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CELLULAR AND MOLECULAR
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska
Received October 29, 2007; accepted February 27, 2008.
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). The EGFR has been shown to be involved in the airway remodeling associated with asthma and pulmonary fibrosis (Boxall et al., 2006; Ingram and Bonner, 2006), which includes hyperplasia and hypertrophy of smooth muscle cells and fibroblasts, goblet cell metaplasia, excess epithelial repair, thickening of the lamina reticularis, and increased angiogenesis (Lazaar and Panettieri, 2003; Boxall et al., 2006); together, these changes contribute to the airway hyperresponsiveness and other aspects of the pathology of chronic airway diseases (Munakata, 2006). In addition, the EGFR is expressed, mutated, and/or abnormally activated in many epithelial tumors, leading to receptor overexpression and/or ligand-independent activation (Mendelsohn and Baselga, 2006) with consequent stimulation of survival and proliferation signaling pathways (Engelman and Cantley, 2006).
The simple lipid mediator lysophosphatidic acid (LPA) acts on diverse cell types to stimulate proliferation, differentiation, tumor metastasis, and cytoskeletal rearrangements involved in chemotaxis, migration, motility, and contraction (Wang et al., 2003; Zhao et al., 2006). LPA has been implicated in multiple airway diseases, particularly in inflammatory diseases such as asthma. LPA is significantly higher in allergen-challenged lungs than in saline controls (Georas et al., 2007) and 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 (Cummings et al., 2004), and interleukin-8 then acts as a neutrophil chemoattractant to enhance inflammation (Saatian et al., 2006). Most recently, it has been shown that LPA is increased in bronchoalveolar lavage fluid after lung injury and that mice lacking the LPA1 receptor are protected from edema, fibrosis, and mortality (Tager et al., 2008). Previous studies from our laboratory have shown that LPA stimulates the proliferation of human lung fibroblasts and human airway smooth muscle (HASM) cells, and it exhibits a marked synergism in stimulating proliferation when added in combination with EGF. LPA also enhances contraction of the airways, enhances fibronectin production and release, and stimulates filopodia extension, processes that are all related to airway remodeling (Toews et al., 2002, and refs. therein).
Five G protein-coupled receptors have been identified for LPA, termed LPA1–5 (Hecht et al., 1996; An et al., 1998; Bandoh et al., 1999; Noguchi et al., 2003; Lee et al., 2006). Through the activation of LPA1–3, the first identified and best characterized LPA receptor subtypes, LPA couples to heterotrimeric G proteins subfamilies Gi/o, Gq/11, and G12/13. It is well known that Gi/o inhibits adenylyl cyclase and also stimulates MEK/ERK activation; Gq/11 stimulates the conversion of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol to activate Ca2+- and phospholipid-dependent protein kinase (PKC) and increase Ca2+ release from the endoplasmic reticulum and to stimulate MEK/ERK activation; and G12/13 activates the small GTP-binding protein Rho.
We showed previously that LPA exhibits diverse and complex regulatory effects on EGFR binding and expression in different airway cell types. In HASM cells, LPA up-regulates EGFR protein and binding over a 24-h time course through a transcriptional and translational mechanism (Ediger et al., 2002). In contrast, LPA has the opposite effect, to decrease binding, in airway epithelial cells. Two "normal" airway epithelial cell lines exhibit a rapid and sustained decrease in EGFR binding, but two lung cancer-derived epithelial cell lines exhibit a rapid decrease that is only transient due to rapid reversal of the decrease in binding, even in the continued presence of LPA (Kassel et al., 2007). In addition, LPA has been shown to transactivate EGFRs in airway epithelial cells (Zhao et al., 2006) but not in HASM cells (Ediger et al., 2002); the rapid decrease in EGFR binding in airway epithelial cells seems to be in part a result of this transactivation of the EGFR by LPA, based on the effects of transactivation inhibitors (Kassel et al., 2007). Because both LPA and EGF are likely to be involved not only in normal cellular homeostasis but also in disease progression, it is important to understand their interactions and the regulation of their receptors in both normal and disease cells (Ingram and Bonner, 2006). Toward a further understanding of the interactions between LPA and EGFRs, the studies presented here define the different LPA signaling pathways that are involved in the rapid and sustained decreases in EGFR binding induced by LPA in BEAS-2B airway epithelial cells.
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). ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technology Inc. (Danvers, MA), and EGFR and pY99 (phosphotyrosine) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat anti-rabbit 800 antibody was from Rockland Immunochemicals (Gilbertsville, PA), and goat anti-mouse 680 antibody was from Invitrogen. Other chemicals, including phorbol-12-myristate-13-acetate (PMA) and U0126, were from Sigma-Aldrich (St. Louis, MO).
Cell Culture. BEAS-2B cells were from American Type Culture Collection (Manassas, VA). BEAS-2B dominant-negative (DN)-PKC
cells were provided by Dr. Anthony Floreani (University of Nebraska Medical Center), and DN-PKC
cells were provided by Dr. Todd Wyatt (University of Nebraska Medical Center) (Wyatt et al., 2007). BEAS-2B cells were cultured in a 1:1 mixture of LHC-9 and RPMI as previously described (Lechner and Laveck, 1985) on 1% Vitrogen in a humidified 5% CO2 incubator at 37°C and passaged weekly. LHC-9 contains bovine pituitary extract, transferrin, EGF, insulin, hydrocortisone, retinoic acid, triiodothyronine, epinephrine, and small amounts of several metals. Cells were starved in RPMI with no additives.
125I-EGF Binding Assays. BEAS-2B cells were plated in six-well plates at 50,000 cells/well, grown to confluence, and starved overnight. Signaling pathway inhibitors were added 30 min before LPA. Regardless of pretreatment, all cells were then treated with LPA or other agents for various times, typically 15 min or 18 h. 125I-EGF binding assays were performed as described previously (Kassel et al., 2007). In brief, cells were washed once with 2 ml of 37°C DMEM-HEPES (20 mM HEPES, pH 7.4) containing 0.1% BSA, washed once with 2 ml of ice-cold DMEM-HEPES containing 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 as the binding in the presence 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 containing 0.1% BSA and then dissolved in 1 ml of 0.2 N NaOH; 125I was quantified in a gamma counter (PerkinElmer Life and Analytical Sciences, Waltham, MA).
ERK1/2 Western Blot. BEAS-2B cells were plated on six-well plates, grown to confluence, and starved overnight. For inhibitor studies, cells were pretreated for 30 min with the inhibitor before stimulation with vehicle or 10 µM LPA for 5 min in the continued presence of inhibitor. Cells were then washed with PBS, lysed on ice for 30 min in lysis buffer (50 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.2% Triton X-100, 5 µl/ml protease inhibitor cocktail, and 0.5 mM dithiothreitol), and scraped. 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 10% Tris-glycine gels and electrophoresed for 105 min at 120 V. Proteins were transferred with a semidry apparatus to Immobilon-FL polyvinylidene fluoride membranes for 1 h at 150 mA. Membranes were blocked for 1 h with a 1:1 mixture of PBS/Odyssey Blocking Buffer (LiCor, Lincoln, NE) and then incubated with phospho-ERK1/2 (rabbit) and total ERK1/2 (mouse) antibodies overnight at 4°C in a 1:1 mixture of PBS/Odyssey Blocking Buffer + 0.2% Tween 20. Blots were washed five times with Tris-buffered saline + 0.1% Tween 20 for 5 min each, then incubated with secondary anti-rabbit (680 nm) and anti-mouse (800 nm) antibodies for 1 h in a 1:1 mixture of PBS/Odyssey Blocking Buffer + 0.2% Tween 20 + 0.02% SDS before washing again as above. Blots were then scanned and quantified using the Odyssey Infrared Imaging System (LiCor). Data were calculated as the ratio of phosphorylated/total protein and then expressed as the ratio of the values for treated/control cells.
EGFR Transactivation Assays. EGFR transactivation was assessed by measuring EGFR tyrosine phosphorylation as described previously (Kassel et al., 2007). BEAS-2B cells were pretreated with various inhibitors for 30 min before stimulation with 10 µM LPA or vehicle 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 β-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 and boiled for 5 min. Samples were loaded on 6% Tris-glycine gels and electrophoresed for 105 min at 120 V. Proteins were transferred with a semiwet apparatus to Immobilon-FL membranes for 2 h at 26 V. Membranes were then blocked for 1 h with a 1:1 mixture of PBS/Odyssey Blocking Buffer (LiCor). EGFR blots were incubated with anti-phospho-tyrosine (pY99) (rabbit) and anti-EGFR (mouse) antibodies overnight at 4°C. EGFR blots were washed, incubated with secondary antibodies, and scanned as detailed above for ERK1/2 Western blots. Data were calculated as the ratio of phosphorylated/total protein and then expressed as the ratio of the values for treated/control cells.
Data Analysis. All data are presented as means ± S.E.M. Data were analyzed using GraphPad Prism 4.00 (GraphPad Software Inc., San Diego, CA). Statistical significance was determined using one-way analysis of variance with the Bonferroni post-test to compare selected pairs of data or Dunnett's post-test to compare treatments to control. All data shown are representative of at least three separate experiments unless otherwise indicated.
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0.05; Fig. 1). In addition, LPA decreased EGFR binding by 30 to 50% after 18 h of treatment (p 0.05; Fig. 1). Pretreating cells with PTx or Y-27632 had no effect on the LPA-induced decrease in EGFR binding at 15 min or 18 h, indicating that LPA does not regulate EGFR binding via Gi/o or Rho kinase (Fig. 1). It is interesting to note that Y-27632 induced a modest increase in binding on its own after 18 h of treatment in BEAS-2B cells, possibly indicating that basal levels of Rho kinase activation are involved in limiting cell surface EGFR expression. Cells were also treated with isoproterenol, a β-adrenergic receptor agonist that activates Gs and increases cAMP, to test whether cAMP production from LPA4 or LPA5 could be a candidate for the LPA effects on EGFR binding. In contrast to LPA, isoproterenol did not decrease EGFR binding at 15 min or 18 h in these cells (data not shown), even though it does decrease EGFR binding in human airway smooth muscle cells (Kassel et al., 2004); thus, Gs and cAMP pathways are probably not involved in the effect of LPA to regulate EGFR binding in BEAS-2B cells.
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Role of PKC in the LPA-Induced Decrease in EGFR Binding. One of the classic effectors activated by LPA downstream of Gq/11 is PKC (Hubbard and Hepler, 2006). Therefore, BEAS-2B cells were treated with 1 µM PMA, a direct PKC activator, to determine whether PMA could mimic LPA regulation of EGFR binding in BEAS-2B cells. Similar to LPA, PMA decreased EGFR binding by 47 ± 3% after 15 min (p 0.001) and by 81 ± 7% after 18 h (p 0.01) of treatment (Fig. 2A). These decreases by PMA were completely blocked by 30 min pretreatment with two pan-PKC inhibitors, RO 31-8220 and bisindolylmaleimide I (Bis I), at both 15 min and 18 h (Fig. 2A; p 0.05).
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0.01), with IC50 values of approximately 2 µM (Fig. 2B); both inhibitors were much less effective in blocking effects of the 15-min LPA treatment (p 0.05), inhibiting this rapid decrease by only 35 to 45% and with an IC50 greater than 10 µM (Fig. 2B).
Because PKC inhibition completely blocks the LPA-induced decrease at 18 h but only partially inhibits the 15-min decrease, BEAS-2B cells were treated with Bis I at different time points during the LPA exposure to determine the time during the 18-h treatment at which the critical PKC-dependent effects occur. In the absence of inhibitor, LPA stimulated a 50 ± 5% decrease after 18 h of treatment (p 0.01; Fig. 3, lower dotted line). Cells pretreated with 10 µM Bis I 30 min before the addition of LPA for 18 h exhibited no decrease in binding compared with cells treated with Bis I alone (p 0.01 compared with LPA). However, if the Bis I was added 1 h after LPA treatment began, LPA decreased EGFR binding by only 24 ± 5% (Fig. 3). If Bis I was added 3 h or more after LPA, the inhibitor had no effect on the LPA-induced decrease in EGFR binding at 18 h (p 0.05 compared with control). These data indicate that the critical PKC effect occurs early during the incubation with LPA, even for the later sustained component of the decrease. These results were not unexpected based on the well established down-regulation of PKC activity that occurs after its activation (Liu and Heckman, 1998).
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To explore which isoform(s) of PKC were responsible for the later decrease in binding, BEAS-2B cells stably transfected with DN forms of PKC (DN-PKC) or PKC (DN-PKC) (Wyatt et al., 2007) were also treated with various concentrations of LPA for 15 min and 18 h. After 15 min of treatment, only DN-PKC significantly inhibited the LPA-induced decrease in EGFR binding compared with nontransfected BEAS-2B cells; EC50 values for LPA were 90 ± 20 nM in DN-PKC cells (p 0.05) and 12 ± 1 nM in DN-PKC cells compared with 5 ± 1 nM in normal BEAS-2B cells (Fig. 4A). After 18 h of treatment, DN-PKC inhibited the LPA-induced decrease in EGFR binding compared with nontransfected BEAS-2B cells (p 0.05); however, the LPA effect was completely absent in DN-PKC cells (p 0.01; Fig. 4B). These data indicate that PKC plays a critical role in the later phase of the LPA-induced decrease in binding in BEAS-2B cells but that PKC may also contribute.
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Role of MEK in the LPA-Induced Decrease in EGFR Binding. LPA has been shown to activate MEK and ERK signaling through either Gi/o or Gq/11 stimulation (Kim et al., 2006; Zhang et al., 2006). Therefore, BEAS-2B cells were pretreated with 10 µM U0126, a MEK inhibitor, before treatment with 10 µM LPA, to determine the possible role of MEK in the regulation of EGFR binding. U0126 inhibited the LPA-induced decrease in EGFR binding at 15 min by 90% (p 0.001; Fig. 5A), suggesting that MEK is critical for the rapid decrease in EGFR binding. U0126 inhibited the 18-h change in binding in BEAS-2B cells by 40% (Fig. 5A), suggesting at least a partial contribution of MEK-dependent mechanisms to the 18-h changes as well.
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0.001; Fig. 5B). Likewise, U0126 inhibited the PMA-induced decrease in EGFR binding at 18 h by approximately 65% (Fig. 5B). These results place MEK downstream of PKC activation in the EGFR regulation pathway.
LPA Time Courses in the Absence and Presence of U0126 and Bis I. LPA time course studies were conducted with MEK and PKC inhibitors to assess in greater detail the contributions of MEK versus PKC to the changes in binding at each time point. BEAS-2B cells were pretreated with 10 µM U0126 or 10 µM Bis I for 30 min, then treated with 10 µM LPA or 1 µM PMA, and binding was then measured at various times from 2 min to 18 h. The MEK inhibitor U0126 completely inhibited the LPA-induced effect for the first 15 min of treatment (p 0.01), but inhibition by U0126 then gradually decreased to less than 50% by 18 h (Fig. 5C). In contrast, the PKC inhibitor Bis I only partially inhibited the LPA effect during the first 15 min of LPA treatment (p 0.01), and its effect gradually increased to essentially complete blockade of the LPA-induced decrease in EGFR binding seen at 18 h (p 0.01; Fig. 5C). It is interesting to note that although the combination of U0126 and Bis I inhibited the LPA-induced decrease in EGFR binding to a greater extent than either agent alone at almost every time point (p 0.01), the inhibition was not complete between 15 min and 6 h. Combined with the ability of U0126 and Bis I to completely inhibit the decrease in EGFR binding at 15 min and 18 h, respectively, this suggests that a possible additional mechanism may play a role during the intermediate time points. In contrast to the results with LPA, the PMA-induced decrease in EGFR binding was completely blocked by Bis I at all time points (p 0.01), whereas U0126 only partially inhibited PMA at all time points (p 0.01; Fig. 5D). These data confirm the requirement of MEK for the rapid decrease induced by LPA and the greater importance of PKC for the later phase of the LPA-induced decrease.
Effects of LPA and Signaling Inhibitors on ERK1/2 Phosphorylation. Because U0126 blocks the rapid decrease in binding, the ability of LPA to stimulate ERK1/2 phosphorylation in these cells was determined. LPA dose-dependently increased ERK1/2 phosphorylation after 5 min of treatment, with an EC50 value of 6 ± 3 nM and a maximal 9 ± 3-fold stimulation with 10 µM LPA (p 0.01; Fig. 6). U0126 completely inhibited ERK1/2 phosphorylation at 5 min by 10 µM LPA (p 0.01) and by 1 µM PMA (Fig. 7A). Bis I completely inhibited the ERK1/2 phosphorylation by PMA but only decreased LPA-stimulated ERK1/2 phosphorylation by approximately 50% (Fig. 7A). These effects of U0126 and Bis I on ERK1/2 phosphorylation are similar to their effects on the decrease in EGFR binding, consistent with a role for ERK1/2 in the LPA-induced decrease in EGFR binding.
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0.001) and 68 ± 10% (p 0.01), respectively (Fig. 7B). These data suggest that Gi/o-independent EGFR transactivation pathways play a role in LPA-stimulated ERK1/2 phosphorylation, in agreement with the previously established contribution of EGFR transactivation to the LPA-induced decrease in EGFR binding (Kassel et al., 2007).
Effects of Signaling Inhibitors on LPA-Induced EGFR Transactivation. To determine whether Gi/o, PKC, and ERK1/2 activation were upstream of EGFR transactivation, BEAS-2B cells were pretreated overnight with 100 ng/ml PTx or for 30 min with Bis I or U0126, then treated for 2 min with LPA before assessing EGFR tyrosine phosphorylation. Similar to previous results (Kassel et al., 2007), LPA stimulated EGFR phosphorylation by 2.3 ± 0.4-fold (p 0.05; Fig. 8). This transactivation was not inhibited by Bis I and was actually enhanced by PTx (Fig. 8). However, U0126 inhibited basal and LPA-stimulated EGFR phosphorylation by 50 ± 20 and 40 ± 10%, respectively (Fig. 8). The lack of an effect of PTx on EGFR phosphorylation is in agreement with the lack of an effect of PTx on both the decrease in EGFR binding and on ERK1/2 phosphorylation. The inability of Bis I to inhibit LPA-stimulated EGFR phosphorylation and the inability of PMA to stimulate EGFR phosphorylation (data not shown) suggest that the actions of PKC in the rapid decrease in binding occur in parallel with EGFR transactivation. Finally, the ability of U0126 to partially inhibit the LPA-induced EGFR phosphorylation suggests that MEK/ERK may be involved both upstream and downstream of EGFR phosphorylation.
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), and this transactivation leads to ERK1/2 phosphorylation. In addition, PKC activation also plays a role in ERK1/2 phosphorylation, but PKC does not contribute to transactivation of the EGFR. MEK and ERK1/2 activation then lead to the rapid decrease in EGFR binding. PKC activation early in the course of treatment also contributes to the sustained decrease in binding observed at 18 h, whereas ERK1/2 phosphorylation seems to contribute to the magnitude of the 18-h decrease primarily by its induction of the rapid decrease.
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LPA stimulates ERK1/2 phosphorylation in the BEAS-2B airway epithelial cell line, similar to previous data with primary cultures of human bronchial epithelial cells (Saatian et al., 2006). ERK1/2 phosphorylation was completely inhibited by the MEK inhibitor U0126 but not by PTx, similar to the effects of these inhibitors on the LPA-stimulated rapid decrease in EGFR binding. However, in two other epithelial cells types, corneal epithelial cells and primary human bronchial epithelial cells, LPA-stimulated ERK1/2 phosphorylation was PTx-sensitive (Wang et al., 2003; Zhang et al., 2006). Likewise, previous studies from our laboratory found that ERK1/2 activation by LPA in HASM cells was also PTx-sensitive (Ediger et al., 2003). This study is the first to show PTx-insensitive ERK1/2 phosphorylation by LPA in epithelial cells. Furthermore, these data suggest that ERK1/2 phosphorylation is mediated by different G protein pathways and possibly by different LPA receptor subtypes in BEAS-2B cells versus HASM cells.
We previously showed that LPA was able to stimulate phosphorylation of the EGFR in BEAS-2B cells, and this "transactivation" was blocked by both the MMP inhibitor GM6001 and the EGFR tyrosine kinase domain inhibitor AG1478 (Kassel et al., 2007). The current studies show that LPA-stimulated ERK1/2 phosphorylation is also decreased by both GM6001 and AG1478, suggesting that transactivation of the EGFR is involved in ERK1/2 activation. These results agree with many studies showing that EGFR transactivation is necessary for ERK1/2 phosphorylation by LPA in other cell types (Shah et al., 2005a,b; Kang et al., 2006). EGFR transactivation has also been linked to a decrease in 125I-EGF binding in several cell types, including rat hepatic C9 cells, human embryonic kidney 293 cells, and COS-7 cells (Pierce et al., 2000; Olivares-Reyes et al., 2005).
ERK1/2 has been shown to be involved in many of the inflammatory processes associated with asthma and COPD. The role of ERK1/2 in proliferation of airway smooth muscle cells, which is inappropriately enhanced in asthmatic airways, is perhaps the most obvious example (Ammit and Panettieri, 2001). Transepithelial migration of neutrophils and the production and secretion of interleukin-8, granulocyte/macrophage colony-stimulating factor, and mucin are stimulated by EGFR activation and ERK1/2 signaling in airway epithelial cells (Hewson et al., 2004; Ogawa et al., 2006; Nakanaga et al., 2007). The ability of LPA to rapidly stimulate EGFR activation and ERK1/2 phosphorylation in airway epithelial cells, together with the known roles of EGFRs and ERK1/2 in regulating proliferation and inflammation in these same cells, are consistent with proposed roles for LPA in diseases such as asthma and cancer.
In contrast to U0126, PKC inhibitors were less effective at inhibiting the early phase but were able to completely block the later decrease in binding induced by LPA. Studies with DN-PKC and DN-PKC cells confirmed this conclusion; both DN-PKCs only shifted the LPA dose-response curve and did not affect the magnitude of the response after 15 min of treatment. However, the later phase of the LPA-induced decrease in binding was completely eliminated in DN-PKC cells, suggesting that PKC plays an essential role in the later decrease in binding in BEAS-2B cells. Although PKC is required for the later phase of the decrease in EGFR binding, this later effect is a result of PKC activation at earlier time points because the PKC inhibitor only inhibited the 18-h decrease when it was present during the first 3 h of LPA treatment. The role of PKC is probably downstream of Gq/11 because the rapid and sustained decreases in binding and ERK1/2 phosphorylation are not inhibited by PTx. It is interesting to note that the direct PKC activator PMA mimicked the effect of LPA to stimulate both a rapid and sustained decrease in EGFR binding in BEAS-2B cells, and this effect was completely blocked by the PKC inhibitors at all time points. Although PMA can mimic LPA at both 15 min and 18 h via PKC activation, PKC seems to be the key mediator of LPA effects for the sustained phase but to be only one of multiple contributors to the early decrease induced by LPA.
PKC contributes to diverse cellular responses that are implicated in lung disease. These include endothelial barrier dysfunction and pulmonary edema, proliferative responses of pulmonary artery smooth muscle cells, and bronchospasm and mucus production in asthma and COPD. Airway epithelial cells induce proinflammatory mediators and MUC5AC via PKC, and PKC is increased in lungs of patients with COPD. In addition, PKC is consistently altered in human tumor cells, and its overexpression is associated with a more malignant phenotype (Dempsey et al., 2007). The possible contributions of PKC-mediated changes in EGFR binding in these processes remain to be established.
LPA time course studies with inhibitors of MEK and PKC confirmed that the early phase is primarily mediated by MEK, although PKC does contribute to ERK1/2 activation as well. The later phase is primarily mediated by PKC, although the early phase contributes to the magnitude of the decrease at longer time points, and all of the PKC-dependent effects seem to occur within the first 3 h. The rapid phase, assessed by U0126 sensitivity, begins immediately upon treatment with LPA and lasts until the maximal decrease at 15 min of treatment. The later decrease in binding between 6 and 18 h of LPA treatment seems to be completely mediated by PKC because it is blocked by the PKC inhibitors and is absent in the DN-PKC cells, but it is not significantly inhibited by U0126. From 15 min to at least 6 h after treatment, both MEK and PKC seem to contribute to the decrease in binding because both inhibitors partially reduce the decrease. It is interesting to note that the time course for LPA in the presence of the PKC inhibitor (Fig. 5C) closely resembles the time courses for H292 and A549 lung cancer cells reported previously (Kassel et al., 2007), suggesting that defective activation of PKC may explain the transient nature of the decrease in these lung cancer cells.
The effects of both U0126 and Bis I on ERK1/2 were similar to their effects on the LPA-induced decrease in EGFR binding. U0126 completely blocked both ERK1/2 phosphorylation and the rapid decrease in binding, whereas Bis I only inhibited both of these effects by approximately half. To the extent that U0126 effects accurately define the role of ERK1/2 activation, these data suggest that ERK1/2 activation may be involved upstream and downstream of transactivation because U0126 partially inhibits transactivation, and GM6001 and AG1478 both partially inhibit ERK1/2 phosphorylation.
In conclusion, these studies suggest that LPA stimulation of EGFR transactivation and PKC activation both contribute to ERK1/2 activation and the subsequent rapid decrease in EGFR binding. These studies also implicate LPA activation of PKC as the mechanism leading to the sustained decrease in EGFR binding. The key pathways involved in these biphasic changes in EGFR binding are summarized in the model shown in Fig. 9, although many details remain to be established. The decrease in EGFR binding induced by LPA after EGFR, ERK1/2, and PKC activation is perhaps mediated by EGFR internalization or desensitization mechanisms that would probably reduce long-term actions mediated by EGFRs. The inability of cancer cells to sustain these decreases in EGFR binding, as reported in our previous studies (Kassel et al., 2007), could contribute to their inappropriate proliferation and migration. Thus, LPA may play multiple roles in airway epithelial cell physiology and pathology that are determined by its ability to activate EGFRs, ERK1/2, and PKC and by its subsequent effects to reduce cell surface EGFR binding and signaling.
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Acknowledgements |
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Footnotes |
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Portions of this work were published as part of a dissertation: Kassel KM (2007) Regulation of epidermal growth factor receptors and mitogenic signaling by lysophosphatidic acid and β2 adrenergic receptors in airway cells. Ph.D. thesis, University of Nebraska Medical Center, Omaha, NE.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; LPA, lysophosphatidic acid; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; PKC, Ca2+- and phospholipid-dependent protein kinase; HASM, human airway smooth muscle; RO 31-8220, 2-[1-(3-(amidinothio)propyl)-1H-indol-3-yl]-3-(1-methylindol-3-yl) maleimide · methanesulfonate; GM6001, N-[(2R)-2-hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; Bisindolylmaleimide I, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; AG1478, 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline; PMA, phorbol-12-myristate-13-acetate; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate; DN, dominant negative; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PTx, pertussis toxin; Bis I, Bisinolylmaleimide I; MMP, matrix metalloproteinase; PAGE, polyacrylamide gel electrophoresis; COPD, chronic obstructive pulmonary disease.
Address correspondence to: Dr. Myron L. Toews, 985800 Nebraska Medical Center, Omaha, NE 68198-5800. E-mail: mtoews{at}unmc.edu
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