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CELLULAR AND MOLECULAR

Lysophosphatidic Acid Decreases Epidermal Growth Factor Receptor Binding in Airway Epithelial Cells

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska

Received January 25, 2007; accepted July 18, 2007.

	Abstract

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We showed previously that treatment of human airway smooth muscle cells and lung fibroblasts with lysophosphatidic acid (LPA) increases the binding of epidermal growth factor (EGF) to EGF receptors (EGFRs). The purpose of this study was to determine whether LPA also regulates EGFR binding in airway epithelial cells. Airway epithelial cells were incubated in the absence or presence of 10 µM LPA for increasing times, and binding of ¹²⁵I-EGF to intact cells on ice was measured. Exposure to LPA for only 15 min caused a 30 to 70% decrease in EGFR binding in a dose-dependent manner, depending on the cell line. This decrease in binding was sustained to at least 18 h in BEAS-2B and primary human bronchial epithelial cells. In contrast, the LPA-induced decrease in binding reversed rapidly in two lung cancer epithelial cell lines, H292 and A549, returning to control levels within 3 h. LPA increased phosphorylation of the EGFR in BEAS-2B cells, and this phosphorylation was inhibited by both 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478; EGFR tyrosine kinase inhibitor) and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001; matrix metalloproteinase inhibitor) but not by CRM197 (heparin-binding EGF inhibitor). AG1478 and GM6001 also inhibited the LPA-induced decrease in EGFR binding but only by 50%, suggesting only partial involvement of EGFR transactivation in the decrease in EGFR binding. In summary, LPA stimulates a decrease in EGFR binding in airway epithelial cells that is sustained in normal cells but that rapidly reverses in cancer cells. LPA-induced transactivation of EGFRs occurs and contributes to the decrease in EGFR binding, but additional pathway(s) may also be involved.

The EGF receptor (EGFR) has been shown to be involved in many of the processes of airway remodeling and asthma, including mucin synthesis and secretion, goblet cell metaplasia, and sustaining a repair phenotype in the injured epithelium (Puddicombe et al., 2000; Nadel, 2001; Holgate et al., 2003). Asthmatic airways exhibit increased EGFRs in bronchial epithelium and smooth muscle as well as in the basement membrane and glandular cells, compared with normal controls (Amishima et al., 1998). Furthermore, the thickness of the lamina reticularis is positively correlated with EGFR expression (Puddicombe et al., 2000). EGF has also been shown to stimulate proliferation of human airway smooth muscle (HASM) cells and lung fibroblasts (Cerutis et al., 1997; Ediger and Toews, 2000).

The simple lipid mediator lysophosphatidic acid (LPA) stimulates proliferation, differentiation, tumor metastasis, and cytoskeletal rearrangement in multiple cell types (Wang et al., 2003; Zhao et al., 2006). LPA is released from activated platelets at sites of injury (Eichholtz et al., 1993) where it then contributes to wound repair (Lee et al., 2000), but it can also be synthesized by epithelial cells via the conversion of phosphatidic acid by phospholipase A₁/A₂ (Cummings et al., 2002). LPA is increased in allergen-challenged lungs compared with saline controls (Georas et al., 2007), and it is 2- to 5-fold higher in injured, prefibrotic mouse lungs (Toews et al., 2004). LPA also increases interleukin-8 production in human bronchial epithelial cells (hBECs) (Cummings et al., 2004), which then acts as a neutrophil chemoattractant to enhance inflammation (Saatian et al., 2006). LPA has been shown to enhance the contractility (Toews et al., 1997) and to stimulate the proliferation of both airway smooth muscle cells (Cerutis et al., 1997) and lung fibroblasts (Ediger and Toews, 2000) as well as to stimulate fibronectin secretion (Romberger et al., 1993) and filopodia extension (Beckmann et al., 1995) by airway epithelial cells. Together, these effects led us to propose an important role for LPA in airway remodeling and disease (Toews et al., 2002). Consistent with this hypothesis, three recent studies have reported significant protection of LPA receptor knockout mice from multiple components of airway disease (Shea et al., 2007; Tager et al., 2007; Zhao et al., 2007).

Studies from our laboratory have shown that LPA stimulates HASM cell proliferation on its own, synergizes with EGF to greatly enhance HASM cell proliferation (Cerutis et al., 1997; Ediger and Toews, 2000), and increases EGFR binding and protein expression (Ediger et al., 2002). LPA does not stimulate transactivation of the EGFR in HASM cells (Ediger et al., 2002), but it does stimulate transactivation of the EGFR in primary human bronchial epithelial cells (Zhao et al., 2006). Two pathways through which LPA can transactivate the EGFR have been established. "Ligand-dependent" transactivation involves the activation of matrix metalloproteinases (MMPs) (Shida et al., 2005; Mori et al., 2006), the cleavage of EGFR proligands (Zhao et al., 2006; Xu et al., 2007), and the subsequent activation of the EGFR. "Ligand-independent" transactivation involves the intracellular activation of EGFRs by kinases such as Src (Daub et al., 1997; Shah et al., 2005).

The purpose of this study was to examine LPA regulation of EGFR binding in airway epithelial cells and to determine whether LPA-mediated transactivation of the EGFR plays a role in this regulation. Because of the contribution of EGF and the EGFR to epithelial repair in asthma, determining how LPA and other factors that are elevated in asthmatic airways regulate the EGFR is critical for understanding the role of the EGFR in airway remodeling. These studies also define additional regulatory interactions between GPCRs and receptor tyrosine kinases in airway epithelial cells. It is noteworthy that these interactions are different for airway epithelial cells than for smooth muscle cells and fibroblasts, and they are also different between the normal and the lung cancer cell lines tested.

	Materials and Methods

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Reagents. Cell culture medium, fetal bovine serum (FBS), penicillin/streptomycin (Pen/Strep), Tris-glycine gels, and polyvinylidene difluoride-FL membranes were obtained from Invitrogen (Carlsbad, CA). Vitrogen was purchased from Angiotech Biomaterials (Palo Alto, CA). EGF was purchased from BioSource International (Camarillo, CA), and LPA, 1-O-octadecyl-2-hydroxy-sn-glycero-3-phosphate (ether LPA), phosphatidic acid, and lysophosphatidylcholine were from Avanti Polar Lipids (Alabaster, AL). GM6001 was from BIOMOL Research Laboratories (Plymouth Meeting, PA), and AG1478 and diphtheria toxin CRM mutant (CRM197) were from Calbiochem (San Diego, CA). EGFR and phosphotyrosine (pY99) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat anti-rabbit 800 antibody was from Rockland (Gilbertsville, PA), and goat anti-mouse 680 antibody was from Invitrogen. Other chemicals were from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Fair Lawn, NJ). ¹²⁵I (Amersham, Piscataway, NJ) was used to synthesize ¹²⁵I-EGF by a chloramine T protocol, and the ¹²⁵I-EGF was purified on a Sephadex G-25 column (Rizzino et al., 1988).

Cell Culture. BEAS-2B, H292, and A549 cells were from American Type Culture Collection (Manassas, VA). Primary cultures of hBECs were kindly provided by Dr. Stephen Rennard (University of Nebraska Medical Center, Omaha, NE). BEAS-2B cells and hBECs were cultured in a 1:1 mixture of LHC-9 and RPMI-1640 medium (Lechner and Laveck, 1985). BEAS-2B cells and hBECs were cultured on 1% Vitrogen to enhance cell attachment. H292 lung cancer epithelial cells were cultured in RPMI 1640 medium with 10% FBS and 1% Pen/Strep, and A549 lung cancer epithelial cells were cultured in Ham's F-12 with 10% FBS and 1% Pen/Strep. All cells were maintained in a humidified 5% CO₂ incubator at 37°C and passaged weekly.

¹²⁵I-EGF Binding Assays. BEAS-2B cells and H292 cells were plated at 50,000 cells/well in six-well plates and grown to confluence. A549 cells were plated at 100,000 cells/well in six-well plates and grown to confluence. Cells were starved in the appropriate basal medium (without FBS or other growth factors) overnight (18–24 h), and then they were treated with LPA or other agents for the indicated times, usually 15 min or 18 h. Control wells received bovine serum albumin (BSA), because LPA was dissolved in 0.25% BSA. If cells were treated with an inhibitor, the inhibitor was added 30 min before stimulation with LPA. Cells were then washed once with 2 ml of 37°C DMEM-HEPES with 0.1% BSA, washed once with 2 ml of ice-cold DMEM-HEPES with 0.1% BSA, and then incubated with ¹²⁵I-EGF for 4 h on ice to selectively label cell surface EGFRs and to prevent EGFR endocytosis and EGF degradation. The ¹²⁵I-EGF solutions contained approximately 300,000 cpm/ml in DMEM-HEPES with 0.1% BSA. Nonspecific binding was defined by the addition of 300 ng/ml nonradioactive EGF. After 4 h of binding on ice, the cells were washed four times with 2 ml of ice-cold DMEM-HEPES with 0.1% BSA, and then they were dissolved in 1 ml of 0.2 N NaOH. ¹²⁵I was then quantified in a gamma counter.

EGFR Transactivation Assays. BEAS-2B cells were plated on 60-mm dishes, and cells were grown to confluence. Cells were then starved in RPMI 1640 medium + Pen/Strep overnight. For experiments with inhibitors, cells were pretreated with the inhibitor for 30 min before stimulation with vehicle or 10 µM LPA for 2 min in the continued presence of inhibitor. Cells were then washed with PBS, incubated on ice for 10 min in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 5 mM -glycerophosphate, 1 mM MgCl₂, 1% Triton X-100, 1 mM sodium orthovanadate, and 10 µl/ml protease inhibitor cocktail), and lysed by scraping. The cell lysate was centrifuged for 5 min at 15,300 rpm at 4°C, and the supernatant was diluted in sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue) and boiled for 5 min. Samples were loaded on 6% Tris-glycine gels and electrophoresed for 110 min at 120 V. Gels for EGFR blots were transferred with a semi-wet apparatus to polyvinylidene difluoride-FL membrane for 2 h at 26 V. Membranes were then blocked for 1 h with a 1:1 mixture of PBS/Odyssey blocking buffer (Li-Cor, Lincoln, NE). EGFR blots were incubated with pY99 (rabbit) and EGFR (mouse) antibodies overnight at 4°C. Blots were washed five times with Tris-buffered saline + 0.1% Tween 20 for 5 min each, and then they were incubated with secondary anti-rabbit (680 nm) and anti-mouse (800 nm) antibodies for 1 h before washing again with Tris-buffered saline + 0.1% Tween 20 as described above. Blots were then scanned and quantified using the Odyssey infrared imaging system (Li-Cor). Data are expressed as the ratio of phosphorylated/total protein, and then further normalized as the ratio of treated/control cells.

Data Analyses. All data are presented as means ± S.E.M. Data were analyzed using GraphPad Prism 4.00 (GraphPad Software, Inc., San Diego, CA). Unless otherwise indicated, all data shown are representative of at least three separate experiments. t tests were used to determine statistical significance.

	Results

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LPA Induces a Rapid and Sustained Decrease in EGFR Binding in Normal Epithelial Cells. BEAS-2B cells, which are virally transformed and immortalized but frequently used as a model for normal airway epithelial cells, and hBECs from primary cultures were treated with 10 µM LPA or with BSA-containing vehicle for various times, and then they were incubated with ¹²⁵I-EGF on ice to quantify cell surface EGF receptor binding. In contrast to the upregulation of EGFRs that occurs in airway smooth muscle cells (Ediger et al., 2002), LPA induced a rapid decrease in EGFR binding in these epithelial cells, with the maximal decrease occurring by 15 min. After 15 min of treatment, BEAS-2B cells showed a decrease of 32 ± 2%, and hBECs showed a decrease of 26 ± 14% (Fig. 1A). This decrease in binding was sustained through 18 h of continuous LPA exposure, with decreases of 45 ± 5 and 44 ± 9% for BEAS-2B cells and hBECs, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 1. LPA decreases EGFR binding in airway epithelial cells. BEAS-2B, hBEC, H292, and A549 cells were starved for 24 h in basal medium before treatment with BSA or 10 µM LPA for the indicated times, followed by washing with DMEM-HEPES and binding of 125I-EGF to intact cells on ice. Data represent the fold increase or decrease relative to binding in control cells treated with the BSA vehicle, and they are from at least three separate experiments, except for hBECs, which are from two separate experiments.

Concentration Dependence of the LPA-Induced Changes in EGFR Binding. The LPA concentration dependence of the changes in EGFR binding was determined at both 15 min and 18 h for BEAS-2B, H292, and A549 cells (Fig. 2, A–C). In BEAS-2B cells, the LPA-induced decrease in EGFR binding at 15 min occurred with high potency; however, the dose-response curve was shifted to the right after 18 h of treatment. The EC₅₀ values were 3.8 ± 0.2 nM at 15 min (Fig. 2A) and 1.1 ± 0.1 µM at 18 h (Fig. 2B). To test whether the lower potency at 18 h was due to deacylation of LPA, we tested the effects of ether LPA, which has a stable ether bond between C1 of the glycerol backbone and the fatty acid chain, preventing phospholipase-mediated deacylation (Prestwich et al., 2005). The dose-response curves for LPA and ether LPA were very similar at both 15 min and 18 h, with EC₅₀ values of 9.8 ± 0.8 nM at 15 min and 1.9 ± 0.1 µM at 18 h for ether LPA (Fig. 2C). Thus, phospholipase degradation of the LPA is apparently not the explanation for the lower potency of LPA for the 18-h decrease.

View larger version (20K): [in this window] [in a new window]   Fig. 2. LPA decreases binding in a dose-dependent manner. BEAS-2B, H292, and A549 cells were treated with BSA or the indicated concentrations of LPA for 15 min (A) or 18 h (B) before incubation with 125I-EGF on ice for 4 h. C, BEAS-2B cells were treated with the indicated concentrations of ether LPA for 15 min or 18 h before incubation with 125I-EGF on ice for 4 h. Data represent the fold decrease relative to binding in control cells treated with BSA, and they are from at least four separate experiments.

Dose-response curves at 15 min for both H292 and A549 cells showed that the LPA-induced decrease in EGFR binding required higher concentrations of LPA in these cancer cells than in the BEAS-2B cells, with EC₅₀ values of 170 ± 10 nM for H292 cells and 24 ± 3 nM for A549 cells (Fig. 2A). Although H292 cells showed no change in binding at 18 h, the A549 cells consistently showed a modest increase in EGFR binding of 31 ± 8%, with an EC₅₀ of 1.4 ± 0.4 µM after 18 h of LPA treatment (Fig. 2B).

The Decrease in Binding Results Primarily from a Decrease in B_max. To assess the nature of the decrease in EGFR binding, saturation and competition binding assays were performed after exposure to 10 µM LPA for 15 min for the two prototype cell lines, BEAS-2B for normal cells and H292 for cancer cells. Saturation curves with both cell lines revealed a prominent decrease in B_max after LPA treatment; a small increase in K_d was also observed, but it was not sufficient to make a major contribution to the decrease in binding (Fig. 3, A and B). Competition curves similarly showed clear decreases in maximal EGFR binding after LPA treatment, with only small increases in IC₅₀ values for both cell lines (Fig. 3, C and D).

View larger version (23K): [in this window] [in a new window]   Fig. 3. LPA decreases Bmax and affinity. BEAS-2B (A) and H292 (B) cells were treated with BSA or 10 µM LPA for 15 min. Cells were then incubated with the indicated concentrations of 125I-EGF on ice for 4 h to generate saturation curves. Data represent total 125I-EGF binding from two separate experiments, except for the two highest concentrations, which were only tested once. BEAS-2B (C) and H292 (D) cells were treated with BSA or 10 µM LPA for 15 min. Cells were then incubated on ice for 4 h with 300,000 cpm/ml 125I-EGF plus the indicated amounts of nonradioactive EGF to generate competition curves. Data represent total 125I-EGF binding, and they are from two separate experiments.

Effects of Other GPCR Agents on EGFR Binding. The effects on EGFR binding were assessed for two additional GPCR agents, sphingosine-1-phosphate (S1P) and thrombin, at both 15 min and 18 h. Both of these agonists are implicated in airway disease, and they can activate similar GPCR pathways as LPA (Jolly et al., 2002; Rosenfeldt et al., 2003; Terada et al., 2004; Kume et al., 2007). With 15-min treatments, the lysophospholipid mediator S1P was similarly potent, but it was slightly more efficacious than LPA in both BEAS-2B and A549 cells, with decreases of 52 ± 4 and 69 ± 3% and EC₅₀ values of 3.6 ± 0.6 and 35 ± 4 nM, respectively (Fig. 4, A and C). S1P was less potent and equally efficacious as LPA in H292 cells, with a decrease of 38 ± 2% and an EC₅₀ value of 2.0 ± 0.1 µM (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Other GPCR mitogens also decrease EGFR binding at 15 min. BEAS-2B (A), H292 (B), and A549 (C) cells were treated with vehicle (CTL) or the indicated concentrations of S1P or thrombin for 15 min. Cells were then washed with DMEM-HEPES, and then they were incubated with 125I-EGF on ice for 4 h. Data represent specific binding expressed as a fold of the appropriate control, and they are from three separate experiments.

S1P and thrombin also induced similar changes as LPA after 18 h of treatment. In BEAS-2B cells, S1P was more potent but slightly less efficacious than LPA, with a decrease of 30 ± 4% and an EC₅₀ value of 200 ± 40 nM (Fig. 5A). In A549 cells, both S1P and thrombin mimicked the small increase in EGFR binding seen after prolonged LPA treatment, with increases of 44 ± 5 and 35 ± 18%, respectively. Thrombin was the most potent agent for this effect, with an EC₅₀ of 5.8 ± 0.4 nM, followed by S1P and LPA, with EC₅₀ values of 1.5 ± 0.3 and 1.4 ± 0.4 µM, respectively (Figs. 2B and 5C). Similar to LPA, S1P and thrombin had no effect on EGFR binding after 18 h of treatment in H292 cells (Fig. 5B).

View larger version (18K): [in this window] [in a new window]   Fig. 5. GPCR mitogens decrease binding in BEAS-2B cells but not H292 or A549 cells at 18 h. BEAS-2B (A), H292 (B), and A549 (C) cells were treated with vehicle (CTL) or the indicated concentrations of S1P or thrombin for 18 h. Cells were then washed with DMEM-HEPES, and then they were incubated with 125I-EGF on ice for 4 h. Data represent specific binding expressed as a fold of the appropriate control, and they are from at least three separate experiments.

Effects of Different Fatty Acid Chain Lengths. Oleoyl-LPA (18:1 LPA) is the most commonly studied form of LPA. We tested both stearoyl (18:0) LPA and palmitoyl (16:0) LPA to determine the LPA structure-activity relationship for the decrease in EGFR binding. The 18:1 LPA was the most potent and most efficacious agent after 15 min of treatment, followed by 18:0 LPA (Table 1). 16:0 LPA was the least potent at 15 min, but it was as effective at decreasing EGFR binding as 18:0 LPA in all cell types. Interestingly, 18:0 LPA was the most potent for the sustained decrease in binding after 18 h of treatment in BEAS-2B cells, followed by 18:1 LPA and then 16:0 LPA (Table 1). This potency order was reversed in A549 cells, where EGFR binding increases rather than decreases after 18 h of treatment, with 16:0 LPA being the most potent, followed by 18:1 LPA, and with 18:0 LPA being the least potent.

LPA Stimulates Tyrosine Phosphorylation of the EGFR in BEAS-2B Cells. LPA transactivates the EGFR in many cells, including primary human bronchial epithelial cells (Zhao et al., 2006). The ability of LPA to transactivate EGFRs in BEAS-2B cells was assessed by measuring EGFR tyrosine phosphorylation (Fig. 6). BEAS-2B cells were treated for the indicated times in the presence of 10 µM LPA. LPA stimulated EGFR phosphorylation 1.7- to 2.0-fold, and this phosphorylation was sustained to at least 15 min. Although LPA stimulated EGFR phosphorylation much less than EGF itself, this magnitude is similar to what has been reported previously (Zhao et al., 2006).

View larger version (32K): [in this window] [in a new window]   Fig. 6. LPA transactivates the EGFR in BEAS-2B cells. BEAS-2B cells were treated with 10 µM LPA for the indicated times. Cells were then lysed, and cell lysates were separated by SDS-polyacrylamide gel electrophoresis and blotted for total EGFR (green) and pY99 (red). Western blots were scanned and quantified using the Odyssey infrared imaging system. Data are shown as phospho-EGFR/total EGFR and then as a fold of time 0. Top, representative blot. Bottom, data from three to four separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 7. LPA stimulates transactivation via MMPs in BEAS-2B cells. BEAS-2B cells were pretreated for 30 min with 2.5 µM AG1478, 25 µM GM6001, or 10 µg/ml CRM197, and then they were treated with vehicle or 10 µM LPA for 2 min. Cells were then lysed, and cell lysate was separated by SDS-polyacrylamide gel electrophoresis and blotted for total EGFR and phosphotyrosine. Western blots were scanned and quantified using the Odyssey Infrared Imaging System. Data are shown as phospho-EGFR/total EGFR and then as a fold of vehicle-treated values. Data are from at least four separate experiments, except for CRM197 (n = 2).

View larger version (25K): [in this window] [in a new window]   Fig. 8. AG1478 and GM6001 inhibit the LPA-stimulated decrease in EGFR binding in BEAS-2B cells. BEAS-2B cells were pretreated for 30 min with 2.5 µM AG1478 (A), 25 µM GM6001 (B), or 10 µg/m CRM197 (C) before treatment with 10 µM LPA for 15 min or 18 h. Cells were then incubated with 125I-EGF on ice for 4 h. Data represent the fold increase or decrease relative to vehicle-treated cells, and they are from at least three separate experiments. *, p 0.05; **, p 0.01; and ***, p 0.001.

	Discussion

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Compounds with structural similarities to LPA, such as oleic acid and LPC, either had no effect on EGFR binding or were much less potent than LPA. PA also had either no effect or was much less potent than LPA, with the exception of 18-h treatment of BEAS-2B cells, where PA and LPA had similar potency and efficacy. The difference in the dose-response curves to LPA at 15 min and 18 h in BEAS-2B cells is not due to deacylation of LPA by phospholipases, but the decreased potency of LPA after 18 h of treatment could result from the metabolism of LPA by other enzymes, thereby decreasing the amount of LPA available to regulate EGFR binding. The specificity of these effects suggests that the decreases in EGFR binding for LPA versus structural analogs is mediated by LPA signaling through one or more of its specific GPCRs and not via a nonspecific lipid-mediated effect.

The decrease in EGFR binding is a result of a decrease in EGF binding sites after 15 min of LPA treatment, as indicated by both saturation and competition curves; there is also a small decrease in the affinity of EGF for its receptor, although this change in affinity contributes minimally to the decrease in EGFR binding. Because the binding studies were conducted with cells on ice to selectively label cell surface receptors, the decrease in binding could be due to receptor internalization or some other decrease in receptor accessibility, without an actual loss of EGFR protein. One obvious possibility is that the rapid decrease in binding is a result of transactivation and subsequent internalization of EGFRs induced by LPA. Previous studies have established that LPA transactivates the EGFR in primary human bronchial epithelial cells (Zhao et al., 2006) and that transactivation of the EGFR results in receptor internalization and a decrease in ¹²⁵I-EGF binding on ice in some cell types (Pierce et al., 2000; Kim et al., 2003; Olivares-Reyes et al., 2005). In rat hepatic C9 cells, angiotensin II stimulates a decrease in EGFR cell surface binding via transactivation and internalization of the EGFR (Olivares-Reyes et al., 2005). Similar results were obtained with serotonin in rat renal mesangial cells (Grewal et al., 2001). Therefore, transactivation and subsequent internalization of the EGFR provide one possible mechanism for the decrease in EGFR binding by LPA in these cells.

In agreement with these previous studies, LPA was also able to stimulate tyrosine phosphorylation of the EGFR in BEAS-2B cells, and this transactivation was completely blocked by both GM6001 and AG1478. However, both GM6001 and AG1478 reduced the decrease in EGFR binding after 15 min of LPA treatment only partially. These data indicate that transactivation of the EGFR, presumably followed by internalization and/or degradation, may contribute to the decrease in EGFR binding after LPA treatment, but it is not the only mechanism through which LPA reduces EGFR binding in these cells. In spite of essentially complete inhibition of transactivation, AG1478 and GM6001 only inhibit the LPA-induced decrease in EGFR binding by about half. These results are very similar to those reported previously with primary hBECs, in which LPA-stimulated EGFR transactivation was completely blocked by AG1478 and GM6001, but the downstream response of interleukin-8 secretion was only inhibited by approximately half (Zhao et al., 2006). However, whereas the HB-EGF inhibitor CRM197 partially inhibited transactivation in hBECs (Zhao et al., 2006), it had minimal effect on LPA-induced transactivation in BEAS-2B cells, suggesting that BEAS-2B cells may use a different EGFR pro ligand to stimulate transactivation.

Five LPA receptors have been identified to date, LPA_1–5; LPA₄ and LPA₅ have only been identified recently (Hecht et al., 1996; An et al., 1998; Bandoh et al., 1999; Noguchi et al., 2003; Lee et al., 2006). The ability of LPA receptors to couple to multiple G proteins greatly expands the number of signaling pathways by which LPA can mediate its effects. LPA, S1P, and thrombin all stimulate common signaling pathways, including G_i/o,G_q/11, and G_12/13 pathways (Sorensen et al., 2003; Radeff-Huang et al., 2004). In addition, S1P and thrombin are mitogenic for HASM cells (Ediger and Toews, 2000), and they have been implicated in airway disease similar to LPA (Jolly et al., 2002; Lan et al., 2002; Kume et al., 2007). Both thrombin and S1P mimicked the effect of LPA to alter EGFR binding in airway epithelial cells; thrombin was much more potent, but it was slightly less efficacious than LPA or S1P for decreasing EGFR binding, and LPA and S1P were comparable in both potency and efficacy. Unexpectedly, thrombin decreased EGFR binding at low concentrations after 18 h of treatment in BEAS-2B cells, but this effect was lost at higher concentrations. The unique activation and complex regulatory mechanisms that are known to occur for thrombin receptors may contribute to these biphasic effects (Lan et al., 2002).

In all airway epithelial cell types tested, LPA induced a rapid decrease in EGFR binding, but a sustained decrease was seen only in a subset of these cells. This is markedly different from the response in airway mesenchymal cells, both smooth muscle cells and fibroblasts, in which LPA increases EGFR binding but only after 12 to 24 h of exposure (Ediger et al., 2002). The up-regulation of EGFRs in mesenchymal cells is via a transcriptional mechanism, leading to an increase in EGFR protein, and it is blocked by both the transcription inhibitor actinomycin D and the translation inhibitor cycloheximide (Ediger et al., 2002). Because of the rapid time course in the epithelial cells, it is unlikely that the initial decrease in binding is a result of transcriptional inhibition of EGFR synthesis, but rather results from more rapid mechanisms such as transactivation, internalization, and perhaps degradation of EGFRs. Several studies have found that transactivation of the EGFR by LPA and other GPCR agents does not occur in HASM cells (Rakhit et al., 1999; Krymskaya et al., 2000; Ediger et al., 2002; Capra et al., 2003), and the lack of this response may be critical to the differences between regulation of EGFRs by LPA in epithelial versus mesenchymal cells. For both the differences among epithelial cell lines and the differences between epithelial versus mesenchymal cells, it is possible that one LPA receptor subtype and signaling pathway mediates one response, whereas a different subtype and signaling pathway mediates the other response. Identification of specific LPA receptors and pathways for the rapid versus sustained component of the changes in EGFRs in normal versus cancer cells is an important goal of ongoing studies.

In summary, LPA rapidly decreases EGFR binding in airway epithelial cells, with a sustained decrease in two models of normal epithelial cells but only a transient decrease in two lung cancer cell lines. The rapid decrease in binding is partially mediated by transactivation of the EGFR, although an additional signaling pathway also seems to be involved. The decrease in EGFR binding is mimicked by other GPCR agents that are implicated in asthma and that use signaling pathways similar to those for LPA, suggesting a broader relevance of these findings to other mediators of asthma and related airway diseases. These and further studies of the regulation of EGFR binding by LPA and other GPCRs and of the different effects in diverse types of airway cells will contribute to our understanding of multiple lung diseases, and they may also point to novel approaches for therapeutic intervention.

	Acknowledgements

 
We thank Dr. Stephen Rennard for providing primary cultures of human bronchial epithelial cells and for helpful discussions. We also thank Bryan Danforth and Jennifer Mihaljevic for technical assistance.

	Footnotes

 
This work was supported by American Heart Fellowship 0415367Z and a Skala Fellowship (to K.M.K.) and GlaxoSmithKline Grant 100909 and Nebraska Department of Health and Human Services Grant 2007-39 (to M.L.T.).

Portions of this work were published in abstract form: Kassel KM, Parker SM, Danforth BL, Lanik AD, Mihaljevic JA, Meeks AS, Schulte NA, and Toews ML (2005) Lysophosphatidic acid decreases epidermal growth factor receptor binding in airway epithelial cells. FASEB J 19:A527 (presented at Experimental Biology 2005, San Diego, CA) and Toews ML, Parker SM, and Kassel KM (2006) Signaling pathways for lysophosphatidic acid (LPA) inhibition of epidermal growth factor receptor (EGFR) binding in lung epithelial cells. FASEB J 20:A695 (presented at Experimental Biology 2006, San Francisco, CA). Portions of this work were published as part of K.M.K.'s dissertation: Kassel KM (2007) Regulation of epidermal growth factor receptors and mitogenic signaling by lysophosphatidic acid and ₂ adrenergic receptors in airway cells. Doctoral dissertation, University of Nebraska Medical Center, Omaha, NE.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.120584.

ABBREVIATIONS: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HASM, human airway smooth muscle; LPA, lysophosphatidic acid; ether LPA, 1-O-octadecyl-2-hydroxy-sn-glycero-3-phosphate; hBEC, human bronchial epithelial cell; MMP, matrix metalloproteinase; GPCR, G protein-coupled receptor; FBS, fetal bovine serum; Pen/Strep, penicillin/streptomycin; AG1478, 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline; GM6001, N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; S1P, sphingosine-1-phosphate; LPC, lysophosphatidylcholine; PA, phosphatidic acid; HB, heparin-binding; CTL, control (vehicle).

Address correspondence to: Dr. Myron L. Toews, 985800 Nebraska Medical Center, Omaha, NE 68198-5800. E-mail: mtoews{at}unmc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Amishima M, Munakata M, Nasuhara Y, Sato A, Takahashi T, Homma Y, and Kawakami Y (1998) Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway. Am J Respir Crit Care Med 157: 1907–1912.[Abstract/Free Full Text]

An S, Bleu T, Hallmark OG, and Goetzl EJ (1998) Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid. J Biol Chem 273: 7906–7910.[Abstract/Free Full Text]

Bandoh K, Aoki J, Hosono H, Kobayashi S, Kobayashi T, Murakami-Murofushi K, Tsujimoto M, Arai H, and Inoue K (1999) Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. J Biol Chem 274: 27776–27785.[Abstract/Free Full Text]

Beckmann JD, Romberger DJ, Rennard SI, and Spurzem JR (1995) Induction of bovine bronchial epithelial cell filopodia by tetradecanoyl phorbol acetate, calcium ionophore, and lysophosphatidic acid. J Cell Physiol 164: 123–131.[CrossRef][Medline]

Capra V, Habib A, Accomazzo MR, Ravasi S, Citro S, Levy-Toledano S, Nicosia S, and Rovati GE (2003) Thromboxane prostanoid receptor in human airway smooth muscle cells: a relevant role in proliferation. Eur J Pharmacol 474: 149–159.[CrossRef][Medline]

Cerutis D, Nogami M, Anderson JL, Churchill JD, Romberger DJ, Rennard SI, and Toews ML (1997) Lysophosphatidic acid and EGF stimulate mitogenesis in human airway smooth muscle cells. Am J Physiol 273: L10–L15.[Medline]

Cummings R, Parinandi N, Wang L, Usatyuk P, and Natarajan V (2002) Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung. Mol Cell Biochem 234/235: 99–109.

Cummings R, Zhao Y, Jacoby D, Spannhake EW, Ohba M, Garcia JG, Watkins T, He D, Saatian B, and Natarajan V (2004) Protein kinase C mediates lysophosphatidic acid-induced NF-B activation and interleukin-8 secretion in human bronchial epithelial cells. J Biol Chem 279: 41085–41094.[Abstract/Free Full Text]

Daub H, Wallasch C, Lankenau A, Herrlich A, and Ullrich A (1997) Signal characteristics of G protein-transactivated EGF receptor. EMBO J 16: 7032–7044.[CrossRef][Medline]

Ediger TL, Danforth BL, and Toews ML (2002) Lysophosphatidic acid upregulates the epidermal growth factor receptor in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 282: L91–L98.[Abstract/Free Full Text]

Ediger TL and Toews ML (2000) Synergistic stimulation of airway smooth muscle cell mitogenesis. J Pharmacol Exp Ther 294: 1076–1082.[Abstract/Free Full Text]

Eichholtz T, Jalink K, Fahrenfort I, and Moolenaar WH (1993) The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J 291: 677–680.[Medline]

Georas SN, Berdyshev E, Hubbard W, Gorshkova IA, Usatyuk PV, Saatian B, Myers AC, Williams MA, Xiao HQ, Liu M, et al. (2007) Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge. Clin Exp Allergy 37: 311–322.[CrossRef][Medline]

Grewal JS, Luttrell LM, and Raymond JR (2001) G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells. J Biol Chem 276: 27335–27344.[Abstract/Free Full Text]

Hecht JH, Weiner JA, Post SR, and Chun J (1996) Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex. J Cell Biol 135: 1071–1083.[Abstract/Free Full Text]

Holgate ST, Davies DE, Puddicombe S, Richter A, Lackie P, Lordan J, and Howarth P (2003) Mechanisms of airway epithelial damage: epithelial-mesenchymal interactions in the pathogenesis of asthma. Eur Respir J Suppl 44: 24s–29s.[Medline]

Jolly PS, Rosenfeldt HM, Milstien S, and Spiegel S (2002) The roles of sphingosine-1-phosphate in asthma. Mol Immunol 38: 1239–1245.[CrossRef][Medline]

Kim J, Ahn S, Guo R, and Daaka Y (2003) Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors. Biochemistry 42: 2887–2894.[CrossRef][Medline]

Krymskaya VP, Orsini MJ, Eszterhas AJ, Brodbeck KC, Benovic JL, Panettieri RA Jr, and Penn RB (2000) Mechanisms of proliferation synergy by receptor tyrosine kinase and G protein-coupled receptor activation in human airway smooth muscle. Am J Respir Cell Mol Biol 23: 546–554.[Abstract/Free Full Text]

Kume H, Takeda N, Oguma T, Ito S, Kondo M, Ito Y, and Shimokata K (2007) Sphingosine 1-phosphate causes airway hyper-reactivity by rho-mediated Myosin phosphatase inactivation. J Pharmacol Exp Ther 320: 766–773.[Abstract/Free Full Text]

Lan RS, Stewart GA, and Henry PJ (2002) Role of protease-activated receptors in airway function: a target for therapeutic intervention? Pharmacol Ther 95: 239–257.[CrossRef][Medline]

Lazaar AL and Panettieri RA Jr (2003) Is airway remodeling clinically relevant in asthma? Am J Med 115: 652–659.[CrossRef][Medline]

Lechner JF and Laveck MA (1985) A serum-free method for culturing normal human epithelial cells at clonal density. J Tissue Cult Methods 9: 43–48.[Medline]

Lee CW, Rivera R, Gardell S, Dubin AE, and Chun J (2006) GPR92 as a new G12/13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem 281: 23589–23597.[Abstract/Free Full Text]

Lee H, Goetzl EJ, and An S (2000) Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing. Am J Physiol Cell Physiol 278: C612–C618.[Abstract/Free Full Text]

Mori K, Kitayama J, Shida D, Yamashita H, Watanabe T, and Nagawa H (2006) Lysophosphatidic acid-induced effects in human colon carcinoma DLD1 cells are partially dependent on transactivation of epidermal growth factor receptor. J Surg Res 132: 56–61.[CrossRef][Medline]

Munakata M (2006) Airway remodeling and airway smooth muscle in asthma. Allergol Int 55: 235–243.[CrossRef][Medline]

Nadel JA (2001) Role of epidermal growth factor receptor activation in regulating mucin synthesis. Respir Res 2: 85–89.[CrossRef][Medline]

Noguchi K, Ishii S, and Shimizu T (2003) Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol Chem 278: 25600–25606.[Abstract/Free Full Text]

Olivares-Reyes JA, Shah BH, Hernandez-Aranda J, Garcia-Caballero A, Farshori MP, Garcia-Sainz JA, and Catt KJ (2005) Agonist-induced interactions between angiotensin AT1 and epidermal growth factor receptors. Mol Pharmacol 68: 356–364.[Abstract/Free Full Text]

Pierce KL, Maudsley S, Daaka Y, Luttrell LM, and Lefkowitz RJ (2000) Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors. Proc Natl Acad Sci U S A 97: 1489–1494.[Abstract/Free Full Text]

Prestwich GD, Xu Y, Qian L, Gajewiak J, and Jiang G (2005) New metabolically stabilized analogues of lysophosphatidic acid: agonists, antagonists and enzyme inhibitors. Biochem Soc Trans 33: 1357–1361.[CrossRef][Medline]

Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, and Davies DE (2000) Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J 14: 1362–1374.[Abstract/Free Full Text]

Radeff-Huang J, Seasholtz TM, Matteo RG, and Brown JH (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. J Cell Biochem 92: 949–966.[CrossRef][Medline]

Rakhit S, Conway AM, Tate R, Bower T, Pyne NJ, and Pyne S (1999) Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem J 338: 643–649.[CrossRef][Medline]

Rizzino A, Kazakoff P, Ruff E, Kuszynski C, and Nebelsick J (1988) Regulatory effects of cell density on the binding of transforming growth factor beta, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. Cancer Res 48: 4266–4271.[Abstract/Free Full Text]

Romberger D, Plevin R, Adachi Y, Rennard SI, Nogami M, and Toews ML (1993) Lysophosphatidic acid stimulates airway epithelial cell fibronectin release. Clin Res 41: 674A.

Rosenfeldt HM, Amrani Y, Watterson KR, Murthy KS, Panettieri RA Jr, and Spiegel S (2003) Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells. FASEB J 17: 1789–1799.[Abstract/Free Full Text]

Saatian B, Zhao Y, He D, Georas SN, Watkins T, Spannhake EW, and Natarajan V (2006) Transcriptional regulation of lysophosphatidic acid-induced interleukin-8 expression and secretion by p38 MAPK and JNK in human bronchial epithelial cells. Biochem J 393: 657–668.[CrossRef][Medline]

Shah BH, Olivares-Reyes JA, and Catt KJ (2005) The protein kinase C inhibitor Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] potentiates agonist-induced mitogen-activated protein kinase activation through tyrosine phosphorylation of the epidermal growth factor receptor. Mol Pharmacol 67: 184–194.[Abstract/Free Full Text]

Shea BS, LaCamera P, Poorvu EC, Hart WK, Chun J, Luster AD, and Tager AM (2007) Pulmonary vascular leak following bleomycin lung injury is dependent on the lysophosphatidic acid receptor LPA1. Am J Respir Crit Care Med 175: A237.

Shida D, Kitayama J, Yamaguchi H, Yamashita H, Mori K, Watanabe T, and Nagawa H (2005) Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells. World J Gastroenterol 11: 5638–5643.[Medline]

Sorensen SD, Nicole O, Peavy RD, Montoya LM, Lee CJ, Murphy TJ, Traynelis SF, and Hepler JR (2003) Common signaling pathways link activation of murine PAR-1, LPA, and S1P receptors to proliferation of astrocytes. Mol Pharmacol 64: 1199–1209.[Abstract/Free Full Text]

Tager AM, LaCamera P, Shea BS, Karimi-Shah BA, Campanella GS, Polosukhin V, Hart WK, Poorvu EC, Chun J, Blackwell TS, et al. (2007) Mice deficient for the lysophosphatidic acid receptor LPA1 demonstrate reduced fibroblast recruitment and reduced pulmonary fibrosis post-bleomycin injury. Am J Respir Crit Care Med 175: A814.

Terada M, Kelly EA, and Jarjour NN (2004) Increased thrombin activity after allergen challenge: a potential link to airway remodeling? Am J Respir Crit Care Med 169: 373–377.[Abstract/Free Full Text]

Toews ML, Ediger TL, Romberger DJ, and Rennard SI (2002) Lysophosphatidic acid in airway function and disease. Biochim Biophys Acta 1582: 240–250.[Medline]

Toews M, Moore BB, Saulnier-Blache JS, Thronson HL, Schulte NA, and Toews G (2004) Elevated concentrations of lysophosphatidic acid in airway lavage fluids from mouse models of pulmonary fibrosis. Am J Respir Crit Care Med 169: A300.

Toews ML, Ustinova EE, and Schultz HD (1997) Lysophosphatidic acid enhances contractility of isolated airway smooth muscle. J Appl Physiol 83: 1216–1222.[Abstract/Free Full Text]

Wang L, Cummings R, Zhao Y, Kazlauskas A, Sham JK, Morris A, Georas S, Brindley DN, and Natarajan V (2003) Involvement of phospholipase D2 in lysophosphatidate-induced transactivation of platelet-derived growth factor receptor- in human bronchial epithelial cells. J Biol Chem 278: 39931–39940.[Abstract/Free Full Text]

Xu KP, Yin J, and Yu FS (2007) Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor. Invest Ophthalmol Vis Sci 48: 636–643.[Abstract/Free Full Text]

Zhao Y, He D, Saatian B, Watkins T, Spannhake EW, Pyne NJ, and Natarajan V (2006) Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase C, Lyn kinase, and matrix metalloproteinases. J Biol Chem 281: 19501–19511.[Abstract/Free Full Text]

Zhao Y, Tong J, Gorshkova I, He D, Stern R, Berdyshev E, Pendyala S, Sperling A, Chun J, and Natarajan V (2007) Lysophosphatidic acid receptor 1 and 2 null mice show resistance to airway inflammation induced by Schistosoma mansoni eggs. Am J Respir Crit Care Med 175: A926.[CrossRef]

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