections were conducted using triplicate cultures.

RNA interference

Small interfering RNA duplexes (siRNA) were purchased from Santa Cruz Biotechnology. The targeted siRNA of LPA1, LPA2, and LPA3 were sc-43746, sc-39926, and sc-37088, respectively. Granulosa-lutein cells were transfected with siRNA at the concentrations of 25 nM in serum-free Opti-MEM by using the Oligofectamine method (Invitrogen Corp., Carlsbad, CA).

Western blotting

The granulosa-lutein 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 (Bio-Rad Laboratories, 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 (AP)-1 decoy oligodeoxynucleotides to granulosa-lutein cells

Synthetic double-stranded oligodeoxynucleotides were used as "decoy" cis-elements to block the binding of NFs to promoter regions of the targeted genes, thus inhibiting gene transactivation. We used the EMSA to examine the specific effect of NF-{kappa}B decoy and AP-1 decoy (29). For transfection of granulosa-lutein cells, the NF-{kappa}B decoy, AP-1 decoy, or scrambled decoy was mixed with the Transfast transfection reagent for 15 min and then incubated with the cells in a serum-free medium.

Human umbilical vein endothelial cells (HUVECs) trans-well migration assay

A total of 2 x 104 HUVECs in 200 µl culture medium was added to the upper chamber of the 24-well Millicell inserts (8 µm pore; Millipore Corporate, Bedford, MA), with 500 µl culture medium added to the lower chamber. When the cells attached to the insert (~6-h incubation), the medium was changed to serum-free medium in the upper chamber and to the indicated conditioned medium (CM) in the lower chamber. After 6 h, the migrated HUVECs were counted.

HUVEC monolayer permeability assay

HUVECs were cultured in trans-well chambers (0.4 µm pore-size polycarbonate filters; Costar Corp., Cambridge, MA). After reaching confluence, the cells were washed, and then 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 kD; 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).

HUVEC capillary tube formation assay

HUVECs (5 x 104) in the medium of indicated conditions were plated on 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 and photographed.

HUVEC proliferation assay

HUVECs were seeded in 96-well plates at densities of 1 x 104 cells per well using 0.2 ml 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 HUVECs 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 a Tukey test for multiple comparisons. The significance level was set as P < 0.05 by a two-tailed test. SAS software version 8.01 (SAS Institute Inc., Cary, NC) was used for calculation.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Human granulosa-lutein cells express LPA-specific receptors

We first detected the mRNA of LPA1, LPA2, and LPA3 receptors using RT-PCR in granulosa-lutein cells, in comparison with the SK-OV3 cell line, which was a well-known ovarian cancer cell line proved to express LPA1, LPA2, and LPA3, and HUVEC, which was proved to express LPA1 only (Fig. 1Go). The results indicated that LPA1, LPA2, and LPA3 mRNA was all expressed in the granulosa-lutein cells of four individual cases.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 1. The mRNA expression of LPA receptors in human granulosa-lutein cells. The total RNA of the cultured granulosa-lutein cells (GLC), HUVEC (H), and SK-OV3 cells (S) was extracted under normal culture conditions. LPA1, LPA2, and LPA3 receptors were detected using RT-PCR with specific primers.

 
LPA enhances IL-8 and IL-6 expression in human granulosa-lutein cells at both protein and mRNA levels

Using EIA (Fig. 2AGo), we found that LPA significantly increased IL-8 (6.7 ± 1.4-fold) and IL-6 (4.9 ± 1.1-fold) protein secretions of granulosa-lutein cells in 24 h. However, LPA did not induce VEGF secretion. Human chorionic gonadotropin (HCG) significantly increased VEGF secretion (1.9 ± 0.2-fold) but did not enhance IL-8 and IL-6 secretions. In 48-h LPA treatment, IL-8 protein level increased to 8.4 ± 1.6-fold and IL-6 to 6.1 ± 1.2-fold. In addition, VEGF remained unchanged. Of HCG treatment, VEGF secretions increased to 3.4 ± 0.4-fold, and IL-8 and IL-6 levels did not increase.


Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 2. LPA induction of IL-8 and IL-6 expressions. A, Different induction patterns of IL-8, IL-6, and VEGF protein secretions between LPA and HCG. Granulosa-lutein cells were serum starved for 24 h and then treated with LPA (10 µM) or HCG (1 IU/ml) for 24 h (left panel) and 48 h (right panel). The supernatant was detected for IL-8, IL-6, and VEGF levels via EIA. Data are the relative folds of induction, comparing with vehicle-treated controls (PBS containing 1% fatty acid-free BSA for LPA groups; 0.5% dimethylsulfoxide for HCG groups; n = 5). B, Granulosa-lutein cells were treated with LPA (10 µM) for various time lengths. The expressions of IL-8 and IL-6 mRNA were determined by RT-PCR (left panel). The real-time quantitative RT-PCR results are shown in the right panel (n = 5). C, Granulosa-lutein cells were transfected with IL-8 reporter plasmids. The transfected cells were treated with indicated doses of LPA. After 4 h, the luciferase activity of the IL-8 promoter was measured by a luminometer. Data are the relative folds of induction, comparing with vehicle-treated controls (left panel). *, P < 0.05 (n = 7). Granulosa-lutein cells were transfected with IL-6 reporter plasmids and assayed as described previously (right panel).

 
Using real-time quantitative RT-PCR (Fig. 2BGo), we found that mRNA expressions of IL-8 and IL-6 increased in a time-dependent manner when treated with LPA. After the 4-h treatment, the levels of IL-8 and IL-6 mRNA (3.2 ± 0.3-fold in IL-8 and 2.4 ± 0.2-fold in IL-6, compared with controls) reached the maximum. We examined the dosage effect of LPA on IL-8 and IL-6 expressions using promoter reporter assays (Fig. 2CGo). The results revealed that the fold of induction of IL-8 promoter luciferase activity increased in a dose-dependent manner from 1–10 µM (6.3 ± 1.5 to 17 ± 4-fold). At the concentration of 20 µM (16 ± 2.6-fold), the elevated level of IL-8 promoter activity was not significantly different from that of 10 µM. A similar pattern was also found in the induction of IL-6 promoter activity from 1–10 µM (3.0 ± 1.5 to 13.3 ± 4-fold). At the concentration of 20 µM (14.6 ± 3.2-fold), the elevated level was not significantly distinct from that of 10 µM.

Signal transduction pathways involved in LPA-mediated IL-8 and IL-6 expressions in granulosa-lutein cells

Using real-time quantitative RT-PCR (Fig. 3AGo), we found that LPA-enhanced IL-8 mRNA expression was significantly diminished by Ki16425, Gi inhibitor (PTX), and epidermal growth factor receptor (EGFR) inhibitor (AG1478). On the other hand, LPA-enhanced IL-6 mRNA expression was significantly reduced by PTX. However, Ki16425 and AG1478 did not have inhibitory effects. We further applied specific LPA receptor siRNA to affect mRNA expressions of LPA receptors in granulosa-lutein cells (Fig. 3BGo). We found that the LPA1 siRNA reduced LPA-inducing IL-8 secretion, and the LPA2 siRNA decreased the LPA-inducing IL-6 secretion. The results indicated an involvement of the PTX-sensitive Gi protein-coupled LPA receptors in the LPA-enhanced IL-8 and IL-6 expressions. LPA induced IL-8 expression through LPA1, and IL-6 expression through LPA2. LPA-enhanced IL-8 expressions were partially through transactivation of the EGFR.


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 3. Receptors involved in LPA-mediated IL-8 or IL-6 expression. A, Granulosa-lutein cells were serum starved for 24 h and then pretreated with Ki16425 (10 µM), PTX (100 ng/ml), or AG1478 (10 µM) for 1 h before LPA (10 µM) treatment. After 4 h, the expressions of IL-8 and IL-6 mRNA were determined by RT-PCR (left panel). The real-time quantitative RT-PCR results are shown in the right panel. Data are compared between the LPA-treated only group and different inhibitor groups. *, P < 0.05 (n = 5). B, Confirmation of the effect of specific LPA siRNA on the mRNA expression of LPA receptors in granulosa-lutein cells using RT-PCR (left panel). The effects of specific LPA siRNA on the LPA-inducing IL-8 and IL-6 protein levels using EIA (right panel). *, P < 0.05 (n = 5).

 
We further explored the signal transduction mediators involved in LPA-induced IL-8 and IL-6 expressions. Using real-time quantitative RT-PCR (Fig. 4AGo), we found that LPA-induced IL-8 mRNA expression was significantly reduced by MAPK/ERK inhibitor (PD98059), phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor (LY294002), and p38 inhibitor (SB203580). However, JNK inhibitor (SP600125) did not have an inhibitory effect. On the other hand, LPA-induced IL-6 mRNA expression was significantly decreased by p38 inhibitor (SB203580). Using Western blotting, we verified that LPA induced phosphorylation of Akt, ERK, and p38. In addition, the phosphorylation was diminished using PTX pretreatment (Fig. 4BGo). LPA also induced EGFR phosphorylation that was inhibited by PTX pretreatment (Fig. 4CGo). The phosphorylation of ERK, but not of Akt and p38, was significantly reduced by the EGFR inhibitor (AG1478) (Fig. 4DGo). Therefore, LPA may transactivate EGFR through the Gi protein and then activate the ERK. The results indicated that LPA induced IL-8 expression via MAPK/ERK, PI3K/Akt, and p38, as well as induced IL-6 expression via p38.


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 4. Signal transduction mediators involved in LPA-mediated IL-8 or IL-6 expression. A, Granulosa-lutein cells were pretreated with SP600125 (10 µM), PD98059 (50 µg/ml), LY294002 (50 µg/ml), or SB203580 (5 µg/ml) for 1 h before LPA 10 (µM) treatment. After 4 h, expressions of IL-8 and IL-6 mRNA were determined by RT-PCR (left panel). The real-time quantitative RT-PCR results are shown in the right panel. *, P < 0.05 (n = 5). B, LPA-induced phosphorylation of Akt, ERK, and p38 was Gi dependent. Granulosa-lutein cells were pretreated with PTX for 1 h before LPA (10 µM) stimulation. After 1 h, the protein levels of phosphorylated forms of indicated protein kinases of the cell lysates were detected using Western blotting. C, LPA induced phosphorylation of EGFR through Gi protein. The examination of EGFR phosphorylation was the same as the aforementioned method. D, LPA induced phosphorylation of ERK through transactivation of EGFR. Granulosa-lutein cells were pretreated with EGFR inhibitor (AG1478), and the other procedures for detection of phosphorylated forms of indicated protein kinases were the same.

 
We verified the specific binding effect of the NF-{kappa}B decoy and AP-1 decoy using the EMSA with antibody supershift analysis (Fig. 5AGo). We further found that the NF-{kappa}B decoy, but not the AP-1 decoy, significantly inhibited LPA-induced IL-8 and IL-6 secretions (Fig. 5Go, B and C). LPA strongly enhanced the NF-{kappa}B binding site-driven luciferase activity (8.7 ± 0.9-fold) that was significantly reduced by inhibitors of Gi (PTX), Akt (LY294002), ERK (PD98059), and p38 (SB203580) (Fig. 5DGo). These results indicate that NF-{kappa}B plays a critical role in LPA-induced IL-8 and IL-6 expressions.


Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		FIG. 5. NF-{kappa}B is critically involved in LPA-induced IL-8 and IL-6 expressions. A, The specific work of the NF-{kappa}B decoy and AP-1 decoy was confirmed by the EMSA. NF-{kappa}B decoy (left panel) or AP-1 decoy (right panel) was biotin-labeled, and then 20 fmol was incubated in a binding reaction containing granulosa-lutein cell nuclear extract. The biotin-labeled decoy and nuclear protein complex were resolved by gel electrophoresis (lane 2). Lane 1, The nuclear extract was omitted from the reaction. Lane 3, The nuclear extract was treated with antibodies to NF-{kappa}B (anti-p65 antibody) or AP-1 (anti-c-Jun antibody) for 30 min before the addition of biotin-labeled decoy. The supershift bands disclosed the specificity of binding reactions of NF-{kappa}B to the NF-{kappa}B decoy and AP-1 to the AP-1 decoy, respectively. Lane 4, The nuclear extract was treated with nonspecific IgG before the addition of biotin-labeled decoy. Lane 5, The reaction contained a 200-fold molar excess of unlabeled decoy. B, Granulosa-lutein cells were transfected with the NF-{kappa}B decoy, AP-1 decoy, or scrambled decoy under serum starvation for 24 h, and then treated with LPA (10 µM) for another 24 h. The following sequences of the phosphorothioate oligodeoxynucleotides were used: NF-{kappa}B decoy, 5'-CCT TGA AGG GAT TTC CCT CC-3' and 3'-GGA ACT TCC CTA AAG GGA GG-5'; scrambled (S) NF-{kappa}B decoy, 5'-TTG CCG TAC CTG ACT TAG CC-3' and 3'-AAC GGC ATG GAC TGA ATC GG-5' (as negative control); AP-1 decoy, 5'-TGT CTG ACT CAT GTC-3' and 3'-ACA GAC TGA GTA CAG-5'; and scrambled AP-1 decoy, 5'-TGT CTC TCT GAT GTC-3' and 3'-ACA GAG AGA CTA CAG-5' (as negative control). IL-8 levels in the supernatant were detected by EIA. Data were compared between the LPA-treated only group and different decoy groups. *, P < 0.05 (n = 5). C, IL-6 levels were examined and compared as described previously. D, NF-{kappa}B binding site-driven luciferase activity assay. Data are compared between the LPA-treated only group and different inhibitor groups. *, P < 0.05 (n = 5).

 
LPA-induced IL-8 and IL-6 protein secretions from granulosa-lutein cells enhance angiogenesis

Using trans-well migration assay (Fig. 6AGo), we observed increased migration of HUVECs when incubated with LPA-treated CM (Fig. 6AGo, left panel, b), compared with vehicle-treated CM (Fig. 6AGo, left panel, a). LPA-treated CM preincubated with IL-8 neutralizing antibodies significantly diminished this enhancing effect (Fig. 6AGo, left panel, c). LPA-treated CM preincubated with IL-6 neutralizing antibodies did not inhibit the enhancing effects (Fig. 6AGo, left panel, d). The quantitative results demonstrated the specificity and direct effect of LPA-induced IL-8 in mediating HUVEC migration. LPA-treated CM preincubated with isotype IgG preserved the enhancing capability (Fig. 6AGo, right panel).


Figure 6
View larger version (53K):
[in this window]
[in a new window]

  		FIG. 6. Angiogenic functions of LPA-induced IL-8 and IL-6 protein secretions from granulosa-lutein cells. A, HUVEC trans-well migration assay. Left panel, a, Vehicle-treated CM. b, LPA (10 µM)-treated CM. c, LPA-treated CM preincubated with IL-8 neutralizing antibody (IL-8 ab). d, LPA-treated CM preincubated with IL-6 neutralizing antibody (IL-6 ab). Right panel, Quantitative results of migrated HUVECs. Comparison is made between the LPA-treated only group and different antibody groups. *, P < 0.05 (n = 5). B, HUVEC monolayer permeability assay. Data are the relative permeability percentage (%) of indicated conditions in that vehicle-treated CM in column 1 is defined as 100%. *, P < 0.05 (n = 5). C, HUVEC capillary tube formation assay. Left panel, a, Vehicle-treated CM. b, LPA (10 µM)-treated CM. c, LPA-treated CM preincubated with IL-8 neutralizing antibody. d, LPA-treated CM preincubated with IL-6 neutralizing antibody. Right panel, Quantitative results of the number of HUVEC tubing. *, P < 0.05 (n = 5). D, HUVEC proliferation assay. Data are the relative cell number percentage (%) of indicated conditions in that vehicle-treated CM in column 1 is defined as 100%. *, P < 0.05 (n = 5).

 
With monolayer permeability assay (Fig. 6BGo), we detected increased permeability of HUVEC monolayer when incubated with LPA-treated CM (Fig. 6BGo, lane 2), compared with vehicle-treated CM (Fig. 6BGo, lane 1). LPA-treated CM preincubated with IL-8 neutralizing antibodies significantly diminished the enhancing effects (Fig. 6BGo, lane 3). LPA-treated CM preincubated with IL-6 neutralizing antibodies also significantly reduced the enhancing effects (Fig. 6BGo, lane 4). LPA-treated CM preincubated with isotype IgG maintained the enhancing capability (Fig. 6BGo, lane 5). These results suggested the specificity and direct effects of LPA-induced IL-8 and IL-6 in increasing permeability of HUVEC monolayer.

In the test of HUVEC capillary tube formation (Fig. 6CGo), we found increased tube formation of HUVECs when incubated with LPA-treated CM (Fig. 6CGo, left panel, b), compared with vehicle-treated CM (Fig. 6CGo, left panel, a). LPA-treated CM preincubated with IL-8 neutralizing antibodies did not enhance HUVEC tube formation (Fig. 6CGo, left panel, c). However, LPA-treated CM preincubated with IL-6 neutralizing antibodies maintained the enhancing effects (Fig. 6CGo, left panel, d). The quantitative data indicated the specificity and direct effects of LPA-induced IL-8 in mediating HUVEC tube formation (Fig. 6CGo, right panel).

In the examination of HUVEC proliferation (Fig. 6DGo), we observed increased growth of HUVECs when incubated with LPA-treated CM (Fig. 6DGo, lane 2), compared with vehicle-treated CM (Fig. 6DGo, lane 1). LPA-treated CM preincubated with IL-8 neutralizing antibodies significantly reduced the enhancing effects (Fig. 6DGo, lane 3). LPA-treated CM preincubated with IL-6 neutralizing antibodies did not diminish the enhancing effects (Fig. 6DGo, lane 4). The results implied the specificity and direct effects of LPA-induced IL-8 in augmenting HUVEC proliferation.

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


Figure 7
View larger version (31K):
[in this window]
[in a new window]

  		FIG. 7. Schematic signaling and possible function of LPA-enhanced IL-8 and IL-6 expressions in granulosa-lutein cells.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study, we first demonstrate that human granulosa-lutein cells express LPA1, LPA2, and LPA3 receptors. In primary culture of granulosa-lutein cells, LPA up-regulates IL-8 and IL-6 expressions in a concentration- and time-dependent mode. We further verify that LPA-induced IL-8 protein functionally enhances multistep processes of angiogenesis, including migration, permeability, tube formation, and proliferation of endothelial cells, and IL-6 enhances permeability. The presence of significant concentrations of LPA in preovulatory follicles may mediate granulosa-lutein cells to secrete IL-8 and IL-6, and regulate neovascularization. Therefore, LPA may play a role in the angiogenesis of the corpus luteum.

High concentrations of VEGF, IL-6, and IL-8 have been found in the plasma and ascites of patients with OHSS (23, 24). These angiogenic cytokines are mainly secreted from multiple corpora lutea after ovarian stimulation (25, 26). Previous studies indicated that the secretion of VEGF of granulosa-lutein cells was principally induced by LH or HCG (5). However, the regulation of the IL-6 and IL-8 secretions remained unclear. These were enhanced by other cytokines, such as TNF-{alpha}, IL-1{alpha}, and IL-1β (30, 31). In our study we found that HCG induced VEGF secretion in granulosa-lutein cells, but not IL-8 and IL-6. LPA induced IL-8 and IL-6 secretions, but not VEGF.

The primary mechanism of OHSS is due to increased capillary permeability induced by angiogenic cytokines that results in acute fluid and albumin loss from the intravascular space into the third space (21, 22). LPA-induced IL-8 and IL-6 in granulosa-lutein cells functionally increased permeability of endothelial cells. Therefore, we suggest that excessive IL-8 and IL-6 secretions from multiple corpora lutea induced by LPA may be a contributing cause of OHSS. Intravenous albumin administration has been applied to prevent and treat OHSS (26). It has been found that serum albumin and other LPA-binding proteins modify the cellular function of LPA (32, 33, 34). The therapeutic effects of albumin administration for OHSS may be partly through reducing LPA function, which deserves further investigation.

Ki16425 is an LPA receptor antagonist, especially on LPA1 and LPA3, that blocks some biological actions of LPA (35). Boucharaba et al. (36) studied the effects of Ki16425 in a human breast cancer cell line using nude mice models. They found that Ki16425 reduced IL-6 and IL-8 secretions of breast cancer cells that diminished osteoclast activity and prevented bone metastasis. In our study we found that Ki16425 blocked IL-8 secretion of granulosa-lutein cells. Gomez et al. (37) used dopamine agonist (cabergoline) to reverse VEGF receptor 2-dependent vascular permeability and treat OHSS in a rat model. It could be suggested that the inhibition of LPA action in granulosa-lutein cells using LPA receptor antagonist may be another therapeutic target for studying the treatment of OHSS.

Inflammatory reaction is one of the processes of ovulation. IL-8 and IL-6 have been found to elevate in the preovulatory follicles and have been implicated in the involvement of ovulation (7, 38). The IL-8 and IL-6 may possess leukocyte chemotactic activity for neutrophils and monocytes that may contribute to tissue degradation in follicle rupture by the release of proteolytic factors (8). Therefore, LPA may play a role in ovulation through induction of IL-8 and IL-6 in granulosa-lutein cells, which deserves further study.

Here, we verify that through LPA1, the Gi, MAPK/p38, PI3K/Akt, and NF-{kappa}B signal pathways are involved in the LPA-induced IL-8 expression. LPA also transactivates EGFR via Gi and then activates ERK to induce IL-8 secretion. Through the LPA2 receptor, the Gi, MAPK/p38, and NF-{kappa}B pathways are involved in the LPA-induced IL-6 expression, and perhaps LPA3 alters something else that remains to be investigated. Recently, LPA was found to induce IL-8 expression in human bronchial epithelial cells and in HUVECs, as well as IL-8 and IL-6 expressions in dendritic cells (17, 39, 40). In addition, these enhancement effects are NF-kB dependent (17, 39). Together, LPA appears to be an important controlling factor for IL-8 or IL-6 expression in human tissues.

Conclusions

We demonstrate that LPA1, LPA2, and LPA3 receptors are expressed in human granulosa-lutein cells. In addition, LPA induces IL-8 and IL-6 expressions through LPA receptors via Gi-dependent NF-{kappa}B signaling pathways, but with different LPA receptors and different signal transduction mediators. Furthermore, LPA induction of IL-8 and IL-6 proteins stimulates angiogenesis. Therefore, the presence of significant amounts of LPA in a preovulatory follicle may play a role in ovulation and the neovascularization of corpus luteum. In addition, excessive IL-8 and IL-6 secretions induced by LPA from multiple corpora lutea of superovulated ovaries may be a cause of OHSS.


   Acknowledgments
 
We thank Ms. Tzu-Hsin Chen for her technical assistance.


   Footnotes
 
This work was supported by grants from the National Science Council (95-2314-B-002-276) and National Taiwan University Hospital (95A707), Taipei, Taiwan.

Disclosure Statement: The authors have nothing to declare.

First Published Online January 2, 2008

Abbreviations: AP, Activator protein; CM, conditioned medium; Edg, endothelial differentiation gene; EGFR, epidermal growth factor receptor; EIA, enzyme immunoassay; HCG, human chorionic gonadotropin; hIL, human IL; HUVEC, human umbilical vein endothelial cell; LPA, lysophosphatidic acid; NF, nuclear factor; OHSS, ovarian hyperstimulation syndrome; PI3K, phosphatidylinositol 3-kinase; PTX, pertussis toxin; siRNA, small interfering RNA duplexes; VEGF, vascular endothelial growth factor.

Received July 9, 2007.

Accepted December 26, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Fraser HM, Wulff C 2003 Angiogenesis in the corpus luteum. Reprod Biol Endocrinol 1:1–8[CrossRef]
  2. Stocco C, Telleria C, Gibori G 2007 The molecular control of corpus luteum formation, function, and regression. Endocr Rev 28:117–149[Abstract/Free Full Text]
  3. von Otte S, Paletta JR, Becker S, Konig S, Fobker M, Greb RR, Kiesel L, Assmann G, Diedrich K, Nofer JR 2006 Follicular fluid high density lipoprotein-associated sphingosine 1-phosphate is a novel mediator of ovarian angiogenesis. J Biol Chem 281:5398–5405[Abstract/Free Full Text]
  4. Motro B, Itin A, Sachs L, Keshet E 1990 Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis. Proc Natl Acad Sci USA 87:3092–3096[Abstract/Free Full Text]
  5. Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA, Marme D, Breckwoldt M 1995 Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 80:1967–1971[Abstract]
  6. Smith GW, Gentry PC, Long DK, Smith MF, Roberts RM 1996 Differential gene expression within the ovine corpus luteum: identification of secreted protein acidic and rich in cysteine as a major secretory product of small but not large luteal cells. Endocrinology 137:755–762[Abstract]
  7. Arici A, Oral E, Bukulmez O, Buradagunta S, Engin O, Olive DL 1996 Interleukin-8 expression and modulation in human preovulatory follicles and ovarian cells. Endocrinology 137:3762–3769[Abstract]
  8. Ujioka T, Matsukawa A, Tanaka N, Matsuura K, Yoshinaga M, Okamura H 1998 Interleukin-8 as an essential factor in the human chorionic gonadotropin-induced rabbit ovulatory process: interleukin-8 induces neutrophil accumulation and activation in ovulation. Biol Reprod 58:526–530[Abstract/Free Full Text]
  9. Bagavandoss P, Sage EH, Vernon RB 1998 Secreted protein, acidic and rich in cysteine (SPARC) and thrombospondin in the developing follicle and corpus luteum of the rat. J Histochem Cytochem 46:1043–1049[Abstract/Free Full Text]
  10. Koga K, Osuga Y, Tsutsumi O, Momoeda M, Suenaga A, Kugu K, Fujiwara T, Takai Y, Yano T, Taketani Y 2000 Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia. J Clin Endocrinol Metab 85:3352–3355[Abstract/Free Full Text]
  11. Drummond AE, Tellbach M, Dyson M, Findlay JK 2007 Fibroblast growth factor-9, a local regulator of ovarian function. Endocrinology 48:3711–3721
  12. Sugino N, Suzuki T, Sakata A, Miwa I, Asada H, Taketani T, Yamagata Y, Tamura H 2005 Angiogenesis in the human corpus luteum: changes in expression of angiopoietins in the corpus luteum throughout the menstrual cycle and in early pregnancy. J Clin Endocrinol Metab 90:6141–6148[Abstract/Free Full Text]
  13. Moolenaar WH 1995 Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270:12949–12952[Free Full Text]
  14. Guo R, Kasbohm EA, Arora P, Sample CJ, Baban B, Sud N, Sivashanmugam P, Moniri NH, Daaka Y 2006 Expression and function of lysophosphatidic acid LPA1 receptor in prostate cancer cells. Endocrinology 147:4883–4892[Abstract/Free Full Text]
  15. Tokumura A, Majima E, Kariya Y, Tominaga K, Kogure K, Yasuda K, Fukuzawa K 2002 Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J Biol Chem 277:39436–39442[Abstract/Free Full Text]
  16. Xie Y, Gibbs TC, Meier KE 2002 Lysophosphatidic acid as an autocrine and paracrine mediator. Biochim Biophys Acta 1582:270–281[Medline]
  17. Cummings R, Zhao Y, Jacoby D, Spannhake EW, Ohba M, Garcia JG, Watkins T, He D, Saatian B, Natarajan V 2004 Protein kinase C{Delta} mediates lysophosphatidic acid-induced NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells. J Biol Chem 279:41085–41094[Abstract/Free Full Text]
  18. Zhang Z, Liu Z, Meier KE 2006 Lysophosphatidic acid as a mediator for proinflammatory agonists in a human corneal epithelial cell line. Am J Physiol Cell Physiol 291:C1089–C1098
  19. Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW, Belinson J, Markman M, Casey G 1998 Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 280:719–723[Abstract/Free Full Text]
  20. Tokumura A, Miyake M, Nishioka Y, Yamano S, Aono T, Fukuzawa K 1999 Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients. Biol Reprod 61:195–199[Abstract/Free Full Text]
  21. Chiba H, Sawada N, Saito T 2006 Hyperstimulation and a gonadotropin-releasing hormone agonist modulate ovarian vascular permeability by altering expression of the tight junction protein claudin-5. Endocrinology 147:694–699[Abstract/Free Full Text]
  22. Villasante A, Pacheco A, Ruiz A, Pellicer A, Garcia-Velasco JA 2007 Vascular endothelial cadherin regulates vascular permeability: implications for ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 92:314–321[Abstract/Free Full Text]
  23. Chen CD, Wu MY, Chen HF, Chen SU, Ho HN, Yang YS 1999 Prognostic importance of serial cytokine changes in ascites and pleural effusion in women with severe ovarian hyperstimulation syndrome. Fertil Steril 72:286–292[CrossRef][Medline]
  24. Abramov Y, Schenker JG, Lewin A, Friedler S, Nisman B, Barak V 1996 Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 11:1381–1386[Abstract/Free Full Text]
  25. Stanek MB, Borman SM, Molskness TA, Larson JM, Stouffer RL, Patton PE 2007 Insulin and insulin-like growth factor stimulation of vascular endothelial growth factor production by luteinized granulosa cells: comparison between polycystic ovarian syndrome (PCOS) and non-PCOS women. J Clin Endocrinol Metab 92:2726–2733[Abstract/Free Full Text]
  26. Chen CD, Chao KH, Yang JH, Chen SU, Ho HN, Yang YS 2003 Comparison of coasting and intravenous albumin in the prevention of ovarian hyperstimulation syndrome. Fertil Steril 80:86–90[Medline]
  27. Contos JJ, Ishii I, Chun J 2000 Lysophosphatidic acid receptors. Mol Pharmacol 58:1188–1196[Medline]
  28. Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR, Mills GB 2002 Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway. Mol Cell Biol 22:2099–2110[Abstract/Free Full Text]
  29. Araujo RS, Mendonca BB, Barbosa AS, Lin CJ, Marcondes JA, Billerbeck AE, Bachega TA 2007 Microconversion between CYP21A2 and CYP21A1P promoter regions causes the nonclassical form of 21-hydroxylase deficiency. J Clin Endocrinol Metab 92:4028–4034[Abstract/Free Full Text]
  30. Machelon V, Nome F, Durand-Gasselin I, Emilie D 1997 Tumor necrosis factor-{alpha} induces interleukin-6 mRNA and protein in human granulosa luteinizing cells via protein tyrosine kinase without involving ceramide. Mol Cell Endocrinol 126:173–184[CrossRef][Medline]
  31. Runesson E, Ivarsson K, Janson PO, Brannstrom M 2000 Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle. J Clin Endocrinol Metab 85:4387–4395[Abstract/Free Full Text]
  32. Goetzl EJ, Lee H, Azuma T, Stossel TP, Turck CW, Karliner JS 2000 Gelsolin binding and cellular presentation of lysophosphatidic acid. J Biol Chem 275:14573–14578[Abstract/Free Full Text]
  33. Hama K, Bandoh K, Kakehi Y, Aoki J, Arai H 2002 Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors. FEBS Lett 523:187–192[CrossRef][Medline]
  34. Osborn TM, Dahlgren C, Hartwig JH, Stossel TP 2007 Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin. Am J Physiol Cell Physiol 292:C1323–C1330
  35. Ohta H, Sato K, Murata N, Damirin A, Malchinkhuu E, Kon J, Kimura T, Tobo M, Yamazaki Y, Watanabe T, Yagi M, Sato M, Suzuki R, Murooka H, Sakai T, Nishitoba T, Im DS, Nochi H, Tamoto K, Tomura H, Okajima F 2003 Ki16425, a subtype-selective antagonist for EDG-family lysophosphatidic acid receptors. Mol Pharmacol 64:994–1005[Abstract/Free Full Text]
  36. Boucharaba A, Serre CM, Guglielmi J, Bordet JC, Clezardin P, Peyruchaud O 2006 The type 1 lysophosphatidic acid receptor is a target for therapy in bone metastases. Proc Natl Acad Sci USA 103:9643–9648[Abstract/Free Full Text]
  37. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, Novella-Maestre E, Alonso-Muriel I, Sanchez-Criado J, Remohi J, Simon C, Pellicer A 2006 Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)-mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model. Endocrinology 147:5400–5411[Abstract/Free Full Text]
  38. Machelon V, Emilie D, Lefevre A, Nome F, Durand-Gasselin I, Testart J 1994 Interleukin-6 biosynthesis in human pre-ovulatory follicles: some of its potential roles at ovulation. J Clin Endocrinol Metab 79:633–642[Abstract]
  39. Lin CI, Chen CN, Chen JH, Lee H 2006 Lysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism. J Cell Biochem 99:1216–1232[CrossRef][Medline]
  40. Oz-Arslan D, Ruscher W, Myrtek D, Ziemer M, Jin Y, Damaj BB, Sorichter S, Idzko M, Norgauer J, Maghazachi AA 2006 IL-6 and IL-8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids. J Leukoc Biol 80:287–297[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S.-U. Chen, H. Lee, D.-Y. Chang, C.-H. Chou, C.-Y. Chang, K.-H. Chao, C.-W. Lin, and Y.-S. Yang
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
Endocrinology, November 1, 2008; 149(11): 5888 - 5896.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
X. Ye
Lysophospholipid signaling in the function and pathology of the reproductive system
Hum. Reprod. Update, September 1, 2008; 14(5): 519 - 536.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, S.-U.
Right arrow Articles by Yang, Y.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, S.-U.
Right arrow Articles by Yang, Y.-S.
Related Collections
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals