Am J Physiol Cell Physiol AJP: Regulatory, Integrative and Comparative Physiology
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Am J Physiol Cell Physiol 291: C1089-C1098, 2006. First published June 7, 2006; doi:10.1152/ajpcell.00523.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Lysophosphatidic acid as a mediator for proinflammatory agonists in a human corneal epithelial cell line

Zhihong Zhang,1,2 Zuguo Liu,2 and Kathryn E. Meier1

1Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington; and 2Zhongshan Ophthalmic Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People's Republic of China

Submitted 18 October 2005 ; accepted in final form 26 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lysophosphatidic acid (LPA) refers to a family of small phospholipid mediators that are generated in response to agonist stimulation in diverse cell types. LPA binds to G protein-coupled receptors to elicit numerous biological responses, including proliferation and inflammation. In this study, LPA production and response were characterized in a human corneal epithelial cell line, 2.040 pRSV-T. LPA levels in cells and medium are increased by exogenous 18:1 LPA (oleoyl-LPA), LPS, IL-1beta, and TNF-{alpha}. LPS, IL-1beta, and TNF-{alpha}, which mediate ocular inflammation, stimulate activation of p38, ERK, and Akt kinases in the corneal cell line. Similar responses are elicited by 18:1 LPA. Pertussis toxin (PTX) blocks LPA-induced activation of p38 and ERK but only slightly inhibits LPA-induced activation of Akt. All of the agonists tested, including LPA, stimulate proliferation of 2.040 pRSV-T cells. In these cells, both Akt and ERK pathways are important for LPA-induced proliferation. Thus PTX only partially suppresses the mitogenic response to LPA. Transcripts for the LPA receptors LPA1/EDG-2, LPA2/EDG-4, and LPA3/EDG-7 are expressed by the corneal cell line. Ki16425, an antagonist for LPA receptors, was used to explore the autocrine role of LPA. LPA-induced activations of p38, ERK, and Akt kinases, as well as proliferation, are inhibited by Ki16425. Ki16425 partially inhibits signal transduction and proliferation induced by the inflammatory agents tested. We conclude that LPA, produced in corneal epithelial cells in response to inflammatory agonists, contributes to mediating the mitogenic responses to these agonists in an autocrine fashion.

phospholipid mediators; protein phosphorylation; G protein-coupled receptors; inflammation

LYSOPHOSPHATIDIC ACID (LPA) is an important lipid mediator that elicits a broad spectrum of biological effects by activating G protein-coupled receptors (GPCRs) (30). The effects of LPA can be broadly classified as growth related (proliferation, antiapoptosis) and cytoskeleton related (aggregation, adhesion, contraction, secretion, chemotaxis). LPA also can participate in pathophysiological processes such as inflammation (48). For example, LPA participates in the motility, polarization, and metabolic burst of human neutrophils (5) and induces haptotactic migration of human monocytes (64). It is now clear that a wide variety of cell types can produce, as well as respond to, LPA (59). This raises the interesting possibility that LPA can act as an autocrine mediator in the same cells in which it is produced.

Previous work in our laboratory (60) has indicated that LPA can be produced in response to treatment of cells with other growth factors and can play a role as an autocrine mediator in cancer cell growth. Similarly, work from another laboratory (17) has suggested that LPA may, in part, mediate proliferation induced by sphingosine-phosphate in mesenchymal stem cells. The recent development of LPA receptor antagonists presents opportunities to test the autocrine role of LPA in a more direct manner than was possible previously. In the current study, we have utilized this approach for the first time to examine the role of LPA in inflammatory responses. A corneal epithelial cell line was used as the model system for these studies.

Inflammation is a significant concern in ocular tissues. Lipid-derived mediators, such as platelet-activating factor (26) and eicosanoids (47), have been implicated in the initiation, propagation, and maintenance of inflammatory and immune responses in the eye. However, the physiological roles of LPA and its receptors in inflammation of the ocular surface have not been fully explored.

Several previous studies have indicated that LPA can play a signaling role in the cornea. Lilliom et al. (25) have shown that LPA participates in corneal wound healing that occurs after corneal injury. Specifically, LPA, alkenyl-glycerophosphate, phosphatidic acid, and lysophosphatidylserine were identified in the aqueous humor and the lacrimal gland fluid. Studies have shown that human corneal cell lines express LPA1 and LPA2 receptors and that sodium dodecyl sulfate (SDS) injury can increase the levels of mRNA for LPA2 (57). The small GTP-binding protein Rho is involved in LPA-induced migration of corneal epithelial cells (31). LPA stimulates tyrosine phosphorylation of focal adhesion proteins in chick corneal epithelial cells (52). Additional studies have shown LPA receptor expression and/or response in corneal endothelial cells (55), keratocytes (18, 40, 56), and lens epithelial cells (34). These data have established a potential role for LPA in the eye. However, the signaling pathways involved in LPA production and response in the cornea remain to be addressed.

It has been established that most LPA actions are mediated through the specific GPCRs of the EDG family (LPA1/EDG-2, LPA2/EDG-4, LPA3/EDG-7) (16, 22, 36, 39) and by LPA4/GPR23 (33). However, it has been difficult to determine which LPA receptor serves as a mediator of a given response. Pertussis toxin (PTX) inhibits many responses to LPA but is not specific for a particular receptor subtype. LPA receptor-selective antagonists, which have recently become commercially available, are important tools for identifying the subtype of LPA receptor that is responsible for mediating a particular downstream effect (14). Ki16425 is an LPA receptor antagonist that shows a preference for LPA1 and LPA3 over LPA2 (35).

In this study, we have addressed the hypothesis that LPA serves as an inflammatory mediator in human corneal epithelial cells. An established cell line was used as a model system. LPA production, signaling responses to LPA and inflammatory agonists, and the effects of LPA receptor antagonists were examined. The results suggest that LPA can act as an autocrine mediator of mitogenic responses to proinflammatory agonists.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 18:1 Oleoyl LPA was obtained from Avanti Polar Lipids (Birmingham, AL). [3H]palmitic acid and EN3HANCE were obtained from PerkinElmer Life Sciences Products (Boston, MA). Antibodies recognizing the phosphorylated active forms of Erk (T202/Y204), p38 (Thr180/Tyr182), and Akt (Ser473) were obtained from Cell Signaling Technologies (Beverly, MA). Anti-focal adhesion kinase (FAK) was obtained from BD Transduction Laboratories (San Jose, CA). LPS, IL-1beta, TNF-{alpha}, Ki16425, U0126, and LY-294002 were obtained from Sigma (St. Louis, MO). PTX was from Calbiochem (La Jolla, CA).

Cell culture. A human corneal epithelial cell line, 2.040 pRSV-T, was obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in keratinocyte serum-free medium (GIBCO Invitrogen, Carlsbad, CA) containing 5 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, 0.005 mg/ml insulin, and 500 ng/ml hydrocortisone and were plated on dishes coated with 0.01 mg/ml fibronectin and 0.01 mg/ml collagen I. Cells were grown at 37°C in 5% CO2-95% air, and were growth factor-starved by incubation in medium lacking EGF, pituitary extract, insulin, and hydrocortisone for 18–24 h before experiments.

Isotopic method for LPA analysis. Production of LPA was assessed in cells and medium as described previously (61). Briefly, cells grown in six-well plates, with triplicate wells for each treatment, were growth factor-starved and simultaneously labeled by incubation for 18 h in medium containing 5 µCi/ml [3H]palmitic acid. Cells were washed and then incubated with agonists in Dulbecco's modified Eagle's medium containing 10 mM HEPES (pH 7.5) at 37°C in a cell culture incubator. Lipids were extracted and separated using TLC on silica gel plates impregnated with 0.5 M oxalic acid. Unlabeled 18:1 LPA was added as standard. 3H-labeled lipids were visualized using autoradiography after being sprayed with EN3HANCE (PerkinElmer). LPA bands, as well as the remainder of each lane, were scraped and quantified using liquid scintillation spectrometry. Results are expressed as percentages of the total radioactivity recovered.

Proliferation assays. Cells were seeded at a density of 1x105 cells/well in triplicate in 24-well plates and then incubated in the plate overnight. After growth factor removal for 18 h, cells were treated with different concentrations of LPA, LPS, IL-1beta, or TNF-{alpha} for various times in fresh medium lacking growth factors. In some experiments, cells were incubated with Ki16425, U0126, or LY-294002 before agonist addition. DMSO was used as the vehicle for all of these agents; the final concentration of DMSO in the incubation was <0.1%. BSA (4 mg/ml final concentration) was used as the vehicle for LPA. Cells were collected using 0.05% trypsin-0.53 mM EDTA (Sigma); Trypan blue was added. Live cells were counted using a hemacytometer.

Immunoblotting. Whole cell extracts, prepared in the presence of protease and phosphatase inhibitors, were subjected to SDS-PAGE and immunoblotting as described previously (10). Protein loading was equalized to 100 µg/lane, as determined using a Coomassie protein assay (Pierce Biotechnology, Rockford, IL). After incubation with antibodies, blots were developed using enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ). Blots were scanned and then quantified using ImageQuaNT software (Amersham Biosciences).

RT-PCR. Total RNA was extracted from harvested cells using TRIzol solution (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Reverse transcription was performed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) in a reaction volume of 20 µl under the conditions recommended by the manufacturer. Total RNA (1 µg) was used as a template for cDNA synthesis. The resulting cDNA was used as a template for PCR. PCR was performed in a 50-µl reaction volume with a buffer consisting of 10x iTaq buffer, 50 mM MgCl2, 10 mM dNTP mix, iTaq DNA polymerase, and 0.25 µM each primer. The primers used were: beta-actin (forward), TGACGGGGTCACCCACACTGTGCCCATCT; beta-actin (reverse), CTAGAAGCATTTGCGGTGG ACGATGGAGGG; LPA1/EDG-2 (forward), TGTCATGGCTGCCATCTC; LPA1/EDG-2 (reverse), CATCTCAGTTTCCGTTCTAA; LPA2/EDG-4 (forward), CCCAACCAACAGGACTGACT; LPA2/EDG-4 (reverse), GAGCCCTTATCTCTCC CCAC, LPA3/EDG-7 (forward), GGACACCC ATGAAGCTAATG; and LPA3/DG-7 (reverse), TCTGGGTTCTCCTGAGAGAA. PCR was performed with initial denaturation at 95°C for 3 min, followed by 30 cycles consisting of denaturation at 95°C for 30 s, annealing for 30 s at 55°C, and extension at 72°C for 30 s. RT-PCR products were separated on a 2% agarose gel with electrophoresis and visualized under UV illumination.

Statistical analysis. Analysis of the significance of differences between two groups was performed by t-test or ANOVA, using Instat software (GraphPad, San Diego, CA). Values are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Production of LPA by 2.040 pRSV-T cells and effects of various mediators on LPA production. LPA production was first assessed using an isotopic assay (61). Corneal epithelial cells were metabolically labeled with [3H]palmitic acid overnight, washed, and then incubated with and without agonists for 40 min. Lipids were extracted from cells and medium, separated using TLC, and then visualized with autoradiography. The results shown in Fig. 1A show that LPA can be produced from endogenous lipids in 2.040 pRSV-T cells and is detected in both cells and medium.


Figure 1
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  		Fig. 1. Radioisotopic assay for lysophosphatidic acid (LPA) production and effects of 18:1 LPA, lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-{alpha} (TNF-{alpha}) on LPA levels in corneal epithelial cells. A: 2.040 pRSV-T human corneal epithelial cells were metabolically labeled by overnight incubation in complete medium containing [3H]palmitic acid. Cells were then washed and incubated in fresh medium without isotope for 40 min. Lipids were extracted from the cells and medium and were separated using thin-layer chromatography (TLC), as described in MATERIALS AND METHODS. The autoradiogram of the portion of the plate containing the LPA band is shown, using triplicate dishes of cells. B: corneal epithelial cells were incubated overnight with [3H]palmitic acid in the absence of growth factors. The cells were then incubated with or without 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 40 min. Lipids from medium were extracted, separated using TLC, and imaged using autoradiography, and the LPA band was quantified using liquid scintillation spectrometry. Radioactivity in LPA was normalized to the total radioactivity recovered from each lane. Each data point represents mean ± SE of values from triplicate well of cells; cpm, counts per minute. Statistical differences between control and treated cells were analyzed using one-tailed t-test: *P < 0.05; **P < 0.01.

 
Having established that corneal epithelial cell produce LPA, we examined whether inflammatory agonists regulate LPA levels. LPS (50), IL-1beta, and TNF-{alpha} (51) are common proinflammatory cytokines that are present in the cornea and that induce inflammation in corneal epithelial cells. The effects of 18:1 LPA were also tested, because LPA induces LPA release in many cell lines (59). LPA in the medium was significantly increased by treatment of corneal epithelial cells for 40 min with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, and 10 ng/ml TNF-{alpha} (Fig. 1B). LPA levels did not change within cells at this time point (data not shown). Because LPS was consistently more efficacious than IL-1beta or TNF-{alpha} (data not shown), this agonist was used for more detailed studies of LPA production in cells and medium.

Effect of LPS on LPA production. The results presented above demonstrated that LPS can increase production of LPA in corneal epithelial cells. The location (cells vs. medium) and time course of LPS-induced LPA production were therefore examined in more detail. Corneal epithelial cells were treated with 10 µg/ml LPS for varying times. LPA generation was assessed in cells and medium, using the isotopic assay (Fig. 2). LPS caused a gradual increase in LPA in corneal epithelial cell medium (Fig. 2A), with a significant (P < 0.05) increase seen at 40 min. The LPS-induced increase persisted for at least 180 min. As shown in Fig. 2B, LPS induced an increase of LPA levels within corneal epithelial cells that was apparent only at 20 min. A dose-response study established that LPA production was maximal with 10 µg/ml LPS (data not shown).


Figure 2
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  		Fig. 2. Effect of LPS on LPA production in corneal epithelial cells. Serum-starved corneal epithelial cells were metabolically labeled with [3H]palmitic acid. Washed cells were incubated with 10 µg/ml LPS for the indicated times. The lipids were extracted from both cells (A) and medium (B), and LPA production was analyzed as described for Fig. 1. Radioactivity in the LPA band is expressed as a percentage of the total radioactivity recovered from each sample. Each data point represents mean ± SE of values from triplicate well of cells. Error bars, when not visible, are included within the data points.

 
Effects of LPA, LPS, IL-1beta, and TNF-{alpha} on 2.040 pRSV-T cell proliferation. The results show that corneal epithelial cells generate LPA in response to inflammatory stimuli and LPA. Whether corneal epithelial cells respond in other ways to LPA is of interest. LPA has been shown to elicit diverse biological actions, including proliferation in a variety of cell types (6, 29, 63). We tested whether LPA enhanced proliferation of the 2.040 pRSV-T cell line, as has been reported for primary corneal epithelial cells (57). Cells were deprived of their usual growth factors and pretreated with or without PTX overnight. They were then incubated in the absence or presence of LPA. PTX can ADP-ribosylate the G proteins Gi/o and G12/13, thereby interfering with many (but not all) GPCR-mediated responses to LPA.

LPA stimulated proliferation of 2.040 pRSV-T human corneal epithelial cells, measured at 48 h, in a dose-dependent manner (Fig. 3A). Cell number was significantly increased by 10, 20, and 30 µM LPA compared with control. When 10 µM LPA was used to treat cells for 24, 48, and 72 h, an increase in cell numbers was observed throughout the time course (Fig. 3B). PTX partially inhibited LPA-induced proliferation but also had an inhibitory effect on cells incubated without LPA. In a separate experiment (Fig. 3C), proliferation was significantly increased after treatment with LPA, LPS, IL-1beta, or TNF-{alpha} for 48 h. PTX was most effective in blocking the response to LPA, although the inhibition was only partial. PTX also significantly inhibited basal and inflammatory agonist-induced proliferation; however, the magnitude of the effect was small. The data indicate that LPA and inflammatory agonists are mitogenic for 2.040 pRSV-T cells. The response to LPA is mediated, in part, by PTX-sensitive G proteins. However, the relative ineffectiveness of PTX in blocking proliferation prompted us to further examine LPA-induced signaling pathways.


Figure 3
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  		Fig. 3. Effects of 18:1 LPA, LPS, IL-1beta, and TNF-{alpha} on proliferation of corneal epithelial cells. For this experiment, 2.040 pRSV-T cells were seeded at a density of 1 x 105 cells/well. A: cells that were 80% confluent were incubated overnight in the absence of growth factors. LPA was then added at the indicated concentrations. Cell counts were performed using a hemacytometer after 48 h. Statistical significance of differences between cells treated with LPA was compared with the value for untreated cells (2.2 ± 0.2 x 105 cells) using one-way ANOVA: **P < 0.01. B: cells were deprived of growth factors overnight in the absence and presence of 100 ng/ml pertussis toxin (PTX) and then incubated with 10 µM LPA for the indicated times. Error bars are included within the data points. C: cells were deprived of growth factors overnight in the absence and presence of 100 ng/ml PTX and then incubated with 10 µM LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha}. Live cells were counted after 48 h. Statistical significance of differences between cells treated with and without PTX, within each data set, was analyzed using one-way ANOVA: *P < 0.05; **P < 0.01. For A–C, each data point represents mean ± SE of values from triplicate wells of cells.

 
Effects of LPA and inflammatory agonists on signal transduction in 2.040 pRSV-T cells. LPA can induce Erk, Akt, and/or p38 activation in a variety of cell lines (22, 39, 43, 54, 60). To assess the ability of LPA to activate Erk, Akt, and p38 in 2.040 pRSV-T cells, we determined the dose-response and time course for LPA effects. Cells were deprived of growth factors overnight and then incubated with 10 µM LPA. Erk, Akt, and p38 activation were determined by immunoblotting for the phosphorylated active forms of the kinases (Fig. 4). All three kinases were activated, with maximal effects at 10–30 µM LPA. Akt phosphorylation was unique in that it declined at LPA doses >10 µM. The kinetics of kinase activation were assessed using 10 µM LPA (Fig. 5). Maximal phosphorylation of Erk, Akt, and p38 occurred at 30 s and decreased subsequently. Akt activation was somewhat more sustained than that of Erk and p38, but all returned to basal levels by 90 min.


Figure 4
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  		Fig. 4. Dose-response for effects of 18:1 LPA on signal transduction in corneal epithelial cells. A: 2.040 pRSV-T corneal epithelial cells were growth factor-starved overnight and then incubated with different concentrations of 18:1 LPA for 5 min. BSA, used as vehicle for LPA, was included at a final concentration of 4 µg/ml in all incubations. Whole cell extracts were immunoblotted for phospho (p)-Erk, p-Akt, and p-p38. Immunoblotting for total focal adhesion kinase (FAK) was done to confirm equal protein loading. B: quantification of the immunoblots is shown. Data are expressed relative to values obtained with untreated cells.

 

Figure 5
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  		Fig. 5. Time course for the effects of 18:1 LPA on signal transduction in corneal epithelial cells. A: growth factor-starved 2.040 pRSV-T corneal epithelial cells were incubated with 10 µM 18:1 LPA for the indicated times. Whole cell extracts, equalized for protein, were immunoblotted for p-Erk, p-Akt, and p-p38. Immunoblotting for total FAK was done to confirm equal protein loading. B: quantification of the immunoblots, for the first 30 min of the time course, is shown. Data are expressed relative to values obtained for untreated cells.

 
Erk, Akt, and p38 have been established as downstream targets of 18:1 LPA (39, 60), LPS (4, 12), IL-1beta (19, 41), and TNF-{alpha} (28, 41) in many cell lines. LPS, IL-1beta, TNF-{alpha}, and 18:1 LPA all caused a more than 10-fold increase in phosphorylation of each kinase in 2.040 pRSV-T cells (Fig. 6). In summary, LPS, IL-1 beta, TNF-{alpha}, and 18:1 LPA activate three of the same signal transduction pathways in 2.040 pRSV-T cells.


Figure 6
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  		Fig. 6. Effects of 18:1 LPA, LPS, IL-1beta, and TNF-{alpha} on signal transduction in corneal epithelial cells. A: growth factor-starved 2.040 pRSV-T corneal epithelial cells were incubated with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 5 min. Whole cell extracts, equalized for protein, were immunoblotted for p-Erk, p-Akt, and p-p38. Immunoblotting for total FAK was done to confirm equal protein loading. B: quantification of the immunoblots is shown. Data are expressed relative to values obtained for untreated cells.

 
We investigated the effects of PTX on the signaling pathways activated by LPA, LPS, IL-1beta, and TNF-{alpha} in corneal epithelial cells (Fig. 7). Cells were incubated with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 5 min in the absence and presence of PTX (18-h preincubation). LPA-induced Erk and p38 activations were blocked with preincubation of the cells with PTX (Fig. 7, B and C). However, PTX did not interfere substantially with activation of Akt in response to LPA (Fig. 7D). These data indicate that LPA may activate Akt through a different receptor or G protein from those used to activate Erk and p38. Activations of Erk in response to the other inflammatory agonists was inhibited by <50% by PTX. Notably, the effects of the inflammatory agonists on Akt activation were relatively resistant to PTX. In view of the fact that basal phosphorylation levels of all three kinases were inhibited to varying extents by PTX, the potential role of G proteins (i.e., via autocrine production of LPA) in the responses to the other agents was difficult to assess. However, the data shown in Fig. 7B suggest that LPA may play a major role in agonist-induced Erk activation. Because none of the other agonists tested binds directly to a GPCR, the strong inhibitory effect of PTX on Erk activation suggests either an autocrine or permissive role for LPA (or another ligand binding to PTX-sensitive GPCRs) in certain signaling responses to inflammatory agonists. This concept was further explored in subsequent experiments.


Figure 7
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  		Fig. 7. Effects of PTX on Erk, p38, and Akt activation induced by 18:1 LPA, LPS, IL-1beta, and TNF-{alpha} in corneal epithelial cells. A: growth factor-starved 2.040 pRSV-T cells were incubated in the absence and presence of 100 ng/ml PTX and then incubated with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 5 min. Whole cell extracts were immunoblotted using antibodies recognizing phosphorylated active Erk, p38, or Akt. Immunoblotting for total FAK was done to confirm equal protein loading. B–D: quantification of the immunoblots is shown. All data are expressed relative to values obtained for untreated cells.

 
LPA receptor expression in 2.040 pRSV-T cells. Gene- and species-specific primers were used to assess the presence of mRNA for LPA receptor subtypes using PCR. Amplification of all three mRNA species was achieved, with the products migrating at the predicted sizes. The results presented in Fig. 8 indicate that LPA1, LPA2, and LPA3 receptors are expressed in 2.040 pRSV-T corneal epithelial cells. Thus this cell line is predicted to be capable of responding to LPA via multiple GPCR-linked signaling pathways.


Figure 8
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  		Fig. 8. LPA receptors in corneal epithelial cells. Total RNA was extracted from 2.040 pRSV-T cells. RT-PCR was performed using the LPA1, LPA2, and LPA3 receptor primers listed in MATERIALS AND METHODS. beta-Actin mRNA was also amplified as a control for loading. PCR products, separated on an ethidium bromide-containing gel, were imaged under UV light.

 
Effect of Ki16425 on agonist-induced Erk, Akt, and p38 activation. Ki16425 is a broad-spectrum LPA receptor antagonist that inhibits LPA-induced responses at LPA1 and LPA3 receptors, with much lower affinity for LPA2 (35). This antagonist has been shown to block LPA response in several different cell types (13, 21, 23, 62). Ki16425 does not inhibit calcium release in response to sphingosine 1-phosphate (17), a lipid mediator that is related to LPA. We first tested the ability of Ki16425 to inhibit LPA-induced kinase activations in a dose-response study. As shown in Fig. 9, the antagonist blocked activations of Akt and Erk in response to 10 µM 18:1 LPA. The IC50 for Erk activation was ~0.033 µM, and that for Akt activation was ~0.4 µM. A dose of 10 µM Ki16425 yielded maximal inhibition of both responses, was equal to the concentration of 18:1 LPA used as agonist, had little effect on basal kinase activity, and is the dose typically used in other studies to inhibit LPA response (23, 62); this concentration of antagonist was therefore used in subsequent studies. We examined the effects of Ki16425 on signaling pathways activated by LPA and other inflammatory agonists. Cells were pretreated with 10 µM Ki16425 for 5 min and then incubated with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 5 min. LPA-induced activations of Erk, Akt, and p38 were inhibited in cells incubated with Ki16425 (Fig. 10). Activations of Erk, Akt, and p38 in response to the other agonists were partly inhibited by Ki16425. However, consistent inhibition of basal kinase activities by Ki16425 complicated interpretation of the partial effects. Because Ki16425 was not cytotoxic in long-term incubations (see below), the significance of these acute effects is not clear.


Figure 9
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  		Fig. 9. Dose-response curve for the effects of Ki16425 on LPA-induced kinase activations in corneal epithelial cells. A: after being deprived of growth factors overnight, 2.040 pRSV-T cells were pretreated with various concentrations of Ki16425 for 5 min and then incubated with 10 µM 18:1 LPA for 5 min. Whole cell extracts were immunoblotted using antibodies recognizing phosphorylated active Erk and Akt. Immunoblotting for total FAK was done to confirm equal protein loading. B: quantification of the immunoblots by densitometry is shown. After subtraction of the control (unstimulated) values and normalization for loading (FAK), data are expressed as a percentage of the maximal response observed with 18:1 LPA alone. Note that the highest concentrations of Ki16425 gave values lower than basal; these are indicated as "0" for the purposes of this graph.

 

Figure 10
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  		Fig. 10. Effects of Ki16425 on Erk, p38, and Akt activation induced by 18:1 LPA, LPS, IL-1beta, and TNF-{alpha} in corneal epithelial cells. A: after being deprived of growth factors overnight, 2.040 pRSV-T cells were pretreated with 10 µM Ki16425 for 5 min and then incubated with 10 µM 18:1 LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} for 5 min. Whole cell extracts were immunoblotted using antibodies recognizing phosphorylated active Erk, p38, or Akt. Immunoblotting for total FAK was done to confirm equal protein loading. B–D: quantification of the immunoblots is shown. All data are expressed relative to values obtained for untreated cells.

 
Roles of Erk and Akt in proliferation of 2.040 pRSV-T cells. The results presented above showed varying efficacies of LPA response inhibitors (PTX, Ki16425) on different cellular responses (kinase activations, proliferation). To reconcile these data, we examined the relative roles of Akt and Erk in proliferation of 2.040 pRSV-T cells. LY-294002 was used as an inhibitor of Akt activation [acting upstream to inhibit phosphatidylinositol 3-kinase (PI3K)], and U0126 was used as an inhibitor of Erk activation (acting upstream to inhibit MEK). Fig. 11A demonstrates that the two inhibitors were effective in blocking LPA-induced Akt and Erk activations, respectively, in this cell line under the conditions used. Proliferation assays were performed in cells treated with LPA, LPS, IL-1beta, or TNF-{alpha} in the absence and presence of Ki16425, LY-294002, or U0126. As shown in Fig. 11B, LPA-induced proliferation was blocked by Ki16425. LY-294002 and U0126 each caused significant inhibition of LPA response, but the inhibition was not complete with either agent alone. The response to LPS was partially but significantly inhibited by Ki16425, LY-294002, and U0126. The response to IL-1beta was significantly inhibited by Ki16425 and U0126 and completely blocked by LY-294002. Finally, the response to TNF-{alpha} was blocked by Ki16425, LY-294002, and U0126. For LPA and TNF-{alpha}, values seen with Ki16425 were not significantly different from untreated control.


Figure 11
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  		Fig. 11. Effects of inhibitors on proliferation induced by 18:1 LPA, LPS, IL-1beta, and TNF-{alpha} in corneal epithelial cells. A: cells were incubated overnight in the absence of growth factors. Inhibitors were added at the following concentrations: 25 µM LY-294002 or 10 µM U0126. After 5 min, 10 µM LPA was added. Whole cell extracts were immunoblotted using antibodies recognizing phosphorylated activated Erk or Akt. FAK was immunoblotted as a control for loading. B: 2.040 pRSV-T cells were seeded at a density of 1x 105 cells/well. Cells were incubated overnight in the absence of growth factors. Inhibitors were added at the following concentrations: 10 µM Ki16425, 25 µM LY-294002, or 10 µM U0126. After 5 min, 10 µM LPA, 10 µg/ml LPS, 10 ng/ml IL-1beta, or 10 ng/ml TNF-{alpha} were added. Live cells were counted using a hemacytometer after 48 h. Values represent means ± SE for triplicate wells of cells. Statistical significance of differences between agonist-treated cells incubated with and without inhibitors, within each data set, was analyzed using one-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

 
The following major conclusions can be drawn from the inhibitor studies. First, both the PI3K/Akt and MEK/Erk pathways play major roles in LPA-induced proliferation in 2.040 pRSV-T cells. Second, the results are consistent with a significant role for autocrine LPA in responses to inflammatory agonists. In all cases, agonist-induced proliferation was inhibited either partially or completely by the LPA antagonist Ki16425 (Fig. 11B). It should be noted that PTX was less effective than Ki16425 in blocking the proliferative effects of LPA and inflammatory agonists (Fig. 3C). This is likely because PTX did not block LPA-induced Akt activation (Fig. 7D), which appears to be important for LPA-induced proliferation in this cell line (Fig. 11). Of responses to the inflammatory agonists, that to TNF-{alpha} was most effectively inhibited by Ki16425 (Fig. 11). The more modest, albeit significant, effects of Ki16425 on LPS- and IL-1beta-induced proliferation (Fig. 11) are consistent with the data presented in Figs. 3, 7, and 9, all of which suggest a contributory role for LPA in responses to LPS and IL-1beta.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although activated platelets were first shown to release LPA (3, 9, 58), many cell types have now been shown to produce this lipid mediator (59). Ours is the first study to show that human corneal epithelial cells can produce LPA. A radioisotopic metabolic labeling method was previously established in our laboratory to quantify LPA production from cultured cells (61). With the use of this method, our data show that LPA can be detected in both 2.040 pRSV-T cells and medium. LPA levels are increased by proinflammatory agonists. It is therefore of interest to address the physiological and pathological role of LPA in these cells.

Corneal epithelial cells play an important role in ocular surface diseases. LPA secreted from corneal epithelial cells constitutes part of the outer layer of tear film (27). As a polar phospholipid and a mediator, LPA may potentially play dual roles in tears, proving an interface between the aqueous and nonpolar lipid layers (27) as well as acting as a proinflammatory agent. Many disorders involve inflammatory changes in the ocular surface. Of these, dry eye (keratoconjunctivitis sicca) is one of the most common (15, 44). Dry eye is a disorder of the tear film caused by tear deficiency or excessive tear evaporation. The outer layer of tear film is a lipid layer responsible for controlling evaporation from ocular surface. This layer is composed of both polar (e.g., LPA) and nonpolar components (27). Histological features of dry eye include abnormal proliferation and differentiation of the ocular surface epithelium (37, 38). It is believed that inflammatory cytokines in the tears contribute to the pathology (32). Increased expression of several inflammatory mediators has been observed in conjunctival epithelium and tear fluid in dry eye (1, 49, 53). Accordingly, treatments of severe forms of dry eye with steroids and topical cyclosporine A have been successful in human trials.

In this study, we focused on the effects of inflammatory agonists (LPS, IL-1beta, and TNF-{alpha}) on 2.040 pRSV-T human corneal epithelial cell lines and the role of LPA in responses to these agents. The proinflammatory cytokines IL-1beta and TNF-{alpha} are important mediators of inflammation and immunity (8). LPS, a major component of the outer membrane of Pseudomonas aeruginosa, exerts many of its biological effects by binding to specific cell surface receptors (24, 45). Binding of LPS to CD14 receptors in human cornea initiates a rapid innate immune response through production of proinflammatory cytokines and chemokines such as IL-6 and -8 (46, 50). Our results show that stimulation of cells with proinflammatory factors (LPS, IL-1beta, and TNF-{alpha}) and LPA results in increased levels of LPA in the medium. LPA appears first in the cells and then in the medium. This general phenomenon was noted in previous studies from our laboratory (60) using a prostate cancer cell line. The time course suggests that LPA is made within cells and then released, but further studies are needed to establish this point. In other words, it remains to be determined whether the increase in LPA in the medium is due to export of agonist-generated LPA or whether LPA is largely produced in the extracellular space (59).

An interrelationship between inflammation and proliferation has been noted in many pathological settings, including cancer (42) and atherogenesis (7). LPA itself has been observed to be involved in both inflammation and proliferation (11). Although LPA has been previously shown to stimulate proliferation of corneal epithelial cells (57), previous data regarding the direct effects of LPS, TNF-{alpha}, and IL-1 on proliferation of corneal epithelial cells appear to be lacking. The current study demonstrates that these agents can stimulate proliferation of a corneal epithelial cell line. Specifically, we found that LPA induced proliferation of 2.040 pRSV-T corneal epithelial cells and that LPS, IL-1beta, and TNF-{alpha} were also mitogenic. We therefore tested for activation of the Erk and Akt pathways, which are commonly involved in mitogenesis, and p38, which is frequently involved in inflammatory responses. LPA induced Erk, Akt, and p38 activation; LPS, IL-1beta, and TNF-{alpha} exerted effects that were similar to these of LPA. PTX inhibited the LPA-induced activation of Erk and p38 but not Akt. The results indicate that LPA can act through PTX-sensitive G proteins to activate Erk and p38 but uses alternate pathways to activate Akt. Activation of both Akt and Erk is important for mitogenesis in this corneal cell line.

Most LPA actions are mediated through GPCRs, i.e., LPA1, LPA2, LPA3 (16, 22, 36), and LPA4/GPR23 (35). In the present study, we found that 2.040 pRSV-T cells express LPA1, LPA2, and LPA3 receptors. All of these receptors can couple to multiple G proteins (2). Previous studies by others showed that human corneal cell lines express LPA1 and LPA2 (57). PTX inhibited some LPA actions in 2.040 pRSV-T cells, indicating that PTX-sensitive G proteins mediate these responses. However, LPA-induced proliferation was not completely inhibited by PTX. The LPA antagonist Ki16425 was used to test the roles of LPA1 and LPA3. Ki16425 blocked LPA-induced proliferation. In addition, LPA-induced activations of Erk and Akt were substantially inhibited by Ki16425, with p38 activation being less sensitive. Because both Akt and Erk are required for LPA-induced proliferation, the mitogenic effects of LPA appear to be mediated by LPA1 and/or LPA3 receptors in this corneal epithelial cell line. Interestingly, the abilities of Ki16425 and PTX to reduce basal levels of activation of Akt and Erk suggest that LPA receptors are constitutively active in this cell line. Whether this is due to constitutive production of LPA or to constitutive activities of LPA receptors in the absence of agonist remains to be determined.

Proinflammatory cytokines and their receptors are components of a complicated signaling network that exists at the ocular surface. Their interactions can result in a persistent inflammatory response. In our study, LPA elicited responses similar to those of other inflammatory mediators (LPS, IL-1beta, and TNF-{alpha}) in 2.040 pRSV-T cells. In addition, the proinflammatory agonists increased LPA production by these cells. An LPA antagonist, Ki16425, blocked the responses of the proinflammatory agonists to varying extents. Partial inhibition of responses likely reflects differences in the downstream signaling pathways utilized by the agonists; these differences resulted in greater dependence on LPA mediation for some agonists (e.g., TNF-{alpha}) than for others (e.g., LPS, IL-1beta). Our data suggest that LPA is an important mediator of the mitogenic effects of proinflammatory cytokines on 2.040 pRSV-T cells. LPA receptors may thus represent "multitasking" therapeutic targets for the control of the proliferation and inflammation in corneal epithelia, as well as in other cell types that are responsive to LPA (e.g., carcinoma cells). LPA receptor-selective antagonists, such as Ki16425, provide a platform for the development of new therapeutic agents.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by US Department of Defense Grant DAMD 17-01-1-0730.


   ACKNOWLEDGMENTS
 
We thank Manpreet Chahal for helpful suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. E. Meier, Dept. of Pharmaceutical Sciences, Washington State Univ., Pullman, WA 99164-6534 (e-mail: kmeier{at}wsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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