Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6260
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ABSTRACT |
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Human airway smooth muscle cells treated with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) exhibit synergistic stimulation of mitogenesis (Ediger TL and Toews ML. J Pharmacol Exp Ther 294: 1076-1082, 2000). The effects of LPA treatment of human airway smooth muscle cells on EGF receptor (EGFR) regulation have now been investigated. LPA treatment for 12-24 h resulted in a twofold increase in 125I-EGF binding and EGFR protein levels as assessed by Western blot analysis. Competition binding assays indicated single-site binding with an affinity of 3 nM, and the affinity was not changed by LPA treatment. EGFR upregulation was blocked by cycloheximide and actinomycin D, suggesting that LPA influences transcriptional regulation of EGFR expression. Inhibitor studies revealed a prominent role for activation of mitogen-activated protein kinase and p70 ribosomal S6 kinase. Both synergism and EGFR upregulation increased with increased cell density, whereas EGFR expression in control cells decreased. The similar requirements for exposure time, LPA concentrations, and cell confluence suggest that EGFR upregulation may be one contributing factor to the synergistic stimulation of mitogenesis seen with LPA plus EGF.
epidermal growth factor receptor regulation; airway remodeling; proliferation; synergism
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INTRODUCTION |
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THE EPIDERMAL GROWTH
FACTOR (EGF) receptor (EGFR) is a single-transmembrane tyrosine
kinase receptor involved in proliferation, differentiation, motility,
and survival (25). The EGFR activates the classic p42/44
mitogen-activated protein kinase (MAPK)/extracellular signal-regulated
kinase (ERK) pathway in which autophosphorylation of the EGFR creates
SH2-binding sites for the adaptor proteins Grb2 and Shc, which then
recruit the Ras guanine nucleotide exchange factor SOS, leading to
sequential activation of Ras, Raf, MAPK kinase (MEK), and ERK. In
addition to the MAPK pathway, activation of the EGFR by EGF also leads
to activation of phosphatidylinositol 3-kinase (PI3K), phospholipase
C-, and the protein tyrosine phosphatase SHP2 (15).
Although these pathways are downstream of ligand binding to the EGFR,
recent studies (7, 8) suggest that the EGFR can also be
involved in EGF-independent proliferative signaling downstream of G
protein-coupled receptor (GPCR) activation. This EGF-independent EGFR
transactivation may be required in some systems for GPCR-mediated ERK
activation. These studies have prompted interest in the potential role
of the EGFR in GPCR signaling.
Lysophosphatidic acid (LPA) is the simplest endogenous phospholipid, released from activated platelets and present in serum at concentrations of 2-20 µM (17). LPA has been shown to mimic the effects of serum in many systems (16). Recently, multiple GPCRs of the Edg or LPA receptor family have been cloned and shown to be LPA receptors (2, 3, 5, 13). LPA receptors can couple to G proteins of the Gi/o, Gq/11, and G12/13 families; activation of LPA receptors can lead to proliferative signaling through the MAPK and Rho pathways as well as to structural changes related to migration and stress fiber formation through Rho-activated pathways (4).
Human airway smooth muscle (HASM) cells treated simultaneously with EGF and LPA exhibit a markedly synergistic activation of mitogenesis (6). This synergistic stimulation of proliferation may be of importance in the pathogenesis of asthma and other diseases of airway remodeling in which airway smooth muscle thickening is a hallmark feature (12, 20). Accordingly, we have explored the molecular mechanism(s) of synergism by investigating potential interactions between the pathways activated by LPA and EGF. Here we report that LPA treatment causes an upregulation of EGFR and investigate the signaling pathways and mechanisms involved. This EGFR upregulation may also be relevant to asthma because increased EGFR levels have recently been demonstrated in airway smooth muscle and epithelial cells in asthmatic tissue (1, 21).
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METHODS |
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Reagents. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY). EGF was purchased from BioSource International (Camarillo, CA) and 18:1(oleoyl)-LPA was from Avanti Polar Lipids (Alabaster, AL). Actinomycin D was from BIOMOL (Plymouth Meeting, PA), pertussis toxin (PTX) was from List Biologicals (Campbell, CA), [3H]thymidine was from NEN (Boston, MA), and 125I-EGF was from Amersham Pharmacia Biotech (Piscataway, NJ). Other chemicals were obtained from Sigma (St. Louis, MO).
Cell culture. HASM cells previously isolated from human trachea by enzymatic dissociation (19) were kindly provided by Dr. Michael Kotlikoff (University of Pennsylvania, Philadelphia, PA) (10, 11). Cells were cultured in high-glucose (4.5 g/l) DMEM with 10% FBS at 37°C in a humidified 5% CO2 incubator and passaged weekly, with cells used between passages 4 and 10. For assays of the amount of receptor expression per cell, cells were counted with a Coulter counter.
[3H]thymidine incorporation assays. [3H]thymidine incorporation assays were performed as previously described (9). Confluent HASM cells were starved in serum-free medium for 24 h before treatment with mitogens for 24 h. [3H]thymidine (2 µCi/ml) was added for the final 2 h of mitogen treatment. Cells were then washed once with PBS and twice with 10% trichloroacetic acid (one 10-min incubation followed by one wash). The precipitated DNA was dissolved with 0.2 N NaOH, and [3H]thymidine incorporation was quantitated by liquid scintillation counting.
Flow cytometric analysis of cell cycle. S phase analysis was performed as previously described (9). Confluent HASM cells were starved in serum-free medium for 24 h before treatment with mitogens for 24 h. Cells that were analyzed before confluence were also plated at 100,000 cells/60-mm dish but were treated with mitogens 2 days after plating, without starving. After treatment, cells were washed once with PBS, removed from the dish by trypsinization, and resuspended in Vindelov's reagent [75 µg/ml of propidium iodide, 3.5 U/ml of ribonuclease A, and 0.1% Nonidet P-40 in Tris-buffered saline (3.5 mM Tris, pH 7.6, and 10 mM NaCl)]. Flow cytometric analysis was performed with a Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer and modeled 10,000 events; data were analyzed with ModFit LT software from Verity Software House (Topsham, ME).
Second messenger assays. According to the cAMP accumulation assay method of Shimizu et al. (23), cells prelabeled with [3H]adenine were treated with agents that altered adenylyl cyclase activity, and the conversion of [3H]ATP to [3H]cAMP was quantitated. Phosphoinositide hydrolysis was measured essentially as previously described (18). HASM cells were labeled with [3H]inositol; after stimulation, labeled compounds were extracted, and [3H]inositol phosphates were separated from [3H]phosphoinositides by ion-exchange chromatography.
125I-EGF binding.
HASM cells were plated at 40,000 cells/well in six-well plates and
grown to confluence. Cells were starved in serum-free medium for
24 h and then treated with growth factors and/or inhibitors for
the indicated time period, typically 12 h. Cells were then washed
once with 2 ml of 37°C DMEM-HEPES, washed once with 2 ml of ice-cold
DMEM-HEPES, and then incubated with 125I-EGF for 4 h
on ice. The 125I-EGF solutions contained 25,000-80,000
counts · min1 · ml
1
(~20-65 pM) in 0.1% BSA-containing DMEM-HEPES. Nonspecific
binding was defined by the addition of 100 ng/ml (17 nM) of
nonradioactive EGF. After 4 h on ice, the cells were washed four
times with 2 ml of ice-cold DMEM-HEPES containing 0.1% BSA, dissolved
in 0.2 N NaOH, and transferred to glass tubes to be counted in a gamma counter.
Western blotting for total and tyrosine-phosphorylated EGFR. HASM cells were plated at 250,000 cells/plate in 100-mm plates and grown to confluence. Cells were starved in serum-free medium for 24 h and then treated with LPA, EGF, LPA plus EGF, or the BSA vehicle for the indicated times before being harvested. Cells were then washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris · HCl, pH 8.0). The lysates were collected and passed through a 25-gauge needle six to eight times. Proteins in the lysates were separated by SDS-PAGE on 7.5% acrylamide gels, transferred to nitrocellulose membrane, and blotted with an anti-EGFR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by chemiluminescence detection with LumiGLO (New England Biolabs, Beverly, MA). For phosphotyrosine analysis, lysates were immunoprecipitated with an anti-EGFR antibody conjugated to agarose beads (Santa Cruz Biotechnology); the immunoprecipitated EGFR was solubilized from the beads and then run on SDS-PAGE. Proteins were transferred to nitrocellulose membrane, blots were probed with the PY99 anti-phosphotyrosine antibody (Santa Cruz Biotechnology), and the signal was detected with LumiGLO chemiluminescence and exposure to film. Films were analyzed by scanning laser densitometry and analyzed with ImageQuant software (Molecular Dynamics/Amersham Pharmacia Biotech).
Data analysis and statistics. Data were analyzed with GraphPad Prism 3.0 software (GraphPad, San Diego, CA). Evaluations of significance employed the two-tailed paired t-test for the comparison of two values or one-way analysis of variance (ANOVA) followed by either the Bonferroni posttest for the comparison of multiple values in one group or Dunnett's posttest for the comparison of values to control. Competition binding curves were analyzed with nonlinear regression analysis.
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RESULTS |
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Lack of effect of EGF on LPA signaling pathways. To determine whether EGF changes functional responses to LPA, the effects of EGF pretreatment of HASM cells on LPA stimulation of inositol phosphate production and inhibition of cAMP accumulation were examined. Pretreatment of HASM cells with 60 ng/ml of EGF for either 4 or 12 h did not change the production of inositol phosphates by 10 µM LPA. Under control conditions, LPA stimulated inositol phosphate production by 3.9 ± 0.4-fold, similar to previous studies (18); with 4 h of EGF pretreatment, LPA stimulated inositol phosphate production by 3.9 ± 0.5-fold. Similarly, after 12 h, LPA stimulated inositol phosphate production by 3.1 ± 0.6-fold for control cultures and 3.3 ± 0.6-fold for cultures after EGF pretreatment. Additionally, no stimulation of inositol phosphate production by EGF alone was detected (0.9 ± 0.1- and 1.0 ± 0.1-fold after 4 and 12 h, respectively). Similarly, pretreatment of HASM cells with EGF did not change the effect of LPA to inhibit cAMP accumulation. LPA decreased forskolin stimulation of cAMP production in HASM cells by 43 ± 8%, similar to that seen in previous studies (18); after pretreatment of HASM cells with 60 ng/ml of EGF for 4 h, LPA decreased forskolin stimulation of cAMP production in HASM cells by 47 ± 10%.
Duration of mitogen treatment required for mitogenesis and
synergism.
To determine the duration of mitogen exposure required for mitogenic
stimulation by LPA, EGF, and the combination of LPA plus EGF, a
mitogen washout experiment was performed (Fig.
1). HASM cells that were treated with LPA
for 6 or 8 h before the mitogen was removed showed stimulation of
[3H]thymidine incorporation at the 24-h time point.
Stimulation of [3H]thymidine incorporation by LPA
appeared to plateau by 12 h of treatment. In contrast, stimulation
of [3H]thymidine incorporation in response to EGF was not
seen at 6 or 8 h but was present and maximal after 12 h of
exposure. Similarly, synergism in response to LPA plus EGF was not seen
until cells were treated with the combination for 12 h; at earlier
time points, the results of stimulation by LPA plus EGF were the same
as those of stimulation by LPA alone. These results suggested that
8-12 h of treatment with both mitogens together was required for
the interaction of the LPA and EGF pathways that results in synergism. Additionally, the earlier effects of LPA in these mitogen washout experiments suggested that LPA might increase the capacity of the EGFR
pathway to be activated in response to EGF.
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Upregulation of EGFR by LPA.
To investigate potential mechanisms by which LPA increases EGF
responsiveness, the effects of LPA pretreatment on EGFR binding were
determined. LPA increased 125I-EGF binding to HASM cells by
approximately twofold but only after 8 h or more of exposure to
LPA (Fig. 2A). EGFR
upregulation was nearly maximal at 12 h, with only a small further
increase at 24 h. Thus LPA increased EGFR binding with a time
course consistent with the effect of LPA in enhancing EGF-stimulated
[3H]thymidine incorporation (Fig. 1). Upregulation was
concentration dependent, with half-maximal upregulation at ~1 µM
and nearly twofold upregulation at 10 µM (Fig. 2B).
Further experiments to characterize LPA upregulation of EGFR levels
focused on the 12-h time point and used 10 µM LPA. Treatment of HASM
cells with the HASM cell mitogen thrombin (2 U/ml) also induced EGFR
upregulation, similar to that seen with 10 µM LPA (Fig.
2B).
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Competition binding analysis of upregulated receptors.
To determine whether the increase in EGFR binding represented an
increase in the total number of cell surface receptors or an increase
in binding affinity of the existing receptors, competition binding
assays were performed (Fig. 3).
Competition binding analysis indicated single-site binding;
pretreatment with LPA for 12 h resulted in a twofold increase in
125I-EGF binding but did not significantly change the
EC50 for EGF competition. The EC50 value for
cells treated with the vehicle BSA was 3.5 nM, whereas after LPA
treatment, the EC50 value was 2.6 nM (P > 0.05 comparing best fit values by two-tailed t-test). Thus
an increase in affinity is insufficient to explain the increased EGFR
binding, suggesting that upregulation is due to an increase in total
receptor number rather than an increase in binding affinity.
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Sensitivity of LPA-induced EGFR upregulation to cycloheximide and
actinomycin D treatment.
To investigate whether the upregulated receptors represented newly
synthesized receptors, the effects of the protein synthesis inhibitor
cycloheximide and the transcription inhibitor actinomycin D on EGFR
upregulation by LPA were assessed (Fig.
4). HASM cells treated with LPA in the
presence of cycloheximide did not show EGFR upregulation. Inclusion of
actinomycin D during the 12-h LPA treatment also blocked EGFR
upregulation. Together, these results suggest that upregulation results
from transcriptional activation and synthesis of additional EGFRs.
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Western blot analysis of upregulated receptors. To confirm that the increase in EGFR levels seen by 125I-EGF binding resulted from an increase in the number of EGFR molecules, EGFR protein levels were assessed with Western blot analysis of whole cell lysates. LPA treatment of HASM cells for 12 h resulted in an increase in EGFR immunoreactivity of similar magnitude (1.8 ± 0.3-fold; Fig. 4C) to the increase seen in binding assays (Figs. 2-4). In contrast, cells treated with EGF for 12 h showed dramatically lower EGFR protein levels, 53 ± 16% of control levels, presumably due to EGF-stimulated downregulation of EGFR. EGFR levels in cells treated with LPA plus EGF were 94 ± 19% of those in control cells, greater than with EGF alone but less than with LPA treatment alone.
Effects of signaling pathway inhibitors on LPA-induced EGFR
upregulation.
Several other HASM cell responses to LPA are PTX sensitive, in
particular, mitogenesis (6). The sensitivity of
LPA-induced EGFR upregulation to treatment with PTX, which inactivates
Gi, was tested. Surprisingly, PTX pretreatment caused
only a 30 ± 4% inhibition of EGFR upregulation by LPA
(Table 1), suggesting that Gi
activation is not the major mechanism for LPA-induced EGFR
upregulation. To further identify LPA signaling pathways for EGFR
upregulation, inhibitors of other pathways were also tested (Table 1).
Both the MEK inhibitor U-0126 and rapamycin, which inhibits the
activation of p70 ribosomal S6 kinase (p70S6k), showed
significant inhibition of LPA-induced EGFR upregulation, implicating
both ERK and p70S6k signaling. Adding both inhibitors
simultaneously did not yield greater inhibition of EGFR upregulation
(n = 2; P > 0.05 by ANOVA; data not
shown). In contrast, neither the PI3K inhibitor LY-294002, the Src
inhibitor PP1, nor the protein kinase C inhibitor calphostin C caused
significant inhibition of EGFR upregulation by LPA.
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Role of EGFR tyrosine kinase activity in LPA-induced EGFR
upregulation.
Because transactivation of the EGFR has recently been implicated in ERK
activation by several GPCR signaling pathways (7, 8), we
used the EGFR tyrosine kinase inhibitor AG-1478 to investigate whether
the tyrosine kinase activity of the EGFR was required for its
upregulation by LPA. However, analysis of the inhibitory effect of
AG-1478 was complicated by the effect of AG-1478 itself to upregulate
EGFR binding. EGFR binding values increased 4.5 ± 0.3-fold after
AG-1478 treatment in the absence of LPA (Fig. 5A), preventing a simple
comparison of the fold increase by LPA in the absence and
presence of AG-1478. However, when cells were treated with LPA in the
presence of AG-1478, a further EGFR upregulation was observed (Fig.
5A), similar in magnitude to that seen in the absence of
AG-1478, suggesting that LPA-induced EGFR upregulation does not require
EGFR tyrosine kinase activity.
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Effect of HASM cell confluence on synergism and on LPA-induced EGFR
upregulation.
To investigate the effect of confluence on EGFR upregulation,
subconfluent cells were assayed for LPA-induced EGFR upregulation. In
these experiments with subconfluent cells, cells were not starved before treatment with LPA. HASM cells assayed on day 2 after being plated did not show EGFR upregulation in response to LPA
treatment; however, there was a progressive increase in EGFR
upregulation with further time in culture (Fig.
6A). Similarly, subconfluent HASM cells showed only additive rather than synergistic mitogenic responses to treatment with LPA plus EGF, whereas confluent cells treated with LPA plus EGF showed strongly synergistic
[3H]thymidine incorporation responses (Table
2). Similar results were obtained with
flow cytometric analysis of S phase progression (Table 2). Thus both
synergism and EGFR upregulation appear to be features of highly
confluent cells. Interestingly, the increase in EGFR upregulation as
cells became more confluent was accompanied by a decrease in
the level of EGFR expression in control cells (Fig.
6B).
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DISCUSSION |
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Synergistic stimulation of HASM cell mitogenesis by the GPCR mitogen LPA and the receptor tyrosine kinase (RTK) growth factor EGF suggests that GPCR pathways and RTK pathways interact to enhance mitogenic signaling. LPA-activated second messenger pathways downstream of both Gi and Gq were not modulated by EGF pretreatment. In contrast, LPA pretreatment of HASM cells increased binding to cell surface EGFR, suggesting that one mechanism for LPA modulation of EGF responsiveness is the upregulation of EGFR levels.
The increased EGFR binding after LPA treatment of HASM cells appeared to be due to an increase in EGFR synthesis because increased EGFR binding was sensitive to cycloheximide and actinomycin D, and Western blot analysis demonstrated increased EGFR protein. Both ERK and p70S6k play a role in EGFR upregulation by LPA, and a Gi-mediated signal may also be involved. EGFR upregulation by LPA does not require activation of protein kinase C, PI3K, or Src.
Interestingly, EGFR upregulation did not require the intrinsic tyrosine kinase activity of the EGFR, suggesting that transactivation of EGFR by LPA is not involved in EGFR upregulation. This observation is consistent with the lack of effect of the EGFR tyrosine kinase inhibitor AG-1478 on other LPA-mediated effects in these cells, including ERK activation and mitogenesis (T. L. Ediger and M. L. Toews, unpublished data). Additionally, we did not observe tyrosine phosphorylation of the EGFR upon LPA treatment. Together, these results suggest that LPA does not cause ligand-independent EGFR transactivation in HASM cells, consistent with the recently reported lack of EGFR transactivation by histamine, carbachol, and thrombin, other GPCR mitogens for HASM cells (14).
The EGFR tyrosine kinase inhibitor AG-1478 also induced a marked upregulation of cell surface EGFR binding. This AG-1478-induced upregulation is clearly distinct from that induced by LPA because it occurred within minutes of AG-1478 exposure, was insensitive to cycloheximide, and was not accompanied by an increase in EGFR protein (T. L. Ediger and M. L. Toews, unpublished data). Thus AG-1478 apparently increases EGFR binding activity by altering the properties of the existing receptors, whereas LPA increases binding by transcriptional mechanisms, leading to increased EGFR expression.
Western blot analysis showed an upregulation in HASM cells treated with LPA alone that was similar to that seen in radioligand binding assays with 125I-EGF, confirming that EGFR upregulation is due to new protein synthesis. In contrast, HASM cells treated with EGF alone showed a marked downregulation of EGFR levels. In the presence of both LPA and EGF, EGFR levels were greater than those with EGF alone, although clearly less EGFR protein was present with LPA plus EGF than with LPA alone. Thus the EGFR level seen when HASM cells were treated with the combination of LPA plus EGF most likely represents a balance between LPA-induced upregulation and EGF-induced downregulation. The ability of LPA to counteract the EGFR downregulation that occurs with EGF treatment provides one potential mechanism for the synergistic mitogenesis seen in HASM cells treated with LPA plus EGF.
The initial goal of these studies was to identify potential molecular mechanisms contributing to the strikingly synergistic mitogenesis observed when HASM cells are treated with LPA plus EGF. Multiple lines of evidence point to a link between EGFR upregulation and the synergism between LPA and EGF. Analysis of the concentration dependence of LPA-induced EGFR upregulation indicated that 10 µM LPA was required to see EGFR upregulation, consistent with previously published data (6) showing a similar LPA concentration dependence for both LPA-stimulated mitogenesis and for its synergism with EGF. More importantly, the time courses for synergism and EGFR upregulation in response to LPA were strikingly similar (Figs. 1 and 2A), with both processes becoming apparent only after 8-12 h of exposure to LPA. Additionally, high cell density was required for both synergism and LPA-induced EGFR upregulation. Finally, EGFR upregulation was also seen when HASM cells were treated with thrombin, a second GPCR mitogen that shows synergism with EGF for HASM cell mitogenesis (9, 14). Together, the correlation of LPA concentrations, time courses, cell growth conditions, and the similar effects of thrombin and LPA to evoke synergism and upregulate the EGFR all suggest a mechanistic link between the two phenomena.
The maximal mitogenic response to LPA plus EGF was not significantly
different between confluent and subconfluent cells. Rather than larger
responses to the combination of LPA plus EGF, confluent cells showed
smaller responses to both LPA and EGF individually. Correspondingly,
the occurrence of synergism with confluent cells may reflect the
smaller responses to individual mitogens. Density-dependent downregulation of EGFR, platelet-derived growth factor receptors, fibroblast growth factor receptors, and transforming growth factor- receptors have been reported in fibroblasts (22).
Similarly, a decrease in the number of EGFRs per cell with increasing
confluence was also observed for HASM cells in our studies. Perhaps
confluent HASM cells undergo a generalized downregulation of GPCRs and
growth factor receptors; synergism may thus occur when confluent, less responsive cells are simultaneously treated with a combination of
mitogens, at least one of which may effect a generalized upregulation of growth factor-mediated responses by upregulating cell surface growth
factor receptors.
Although upregulation of EGFR expression may represent one component of the mechanism for synergism, other potential mechanisms have been proposed. In another study (14) with HASM cells, greater activation of p70S6k was seen in response to thrombin plus EGF than to either agent alone, suggesting that a p70S6k-mediated response may be involved in synergistic proliferative signaling. In our studies, rapamycin sensitivity implicated p70S6k activation in EGFR upregulation by LPA, suggesting EGFR upregulation as one potential mechanism by which p70S6k contributes to synergistic signaling.
Based on the known effects of LPA on HASM cell proliferation (6) and airway smooth muscle contractility (24), we hypothesized that LPA may be a mediator of asthma and airway remodeling. Because EGFR upregulation in epithelial and smooth muscle cells has been reported in asthmatic lung tissue (1, 21), the ability of LPA to upregulate the EGFR provides additional support for our hypothesis. More importantly, our results with both LPA and thrombin suggest GPCR-mediated transcriptional regulation as an important mechanism contributing to this increase in EGFR expression. Both LPA and thrombin are released from activated platelets and thus may be present at sites of injury and inflammation. Upregulation of smooth muscle cell EGFRs by these agents could potentially contribute to the increased smooth muscle mass and proliferation that occur in airway remodeling, and strategies targeted at preventing GPCR-mediated upregulation of EGFR may provide new avenues for treatment of airway remodeling and asthma.
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ACKNOWLEDGEMENTS |
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This work was supported in part by research seed grants from the University of Nebraska Medical Center (to M. L. Toews), a University of Nebraska Medical Center Emley Fellowship (to T. L. Ediger), and by the American Society for Pharmacology and Experimental Therapeutics Summer Research Program (B. L. Danforth).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. L. Toews, Dept. of Pharmacology, Univ. of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198-6260 (E-mail: mtoews{at}unmc.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.
Received 31 May 2001; accepted in final form 22 August 2001.
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